JP2002522061A - Vanadate-resistant glycosylation 4 gene - Google Patents

Abstract

  (57) [Summary] The present invention is a vanadate-resistant glycosylation (VRG4) gene and gene product from pathogenic yeasts and fungi that encode Golgi-nucleotide-sugar transporters. Methods for detecting transporter activity and screening for candidate antifungal compounds are disclosed.

Description

DETAILED DESCRIPTION OF THE INVENTION

FIELD OF THE INVENTION [0001] The present invention is directed to identifying inhibitors of the GDP-mannose transporter for use as the yeast vanadate-resistant glycosylation 4 (VRG4) gene and homologs thereof and antifungal compounds. The present invention relates to proteins encoded therein useful in the method.

BACKGROUND OF THE INVENTION The Golgi apparatus is the site where terminal glycosylation of both proteins and lipids occurs. Unlike mammalian cells, yeast S. In cerevisiae, glycosylation and sphingolipids are exclusively modified by the addition of mannose residues in the Golgi. Glycosylation undergoes two types of modifications, oligosaccharides either bind to asparagine residues (N-linked) or to serine / threonine residues (O-linked) (for review, 1,2 reference).
Both of these pathways begin at the endoplasmic reticulum (ER) and end at the Golgi. After transport of the protein to the Golgi apparatus, most N-linked oligosaccharides are extended by a series of different mannosyltransferases to form glycoproteins containing outer chains of more than 50 mannose residues. α1,6-linked outer chain is α1,2-
And highly branched by α1,3-linked mannose. As in higher eukaryotes, the various mannosyltransferases that catalyze these sequential reactions appear to be compartmentalized within each Golgi capsule. In O-linked sugars, up to 5 mannoses are added after the first mannose in the ER (3
, 4). Sphingolipids containing phosphoinositol in yeast also undergo mannoseization in the Golgi apparatus. S. In cerevisiae, there are three main classes of sphingolipids. These include inositol-phosphorylceramide (IPCs) and mannosyl-inositol phosphorylceramide (MIC).
PC and M (IP) ₂ C) (for review see 5). MIPC and M
(IP) ₂ C contains a single mannose linked to inositol, but little is known about mannosyltransferases that catalyze this reaction.

[0003] The mannosyl donor for all of these Golgi localization reactions is the nucleotide sugar GDP-mannose, the site of synthesis of which is the cytoplasm. GDP-mannose must be transported to the Golgi by a specific nucleotide sugar transporter before it can be utilized by different rumen mannosyltransferases (7). If a sugar is added to the rumen mannosyltransferase acceptor, diphosphate GDP is converted to monophosphate by nucleoside diphosphatase (7). As in the mammalian Golgi apparatus, transport of nucleotide sugars to the rumen is coupled to the monophosphate surface exit in yeast. The yeast GDPase-encoding gene GDA1 has been isolated (8). As expected, deletion of GDA1 results in low glycosylation of proteins and lipids, while null alleles have no effect on growth.

[0004] The activities of many nucleotide sugar transporters have been reported, differing from each other in their substrate specificity and subcellular localization (9). Because cytoplasms are the only sites where nucleotide sugars are synthesized, they must be transported to various organelles where glycosylation takes place. Mammalian cells require the transport of many different nucleotide sugars due to the diversity of carbohydrate processing within the ER and Golgi. The carbohydrate chains may include galactose, sialic acid, fucose, xylose, N-acetylglucosamine, and N-acetylgalactosamine. In contrast, S.D. In S. cerevisiae, glycosylation of the Golgi apparatus is largely limited in principle to mannoseylation, which requires only a single transporter.

[0005] The VRG4 gene is an essential gene required for many different Golgi-specific functions, including N-linked glycosylation (10-12), secretion, protein classification and maintenance of the normal inner membrane system ( 12).

[0006] The present invention discloses that the transport of GDP-mannose to the Golgi is a major function of the VRG4 gene product in yeast. The present invention also relates to the use of VRG4
It is disclosed that it contains a gene homolog. The VRG4 gene is essential for survival. GD
A simple system for assaying P-mannose transport is disclosed using permeabilizing yeast sphingolipids. Methods for identifying putative inhibitors and gene products of the VRG4 gene are disclosed.

SUMMARY OF THE INVENTION The present invention provides a nucleotide-sugar to a source comprising a nucleotide-sugar transporter bound to a phospholipid membrane and measures the amount of nucleotide-sugar bound or transported across the membrane. A method for measuring the activity of a nucleotide-sugar transporter derived from yeast.

[0008] The invention further provides a nucleotide-sugar to a source comprising a nucleotide-sugar transporter bound to a phospholipid vesicle, and measures the amount of nucleotide-sugar transported or accumulated in the vesicle. And a method for measuring the activity of a yeast-derived nucleotide-sugar transporter.

[0009] It is an object of the present invention to provide nucleotide-sugars to sources from permeabilized yeast spheroplasts, including nucleotide-sugar transporters and Golgi apparatus, and as indicators of nucleotide-sugar transporter activity. An object of the present invention is to provide a method for measuring the activity of a Golgi nucleotide-sugar transporter from yeast, which comprises measuring the amount of nucleotide-sugar bound to the Golgi.

[0010] Another object of the invention is to provide a putative inhibitor with a nucleotide-sugar-derived source from permeabilized yeast spheroplasts, including nucleotide-sugar transporters and Golgi apparatus. Measuring the amount of Golgi-linked nucleotide-sugar in the presence of the inhibitor, as compared to the amount of Golgi-linked nucleotide-sugar in the absence of the candidate inhibitor, the Golgi-nucleotide-sugar transporter in yeast To identify inhibitors of the activity of

[0011] One aspect of the present invention is a permeabilized yeast cell useful in a method for evaluating a nucleotide-sugar transporter. The present invention includes permeabilized yeast cells that are useful in a method of assessing a GDP-mannose transporter and useful in a method of identifying an inhibitor of a GDP-mannose transporter. The present invention provides permeabilizable yeast cells containing a dpm1 mutation useful in a method for measuring transporter activity.

[0012] One aspect of the invention is a kit that optionally comprises a permeabilized yeast cell and a nucleotide sugar useful in a method for measuring Golgi nucleotide-sugar transporter activity.

[0013] One aspect of the invention is an antifungal compound that inhibits GDP-mannose transporter activity in yeast. Another aspect of the invention is an isolated VRG4 gene and a portion thereof encoding a Golgi GDP-mannose transporter in pathogenic yeast.

[0014] Yet another aspect of the invention is an isolated VRG4 protein, or a portion thereof, encoded by a VRG4 gene from a pathogenic yeast. Another object of the present invention is to provide a recombinant method for producing RNA or protein encoded by the VRG4 gene encoding Golgi GDP-mannose transporter from pathogenic yeast.

[0015] Another aspect of the invention is an antibody that specifically reacts with the VRG4 protein or an immunogenic portion thereof. A further aspect of the present invention is a pharmaceutical composition comprising an antibody specifically reactive with a VRG4 protein or an immunogenic portion thereof useful for inhibiting nucleotide-sugar transporter function.

Another object of the present invention is to provide a nucleic acid probe for detecting a wild-type VRG4 gene or a change or mutation in the VRG4 gene. According to the present invention, such a nucleic acid probe is complementary to the wild-type VRG4 gene coding sequence and capable of forming a mismatch with the mutated or altered VRG4 gene, ie, enzymatic resolution, chemical resolution, Or a shift in electrophoretic mobility allows their detection.

Yet another object of the present invention is to provide a method for diagnosing human cells and tissues infected with yeast. According to the present invention, the method comprises isolating cells and / or infected tissues from a human and detecting normal or wild-type VRG4 gene, mRNA or their expression products from the cells and / or infected tissues,
At that time, detection of genes, mRNA or their expression products in cells and / or infected tissues is an indicator of yeast infection.

Another aspect of the present invention is to provide a method for determining the therapeutic effect of a yeast infection in a human, comprising isolating cells and / or infected tissue from a human and normal or wild-type VRG4 gene, It is an indicator of the therapeutic effect of an antifungal compound, comprising detecting a change, decrease or absence of mRNA or expression product.

It is a further object of the present invention to provide a kit for identifying and measuring a genomic nucleotide sequence of the VRG4 gene using an oligonucleotide probe.

Another object of the present invention is to provide a diagnostic probe for detecting pathogenic yeast in humans. It is a further object of the present invention to provide a method of providing normal or wild-type VRG4 gene function to cells that have lost such normal gene function due to mutations or alterations in the endogenous wild-type VRG4 gene. Expressing the wild-type gene in the cell by introducing the exogenous wild-type VRG4 gene or a functional part thereof into a cell that has lost the gene function or a cell that contains an abnormal gene.

Another object of the present invention is to provide a method of supplying a VRG4 gene function to a cell lacking such a gene, wherein a part or a part of a wild-type VRG4 gene is replaced with a cell lacking the gene function. To express a part or a part of the gene in the cell.

DETAILED DESCRIPTION OF THE INVENTION The present invention provides that the vanadate-resistant glycosylation 4 (VRG4) gene isolated from Saccharomyces cerevisiae encodes a protein that transports GDP-mannose from the cytoplasm to the Golgi lumen. Disclose. GDP-mannose is a mannosyl donor for all glycosylation events that occur in the Golgi apparatus of yeast. As such, the VRG4 protein may be said to be the master regulator of glycosylation in yeast. The VRG4 protein of the present invention is essential for survival of the Golgi apparatus of yeast. Mammalian cells do not have a GDP-mannose transporter. The VRG4 protein is a yeast-specific gene product and therefore represents a specific antifungal drug target. That is, VRG
Antifungal compounds specifically targeted to the four genes or gene products have little or no side effects on mammalian cells.

The present invention encompassed the VRG4 gene isolated from yeast. Of particular interest is the VRG4 gene isolated from pathogenic yeast. Pathogenic yeasts include, but are not limited to, Candida albicans, Candida tropical.
is, Torulopsis glabrata, Cryptococcus
neoformans, Aspergillus, Microsporium,
Trichophyton, Epidermophyton, Pytyrosp
orum, Histoplasma, Blastomyces, and the like. Preferably, the VRG4 gene is from Candida albicans. VRG
Homologs and functional portions of the four genes are included in the scope of the present invention. A functional moiety as referred to herein is any moiety that has nucleotide-sugar transporter activity, preferably GDP-mannose transporter activity. The nucleotide sequence of the VRG4 gene is shown in FIGS. However, the nucleotide sequence of the VRG4 gene of the present invention is not limited to the nucleotide sequences shown in FIGS. 6 and 7; May be included as known in the art.

The present invention includes a mutant VRG4 gene. Included in the mutant VRG4 gene are mutations that render the gene completely non-functional or render selected portions of the gene non-functional. In one embodiment, the mutant VRG4 gene has one or more mutations in the GDP-mannose domain. The mutation may be one or more substitutions or deletions, etc.
Disables binding to mannose. In one embodiment, the mutant VRG4 gene contains a single base pair change in the region encoding the GDP-mannose binding domain. In one particular embodiment, the mutant VRG4 gene comprises a single C to A base pair change at nucleotide position 857 of the wild-type VRG4 gene.

The VRG4 gene from yeast can be obtained by methods known in the art, for example, Saccha.
may be cloned by the method used to clone the VRG4 gene of R. cerevisiae (12). Of particular interest is the cloning of the full-length VRG4 gene from a pathogenic yeast, such as Candida albicans. Full length VRG4 from Candida albicans
The gene can be obtained using standard methods known in the art, Candida albi.
may be isolated from a library of C. cans. In one embodiment, the full-length gene may be isolated by using a Candida genomic library transformed into E. coli. Full-length clones are obtained by screening E. coli colonies using a portion of the Candida gene as a probe by conventional methods of colony hybridization.

The isolated VRG4 gene may be inserted into a vector including, but not limited to, a yeast vector, a yeast shuttle vector, a plasmid vector for expression in mammalian cells and the like. The vector contains a suitable promoter and a selectable marker, as is known in the art for expression in a host cell. In one embodiment, the vector has accession number ATCC 20313 under the terms of the Budapest Treaty.
C.7, deposited at the American Type Culture Collection, Manassas, 10801 University Boulevard, Aug. 11, 1998. albica
Plasmid SK ^- Ca VRG4 containing the ns partial VRG4 gene.

The present invention includes host cells transformed or transfected with the VRG4 gene or a portion thereof. Host cells include both eukaryotic and prokaryotic host cells, as long as they contain the correct elements for host-specific expression. Such host cells include, but are not limited to, bacterial cells, yeast cells, mutant yeast, mammalian cells such as BH
K-21, COS-7, CV-1, healer, etc. In one embodiment, Sa
ccharomyces cerevisiae shows that the endogenous VRG4 gene is Ca
This is a host cell replaced with a homologous VRG4 gene isolated from ndida albicans.

[0028] The present invention includes the VRG4 gene product from yeast. In one embodiment,
The VRG4 gene product is a protein of about 36.9 kDa and its peptide. The VRG4 gene product of the present invention binds to the Golgi GDP-mannose transporter in yeast. VR from Saccharomyces cerevisiae
The amino acid sequence of the G4 gene product is shown in FIG. Functional portions of the VRG4 protein are within the scope of the present invention. Such isolated moieties can extend beyond the Golgi to GDP-
It is the part that promotes the transport of mannose.

The full length nucleic acid sequence of the VRG4 gene from Candida albicans and the predicted amino acid sequence of the VRG4 protein are shown in FIGS. 7A and 7B. C. alb
icans amino acid sequence and S. FIG. 8 shows the alignment of cerevisiae.
To provide. The two proteins show 65% homology and 78% similarity along their full length.

[0030] In one embodiment, the functional portion of the VRG4 protein includes a GDP-mannose binding domain. In another embodiment, the GDP-mannose binding domain comprises a consensus sequence:
W Xaa Xaa Xaa Xaa T Xaa Xaa TTYS 1510 Xaa V G Xaa LNK Xaa P Xaa Xaa 15 20 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa at position 20 is Leu or Ile; Xaa at position 22 is Leu or Ile; and 2-5, 7-8, 22-25, 27-29 and 31 Xaa at position is any one of the naturally occurring amino acids (SEQ ID NO: 23).

In other examples, the GDP-mannose binding domain comprises SEQ ID NO: 7, SEQ ID NO: 9, or SEQ ID NO: 11. Another aspect of the invention is a mutant GDP-mannose binding domain. The mutation may be the result of one or more substitutions, deletions, etc., affecting the function of the domain. In one embodiment, the mutant GDP-mannose binding domain comprises SEQ ID NO: 8 and comprises a single amino acid substitution in the consensus sequence.

[0032] The full-length VRG4 protein from Candida albicans may be readily obtained by recombinant techniques, or the protein may be obtained from yeast cells by chromatographic techniques known in the art, such as immunoaffinity chromatography.

[0033] In one embodiment, the recombinant VRG4 protein or a portion thereof is an isolated VRG4 protein.
The RG4 gene or its functional nucleic acid sequence is inserted into a vector, the host cell is transformed or transfected with the vector, and the host cell is cultured under conditions that permit expression of the recombinant VRG4 protein or a portion thereof. It is created by a method consisting of

[0034] The VRG4 protein or a portion of its functionality may be used in assays that measure VRG4 transporter activity and assays that identify inhibitors of GDP-mannose transport activity. Such inhibitors may be used as anti-fungal compounds in the treatment of yeast infections. The VRG4 protein or an immunogenic portion thereof is also useful for eliciting anti-VRG4 antibodies. Such antibodies are useful in diagnostic assays and in therapy to inhibit transporter activity.

The present invention includes a method for measuring nucleotide-sugar transporter activity. Under natural conditions, nucleotide-sugar transporters are unique to yeast cells and are not present in mammalian or bacterial cells under natural conditions. Of particular interest are pathogenic yeasts such as Candida albicans, Cryptococcus, A
spergillus and other nucleotide-sugar transporters.

In a method for measuring nucleotide-sugar transporter activity, the transport of a significant amount of exogenous added nucleotide-sugar is measured, which is bound to the Golgi apparatus. The nucleotide-sugar transporter of the present invention can cause the transport of nucleotide-sugar to the Golgi apparatus. The amount of nucleotide-sugar that accumulates in the Golgi, especially the Golgi lumen, is an indicator of transporter activity.

By using the method of the present invention, the activity of any nucleotide-sugar transporter bound to the Golgi apparatus may be measured. In one embodiment, the activity of the GDP-mannose transporter is measured. GDP
-The method of assaying mannose transporter activity consists in adding a considerable amount of GDP-mannose to the source of the GDP-mannose transporter and to the source of the membrane. GDP-
The mannose transporter is bound to the source of the membrane in such a way that GDP-mannose binding and transport is measured from the source of the membrane.

In one embodiment, GDP-mannose is detectably labeled in such a way that when bound to the GDP-transporter it is easily detected and quantifiable.
Such labels are not limited, but do not interfere with the transporter, enzymes, radioisotopes,
It includes a chemiluminescent compound, a bioluminescent compound and the like. Or,
A second reagent may be added to detect GDP-mannose. A second reagent, eg, an antibody, may be labeled.

The source of the GDP-mannose transporter is from natural or recombinant sources. GDP-
The mannose transporter may be endogenous GDP-mannose transporter gene or exogenous GDP-
It may be expressed from a mannose transport gene or a functional part thereof. In a preferred embodiment, the GDP-mannose transporter is attached to a membrane, eg, a Golgi membrane, or a synthetic membrane, eg, a liposome membrane. Liposomal membranes useful in the present invention can be prepared by methods known in the art, for example, US Pat. Nos. 4,663,161 and 5,766.
626.

[0040] Membrane sources for use in the methods of measuring transporter activity and inhibitors of transporter activity include, but are not limited to, permeabilized yeast cells, mammalian cells, binding GDP-mannose transporter. Liposomes and the like.

In one embodiment, the source of the membrane is a mammalian cell transformed or transfected with a yeast nucleotide-sugar transporter. In a preferred embodiment, the permeabilized yeast cells are used in a method for measuring nucleotide-sugar transporter activity. Yeast cells for use in the method of measuring transporter activity are yeast spheroplasts that lack cell walls. Yeast spheroplasts are permeabilized spheroplasts that contain a leaky plasma membrane containing a complete Golgi apparatus, an inner membrane system, and a Golgi-linked nucleotide-sugar transporter.

[0042] Permeabilization of yeast spheroplasts provides a leaky plasma membrane that allows nucleotide-sugar access to cells. Yeast cells may be permeabilized by means that slightly disrupt the integrity of the plasma membrane, while having little or no effect on the nucleotide-sugar transport system. In a preferred embodiment, yeast spheroplasts are permeabilized using liquid nitrogen.

The permeabilized yeast spheroplasts for use in the method of measuring Golgi nucleotide-sugar transporter activity utilize GDP-mannose or G
Lacks any other systems that might compete with DP-mannose transport.

In a preferred embodiment, the permeabilized spheroplast has been genetically modified to lack or inactivate dolichol phosphate-mannose synthase. In one preferred embodiment, the permeabilized spheroplast comprises:
It has a dolichol phosphate-mannose synthase mutation that renders the synthase inactive.

A permeabilizing spheroplast for use in a method of measuring Golgi nucleotide-sugar transporter activity, preferably Golgi GDP-mannose transporter activity, comprises a functionally active endogenous Golgi GDP- Includes exogenous sources of mannose transporter or functionally active Golgi GDP-mannose transporter. Golgi GDP
-In the embodiment where spheroplasts utilize a foreign source of active Golgi GDP-mannose transport provided by the mannose transporter, the endogenous encoding Golgi GDP-mannose transporter, if present, is present. Mutating the sex gene inhibits expression of the endogenous Golgi GDP-mannose transporter.

In one embodiment, the permeabilized spheroplast has a functional VRG4 gene from a pathogenic yeast, preferably Candida albicans. In a preferred embodiment, the permeabilizing spheroplasts have a Sac having a dpm1 gene mutation (dpm1 ⁻ ) and an endogenous VRG4 gene mutation (VRG4 ⁻ ).
from the Charomyces strain, each mutation inactivating each gene. In one preferred embodiment, the yeast strain has accession number AT under the conditions of the Budapest Treaty.
Saccharomyces cerevi with genotype dpm1 ⁻ / VRG4 ⁻ deposited on Aug. 11, 1998 with the American Type Culture Collection as CC74461, 10801 University Boulevard, Manassas.
siae JPY263D. The functionally active exogenous VRG4 gene is incorporated into the JPY263D yeast strain by techniques known in the art, and transporter activity may be measured using the methods of the invention.

The method of the present invention is an improvement over previously described methods because of its simplicity and efficiency. Past methods have relied on the use of abundant and partially purified Golgi membranes with low specific activity. The conventional method involved the cultivation of liter (10) of cell culture, resulting in the recovery of only a small amount of active membrane. Typically, conventional methods started with one liter of cells and provided only enough material for about 4-5 reactions. The method of the present invention allows for the initiation with only a small cell culture volume, but nevertheless allows the Golgi membrane encapsulated in permeabilized spheroplasts to retain a high level of activity. It provides enough material to perform many assays. In one embodiment of the invention, one liter provided sufficient material for about 100-200 assays. For applications testing Golgi nucleotide-sugar transport inhibitor inhibitors, a fast and reliable quantification assay is essential so that candidate inhibitors can be screened at the same time.

Vectors suitable for use in the present invention include at least one expression control element operably linked to the above nucleotide sequence or a portion thereof. Insertion of expression control elements into the vector controls and regulates expression of the nucleic acid sequence. Non-limiting examples of expression control elements include, but are not limited to, a lac system,
Phage lambda operator and promoter regions, yeast promoter,
And vaccinia virus, adenovirus, retrovirus, or SV40
Includes promoter of origin. Other manipulation elements include, but are not limited to, suitable leader sequences, termination codons, polyadenylation signals, and other sequences necessary for proper transcription and subsequent translation of the nucleic acid sequence in a given host cell. Of course, the precise combination of expression control elements will depend on the host system used. In addition, expression vectors contain any additional elements necessary for the transfer and subsequent replication of the nucleic acid-containing expression vector in a host system. Examples of such elements include, but are not limited to, the origin of replication and appropriate markers. Such expression vectors are commercially available or are readily constructed using methods known in the art (see, eg, F. Ausubel et al., 1987, "Current."
Protocols in Molecular Biology ", John
Wiley and Sons, in New York, New York).

[0049] A recombinant expression vector containing all or part of a VRG4 nucleic acid sequence is transformed, transfected, or otherwise inserted into a host organism or cell. Host cells transformed with the VRG4 nucleic acid sequences of the present invention include eukaryotic and mammalian cells such as animal, plant, insect and yeast cells, as well as prokaryotic organisms such as E. coli or algal cells, as known in the art. It is. The means by which the vector containing the gene may be introduced into the cell includes, but is not limited to, microinjection, electroporation, transduction, or transfection, DEAE-dextran, lipofectin, calcium phosphate, or other known to those skilled in the art. Methods (Sambrook et al.
(1989) "Molecular Cloning. A Laboratory.
y Manual ", Cold Spring Harbor Press, P
rainview, new York). In one embodiment, a eukaryotic cell,
Preferably, eukaryotic expression vectors that function in mammalian cells are used. In a preferred embodiment, a yeast expression vector that functions in a yeast host cell is used. Non-limiting examples of vectors include adenovirus vectors, herpes virus vectors, and baculovirus vectors. Preferred eukaryotic cell lines include, but are not limited to, COS cells, CHO cells, HeLa cells, and NIH / 3T3 cells. Particularly preferred host cells are yeast cells.

The expressed recombinant VRG4 protein or a portion thereof may be measured by methods known in the art, some of which are Coomassie blue stained, silver stained, and VRG4 protein specific antibodies further discussed below. Includes Western blot analysis using Further, the recombinant protein expressed by the transformed host cells can be obtained as a crude lysate or fractionated precipitate, molecular sieve chromatography, ion exchange chromatography, isoelectric focusing, gel electrophoresis, It can be purified by standard protein purification methods known in the art, including affinity and immunoaffinity chromatography (Ausubel et al., 1987, "C.
current Protocols in Molecular Bilog
y ", John Wiley and Sons, New York, New
In York). In the case of immunoaffinity chromatography, the recombinant protein is VRG
The protein specific antibody may be purified through a column containing a resin bound thereto (
Ausubel et al. , 1987, "Current Protocol.
ls in Molecular Biology ”, John Wiley
and Sons, in New York, New York).

In one embodiment, the active VRG4 protein was purified using an HA-epitope added gene that was constructed and tested for activity by complementation of the VRG4 mutant phenotype. This HA-added protein was then overexpressed and purified by affinity chromatography using a 12CA5 binding resin.

According to the diagnostic method of the present invention, a wild-type VRG4 gene or a change in the wild-type VRG4 gene is detected. Alterations of the wild-type gene according to the invention include all forms of mutation, including deletions. Changes may also be due to rearrangements, such as insertions, inversions, and deletions or point mutations. The deletion may be of the whole gene or a part of the gene. The method of the invention is used to determine the efficiency of an antifungal treatment of a mammal, wherein altering, reducing or eliminating a wild-type VRG4 gene, mRNA or gene product is an indicator of the efficiency of reducing or eliminating yeast infection. May do it. Mutations induced by antifungal therapy lead to non-functional or absent gene products which also lead to inhibition and loss of yeast viability.

Detection of point mutations may be achieved by molecular cloning and sequencing of alleles present in yeast, using techniques known in the art. Alternatively, gene sequences can be amplified directly from genomic DNA preparations from yeast by using polymerase chain reaction (PCR). The DNA sequence of the amplified sequence can then be determined by conventional methods. The polymerase chain reaction itself is well known in the art (eg, Sa
iki et al. , 1988, Science, 239: 487; U.S. Patent No. 4,683,203; and U.S. Patent No. 4,683,195). Specific primers can be used to amplify the gene. As will be appreciated by those skilled in the art, the primers provided herein, particularly Candida a
By using primers from the Ivicans VRG4 gene, a particular VRG4 gene may be amplified to screen a sample of the population for mutations. Ligation chain reactions known in the art can also be used to amplify the VRG4 gene (see, eg, Wu et al., 1989,
Genomics, 4: 560-569). In addition, allele-specific P
A technique known as CR can be used (eg, Ruano and
Kidd, 1989, Nucl. Acids Res. , 17: 8392).
According to this technique, primers are used that hybridize at their 3 'end to a particular VRG4 mutation. If no particular VRG4 mutation is present, no amplification product is observed.

Also, by using a combination of oligonucleotide pairs based on the VRG4 nucleotide sequence as PCR primers, a reverse transcriptase polymerase chain reaction (RT-PCR) process for amplifying selected RNA nucleic acid sequences can be used. VRG4 mRNA in a biological sample may be detected,
subel et al. , 1987, "Current Protocols.
in Molecular Biology ", Chapter 15, John Wile
Y and Sons, New York, New York. Oligonucleotides can be synthesized by automated machines sold by various manufacturers, or can be produced commercially based on the nucleic acid sequences of the present invention. Biological samples to test include cells, tissues, organs,
May include blood, serum, feces, saliva, amniotic fluid, mucus secretions and urine.

In one preferred embodiment, insertions and deletions of the VRG4 gene are detected using a mutation non-complementation assay, as is known in the art (Curr.
ent Protocols in Molecular Biology, V
ol. Chapters 2, 13; Compilation, Ausubel, F .; M. et al. , John
Wiley & Sons, Inc. 1998).

The present invention provides a method for immunoreactive specifically with the GDP-mannose transporter, preferably VR
It also relates to antibodies immunoreactive with the G4 protein or an epitope thereof. The invention includes antibody preparations or antibodies that are immunoreactive with the amino acid sequence shown in FIG. 6, 7, or 8, or only a portion thereof, or a peptide thereof. In this aspect of the invention, the antibodies are of either monoclonal or polyclonal origin. The antibody may be a natural VRG4 protein or peptide, a VRG4 functional protein or peptide,
Alternatively, it may be raised against a mutant VRG4 protein or peptide. VRG4 proteins or peptides used to raise antibodies may be derived from natural or recombinant sources, or may be produced by chemical synthesis using synthetic techniques known in the art. The native VRG4 protein can be isolated from yeast cultures, isolated Golgi apparatus, biological samples of mammals containing or suspected of containing yeast, and the like. Recombinant VRG4 protein or peptide may be produced and purified by conventional methods. Based on the predicted amino acid sequences of the present invention, synthetic VRG4 peptides may be custom-ordered or made commercially (FIGS. 6, 7 or 8), or may be synthesized by methods known in the art (Merrifield) ,
R. B. 1963, J.A. Amer. Soc. 85: 2149). If the peptide is not large enough to be antigenic, it may be conjugated, complexed, or otherwise covalently linked to a carrier molecule to increase the antigenicity of the peptide. The carrier molecule can be, but is not limited to, albumin (eg, human,
Cattle, fish, sheep, and keyhole limpet
Contains hemocyanin ("Basic and Clinical Immuno"
Logic ", 1991, edited by DP States, and AI Ter.
r, Appleton and Language, Norwalk Connect
icut, San Mateo, California).

The antibodies should be specific and immunoreactive with an epitope of VRG4, preferably an epitope that is not present on other yeast or human proteins to avoid cross-reactivity. However, if one intends or needs to detect related structures or molecules, one can raise antibodies to certain epitopes commonly found in other proteins. In certain embodiments of the invention, the antibody will immunoprecipitate the VRG4 protein from solution and will react with the VRG4 protein on a Western or immunoblot of a polyacrylamide gel on a membrane support or substrate. In other preferred embodiments, the antibodies are immobilized on paraffin or frozen tissue sections, or immobilized or unimmobilized for use in immunocytochemistry, immunohistochemistry and immunofluorescence techniques and slides or coverslips. The VRG4 protein in the cell prepared above is detected.

Exemplary antibody molecules for use in the detection method or as a diagnostic method of the present invention are intact immunoglobulin molecules, substantially intact immunoglobulin molecules, or F (ab) in the art. , which is part of the F (ab) ¹ _2, and F (v) an immunoglobulin molecule comprising an antigen-binding site known as immunoglobulin fragments. Polyclonal or monoclonal antibodies may be produced by methods commonly known in the art (eg, Kohler and Milste).
in, 1975, Nature, 256: 495-497; Campbell "
Monoclonal Antibody Technology, the P
production and Characterization of Ro
dent and Human Hybridomas ”, 1985,“ Lab
oratory Technologies in Biochemistry a
nd Molecular Biology ", Compilation, Burdon et a
l. , Volume 13, Elsevier Science Publishers, A
msterdam). The monoclonal antibody may be a human monoclonal antibody, a chimeric monoclonal antibody, or a humanized monoclonal antibody made using techniques known in the art (Takeda 1985 Nature).
314: 452; U.S. Patent No. 5,585,089, U.S. Patent No. 5,530,
No. 101). Antibodies or antigen-binding fragments may be made by genetic engineering. Techniques for expression of both heavy and light chain genes in E. coli are described in PCT Patent Application,
Publication numbers WO901443, WO901443 and WO9014424 and Huse et al. , 1989 Science, 246: 1275-12.
81 subjects. An antibody molecule of the present invention can be a complete immunoglobulin molecule, or a portion thereof that includes an antigen binding site. Single chain antibodies may be constructed by methods known in the art (US Pat. No. 4,946,778; Davis, GT).
. et al. 1991 Biotechnology 9: 165-169;
Pluckthun, A .; 1990 Nature 347: 497-498).
. Antibody molecules can be of any class, including IgG, IgM and IgM.

The antibodies of the present invention may be used as diagnostic reagents for detecting and quantifying Golgi GDP-mannose transporters and for measuring Golgi GDP-mannose transporter activity. The standard immunoassay is for Golgi GDP in biological samples.
-May be used with anti-GDP-mannose transporter antibody for detection and quantification of the mannose transporter.

In another embodiment, a VRG4 protein-specific antibody is used in an immunoassay to detect novel VRG4 protein in a biological sample. In this method, an antibody of the invention is contacted with a biological sample, and complex formation between the VRG4 protein and the antibody is detected. As described, suitable immunoassays include radioimmunoassays, western blot assays, immunofluorescence assays,
Including enzyme-linked immunoassay (ELISA), chemiluminescence assay, immunohistochemical assay, immunocytochemical assay and the like (for example, ""
Principles and Practice of Immunoass
ay ", 1991, compiled by Christopher P. Price and
David J. et al. Neoman. Stockton Press, New Yo
rk, New York; "Current Protocols in Mo
recital biology ", 1987, Ausubel et al.
al. , John Wiley and Sons, New York, New
York). Known standard techniques for ELISA are Methods
in Immunodiagnosis, 2nd edition, compilation, Rose and B
igazzi, John Wiley and Sons, New York
1980; and Campbell et al. , 1984, Methods
in Immunology, W.C. A. Benjamin, Inc. ). Such assays are direct, indirect, competitive, or non-competitive and are as described in the art (eg, “Principles and”
Practice of Immunoassay ", 1991, compiled, Chr
istopher P. et al. Price and David J. Neoman,
Stockton Pres. NY, NY; and Oellrich, M .; , 1
988, J.C. Clin. Chem. Clin. Biochem. , 22: 895
-904). Proteins may be isolated from test specimens and biological samples by conventional methods, and are described in Current Protocols in Molec.
ullar Biology ", 1987, edited by Ausubel et al.
, John Wiley and Sons, New York, New Yo
rk.

[0061] The antibodies of the present invention may be provided in kit form and may also include GDP-mannose and / or other assay reagents. The antibody of the present invention may be used as a therapeutic agent that specifically inhibits the function of the Golgi GDP-mannose transporter. Binding of the antibody to the transporter inhibits glycosylation in yeast, resulting in a loss of yeast cell viability. Antibodies may be administered intraperitoneally, intravenously, intramuscularly, subcutaneously, transmucosally, or topically. Antibodies are administered to a yeast-infected patient for a period of time sufficient to reduce or eliminate the infection.
For use as therapeutics, the antibody may be modified to enhance its transport across the yeast cell wall, thereby assisting its access to the transporter in the Golgi apparatus. For topical administration or as an oral mouse wash, the antibody may be administered in combination with a detergent to allow entry of the antibody across the cell wall.

In another aspect of the present invention, the primer pair of the present invention is useful for determining the nucleotide sequence of the VRG4 gene using polymerase chain reaction. The pair of single-stranded DNA primers is annealed to or around the VRG4 gene or to another segment of the gene to create the DRG4 gene itself.
NA synthesis amplification can be started. Generally, the PCR primers are about 100-600b
p, more preferably about 1 to about 100-200 bp of stretched DNA.
It is of the order of 5-40 bp, more preferably of the order of about 18-30 bp.
The complete set of primers allows the synthesis of all nucleotides of the VRG4 gene coding sequence. Allele specific primers can also be used.
Such primers will anneal only to the particular VRG4 mutant allele, ie will amplify the product in the presence of the mutant allele as a template. A non-limiting example of a VRG4 sequence primer for use in the present invention may be a nucleic acid sequence as shown in FIGS. 6, 7A and 7B, or a portion thereof.

[0063] To facilitate subsequent cloning of the amplified sequences, the primers may have an enzyme restriction site sequence added to their 5 'end. That is, all nucleotides of the primer are derived from the VRG4 sequence or a sequence adjacent to VRG4, except for some nucleotides necessary to form a restriction enzyme site. Such enzymes and enzyme restriction sites are known in the art. Primer itself (DN
For each chain of A) can be synthesized using techniques known in the art. Generally, the primers can be made using commercially available synthesis machines. Given the sequence of the VRG4 open reading frame shown in FIGS. 6 and 7A and 7B, the design of a particular primer is within the skill of the art, and there are no exons in the VRG4 gene.

The nucleic acid probes provided by the present invention are useful for many purposes. They are,
It can be used in Southern hybridization assays for genomic DNA, and in RNase protection methods to detect point mutations. By using the probe, a PCR amplification product can be detected,
Other techniques can also be used to detect mismatches with the VRG4 gene or mRNA. Examples of nucleic acid sequences that can be used as a probe include, but are not limited to,
Includes natural DNA, recombinant DNA, and synthetic oligonucleotides. Nucleic acid probes can be prepared and labeled using methods known in the art. As an example, the DNA sequence can be labeled with ³² P using Klenow enzyme, polynucleotide kinase or polymerase such as TAQ used in PCR reactions. Non-radioactive labeling techniques are also available for signal amplification, and methods of linking chemical moieties to pyrimidine and purine rings (Dale, RKK et al.,
1973, Proc. Natl. Acad. Sci. USA. 70: 2238-
2242; and Heck, R.A. F. 1986, S.M. Am. Chem. Soc
. , 114: 8736-8740); A method enabling detection by chemiluminescence (Barton et al., 1992, J. Am. Chem. Soc.
. , 114: 8736-8740); and a method utilizing a biotinylated nucleic acid probe (Johnson, TK et al., 1983, Anal. Bioc).
hem. Erickson, P., 133: 125-131; F. et al.
, 1982, J.A. Immunol. Meths. , 51: 241-249; and Matthaei, F .; S. et al. 1986, Anal. Bioch
em. , 157: 123-128); and methods that allow detection by fluorescence using commercially available kits.

Using a complete battery of nucleic acid probes, a kit for detecting the wild-type VRG4 gene and its variants is formulated. The kit has a complete V
It allows hybridization to the RG4 gene or a particular region thereof.
The probes may overlap each other or may be adjacent. The kit may include other reagents for performing the assay, such as buffers, enzymes, control samples, and the like.

The nucleic acid probe may be complementary to the VRG4 gene or a mutant allele of the VRG4 gene. Such probes are useful for detecting similar homologs or mutations based on hybridization. As described, VRG4
Probes can be used in Southern hybridizations to genomic DNA to detect large chromosomal changes, such as deletions and insertions. Furthermore, by using the above-mentioned probe, VRG from yeast in tissue can be obtained.
4 mRNA can be detected to determine if the expression of the wild-type VRG4 gene is reduced as a result of antifungal therapy.

The antisense oligonucleotide was derived from the VRG4 gene as shown in FIGS. 6, 7A and 7B or from the nucleic acid sequence encoding the GDP-mannose binding domain as shown in FIGS. 14A and 14B. Good. Antisense oligonucleotides are useful for specifically inhibiting the formation of the VRG4 gene product. Antisense oligonucleotides may be made by methods known in the art.

According to the present invention, there is also provided a method for providing wild-type VRG4 function to cells lacking the VRG4 gene or having a mutant VRG4 allele. Wild type V
The RG4 gene or a part of the gene may be introduced into a cell in a vector. Gene transfer vectors for both recombination and extracellular maintenance are known in the art and may be used in the present invention. Methods for introducing DNA into cells, such as electroporation, potassium phosphate co-precipitation, and virus transduction, are known in the art as described above; thus, the choice of method depends on the skills and preferences of the skilled practitioner. I do. By using cells transformed with the wild-type VRG4 gene as a model system, nucleotide-sugar transporters are studied to screen antifungal agents for treatment against yeast infection.

The VRG4 protein or peptide may be used as a competitive inhibitor of the native VRG4 protein, and as such, may be used as an antifungal compound for treating infections caused by yeast. Proteins and peptides can be produced by expressing isolated DNA sequences, especially genomic DNA, in yeast using known expression vectors. Alternatively, VRG4 can be extracted from VRG4-producing cells, such as yeast cells. The protein may be cleaved using an enzyme or formed into a peptide using a reducing agent. Inhibitor peptides may be selected using the methods described herein. Further, VRG4 proteins or peptides may be synthesized by employing synthetic chemistry techniques.

The active inhibitory VRG4 molecule may be introduced into cells by, for example, microinjection or the use of liposomes. Alternatively, some active molecules are actively or passively taken up by cells by diffusion. Extracellular application of the VRG4 gene product may be sufficient to affect yeast growth. Addition of the agent may facilitate entry of the VRG4 protein or peptide across the cell wall of the yeast, and may facilitate ligation of the exogenously supplied VRG4 body or peptide to the Golgi body. Such agents include, but are not limited to, liposomes and the like.

The isolated VRG4 protein or a portion thereof can be used in the form of a pharmaceutical composition with standard excipients as is known in the art for the inhibition of the yeast endogenous VRG4 gene product. Good. The inhibitors of the present invention are useful for preventing and treating yeast and fungal infections in mammals, including humans. Yeast and fungal diseases treated with the inhibitors of the present invention include, but are not limited to, candidiasis, aspergillosis, mucormycosis, nocardiosis, cryptococcosis, histoplasmosis, blastmycosis, coccidioidomycosis, parasitosis Coccidioid mycosis,
Including onycholysis, dermatophyte infection, etc.

The inhibitors of the present invention may be administered alone or as a mixture in the form of a pharmaceutical composition,
It may then be administered in combination with one or more other fungicidal and bacteriostatic compounds. Fungicidal and bacteriostatic compounds include, but are not limited to, ketoconazole, flucytosine, fluconazole, itraconazole, amphotericin B, and the like.

Means for administering the VRG4 protein or a portion thereof include, but are not limited to, oral, sublingual, intraperitoneal, transdermal, intranasal, subarachnoid, subcutaneous, transmucosal, or intestinal. Topical administration to the site of distress may be performed using means known in the art, including, but not limited to, topical application, injection, and implantation. In a method of treating a yeast or fungal infection in a mammal, the inhibitor is present in a dosage sufficient to inhibit transport of the nucleotide-sugar transporter to the Golgi apparatus of the yeast or fungus, preferably in the yeast or fungus. Provided at dosages sufficient to inhibit the Golgi GDP-mannose transporter. Such inhibition in transport results in inhibition of yeast or fungal growth causing infection and loss of viability.

Other inhibitors of the Golgi nucleotide-sugar transporter include, but are not limited to, the inhibitor specifically inhibits the Golgi nucleotide-sugar transporter in a fungus or yeast and is non-toxic to mammalian cells Or a nucleotide-sugar analog, stilbane or a derivative thereof, provided that it has no or minimal effect. The inhibitors of the present invention may be competitive or non-competitive inhibitors.

The inhibitors of the present invention are useful for inhibiting VRG4 transporter activity in fungi and yeast. Such inhibition of VRG4 transporter activity in the Golgi results in fungal and yeast growth inhibition and the ultimate lack of viability. Fungi and yeasts that are subject to inhibition by inhibitors of VRG4 transporter activity include, but are not limited to, Candida
, Torulopsis, Cryptococcus, Aspergillus
, Nocardia, Histoplasmosis, Trycophyton
And so on.

[0076] All publications, patents, and literature cited herein are fully incorporated herein by reference. The following examples are presented for the purpose of illustrating the invention,
It is not intended to limit the scope of the invention. Those skilled in the art will recognize that many modifications and substitutions may be made without departing from the spirit and scope of the invention.

Example 1 Cloning of the VRG4 Gene of Saccharomyces cerevisiae Cloning and Sequencing of the Wild-Type VRG4 Gene—Strain NDY5 (ura3-
52 leu2-211 vrg4-2) was transformed with a yeast genomic CEN-based library with a LEU2 selectable marker. Prototroph transformants were selected on leucine-deficient media and replicas were plated on media containing 50 μg / ml hygromycin B. Plasmid DNA from hygromycin B resistant colonies was isolated, amplified in E. coli, and confirmed for complementation activity by re-transformation with a VRG4 mutant.

A 2.1 kb E that can complement the hygromycin B sensitivity of VRG4
The coRI / HindIII fragment was sequenced by the dideoxy method (43) and
A nested deletion series was generated using the xoIII / ExoVII method (44). Both DNA strands were sequenced. Comparisons of DNA and predicted amino acid sequences to the database were made using the BLAST algorithm (41) and analyzed using the GCG program.

Plasmid Construction-All DNA manipulations were performed using standard protocols (45). 2.1 kb EcoRI / Hi containing complete VRG4 gene and regulatory sequences
Subcloning the ndIII fragment into the vector pRS316 (46) generated the CEN-based plasmid pRHL containing the selectable marker URA3. This plasmid was labeled with [ ³² P] dCTP (Amersham) using a random priming method and used to probe a nitrocellulose filter containing the isolated yeast chromosomes (47).

The disrupted plasmid pG5 :: Leu was constructed by inserting the SmaI / SalI fragment containing the LEU gene (blunted with Klenow) into the unique HpaI site present in the VRG4 gene.

The integrated plasmid pG5i was constructed using the HindII containing the complete VRG4 gene.
It was constructed by inserting the I / EcoRI fragment into URA3-containing pRS306. This plasmid was linearized at the unique HpaI site in the VRG4 gene to transform strain NDY5.

Cloning and Analysis of the VRG4 Gene—Vanadate Resistant Mutants Do Not Grow on Hygromycin B-Containing Media at Concentrations Where Wild-Type Grow Normally (10).
This drug sensitivity was developed as a means of cloning the wild-type VRG4 gene. Mutants were transformed with a CEN-based yeast genomic library containing the Leu2 gene as a selectable marker. Select leucine auxotroph and 50
Replicas were plated on media containing μg / ml hygromycin B. The plasmid isolated from each of the six hygromycin B resistant colonies from which the six hygromycin B resistant colonies were isolated contained different but overlapping restriction fragments. All six plasmids conferred hygromycin B resistance when re-transformed into the VRG4 mutant. Further subcloning will result in complementation of the 2.1-kb EcoRI
/ HindIII fragment. ³² P-labeled EcoRI / HindIII
Hybridization of the fragment to the isolated yeast chromosome mapped the gene on chromosome XV (data not shown). Putative V in VRG4 mutant
Expression of the cloned fragment containing the RG4 gene restores the ability of these cells to retain the ER protein and rescue the invertase glycosylation deficiency. A small amount of invertase lacking glycosylation could still be detected in the mutant cells harboring the cloned gene. Cell growth during invertase induction was not performed under conditions that favored plasmid selection. Thus, this apparent leakiness was likely due to plasmid loss in some of the cells assayed.

To confirm that the cloned fragment contained the VRG4 locus,
The oRI / HindIII fragment was cloned into an integrative plasmid (pRS306) containing the selectable marker URA3. Linearizing the plasmid at the unique restriction site in the VRG4 portion to allow homologous recombination at the VRG4 locus,
VRG4 was used to transform ura3 cells. Ura ⁺ transformants were then crossed with the VRG4 ura3 strain. The resulting diploids sporulated and totally cleaved into tetrads. This analysis, Ura ⁺ / Ura ^- about 2: For the second separation patterns and hygromycin resistance / hygromycin sensitive 4: 0 since showing the separation patterns of the cloned fragments are linked to firmly VRG4 VRG
It is likely to contain four loci (data not shown).

DNA sequence analysis of the 2.1-kb fragment revealed the presence of two open reading frames. From further analysis, a 1.6-kb HindIII / E
Complementary activity to the large open reading frame within the coRV fragment was mapped. FIG. 6 shows the nucleotide sequence and the predicted amino acid sequence of this region.

The VRG4 DNA sequence encodes a predicted protein of about 36.9 kDa.
There are five potential recognition sites for N-linked glycosylation (indicated by stars in FIG. 6). Hydrophobicity analysis (33) suggests that the protein is hydrophobic and contains multiple membrane spatial domains.

Example 2 Cloning of the VRG4 Gene of Candida Albicans SK ^- Ca Partially Containing the VRG4 Gene of Candida albicans
The plasmid designated VRG4 was cloned under accession number ATCC under the terms of the Budapest Treaty.
Deposited with the American Type Culture Collection, 18801 University Boulevard, Manassas on August 11, 1998 as 203137. SK ^-
The physical map of the Ca VRG4 plasmid is shown in FIG. Saccharomyces cer lacking dpm1 and having a mutation in the endogenous VRG4 gene
Evisiae yeast strain JPY263D was transformed into Candida albicans.
Was used as a host cell for the uptake of the VRG4 gene. The yeast strain is a member of the American Type Culture Collection, 10801 University Boulevard, Manassas under Accession No. ATCC 74461 under the terms of the Budapest Treaty.
Deposited on August 11, 998.

The use of the partial VRG4 gene allows the genomic Candida
The full-length genomic VRG4 gene from Albicans was isolated and a clone containing the full-length genomic VRG4 gene was isolated.

Example 3 GDP-Mannose Transport Function of VRG4 Materials and Methods Yeast Strains, Media and General Genetics Methods-The yeast strains used in this study are listed in Table 1. Preparation of media and standard yeast genetic methods for sporulation, quadruple splitting and strain construction have been described (13).
. 0.5M KCl was added to YPAD liquid medium for growth of vrg4 mutant (10,12). Hygromycin B (final concentration 30 μg / ml)
Boehringer Mannheim) was added to the YPAD agar after autoclaving. The yeast strain was transformed using the lithium acetate method (14).

[0089]

[Table 1]

Table 1. Alleles of VRG4 with the addition of the strain plasmid epitope used in this study were created in several steps. First, the VRG4 gene was amplified by using PCR,
The stop codon was replaced at the I site. 1.5 kb HindI containing VRG4 gene
The II / NsiI fragment was then converted to HindIII / P of SK ^- P / X HA3 (15).
Ligation to the stI site generated pSK ^- RHL HA3. This results in an in-frame fusion of VRG4 with three tandem copies of the HA epitope at the 3 'end followed by a sequence encoding a stop codon. HA addition 3 'of VRG4
The 0.5 kb HpaI / XbaI fragment from pSK-RHL HA3, including the termini, was ligated to the pRG containing the VRG4 gene and 5 ′ and 3 ′ flanking control sequences.
Replaced with the 3 'end of VRG4 in HL (12). It contains the HA addition allele of VRG4 under its own promoter in the URA3 / CEN yeast expression plasmid.

PYRHL-HA3 contains HA3-added VRG4 under its own promoter in a 2μ URA3 plasmid. It is the Hind from pRHL-HA3
It was constructed by inserting a III / XbaI fragment into the HindIII / XbaI site of YEp352.

A DNA fragment containing the complete HVG1 gene and flanking sequences was cloned by PCR amplification of genomic yeast DNA. This 1.3 kb fragment flanking the BamHI / HindIII sites was cloned into pRS316 to generate the plasmid pHVG1. Similarly, PC of yeast genomic DNA
By using R amplification, a 1,026 bp fragment containing only the HVG1 ORF was generated. This fragment flanking the BamHI / HindIII sites was
It was placed under the control of the TP1 promoter by cloning into p56X (16). Deletion of the HVG1 gene is cerevisiae HIS3 gene (17). Pombe HIS5 gene, complete HVG
This was performed by replacing one open reading frame. An S.H. fragment containing the HIS5 gene of Pombe (Sean
A linear fragment containing the HIS5 gene flanked by a 50 bp HVG1 homologous sequence was generated by using PCR amplification (kindly provided by Munro, MRC, LMB). Strain SEY6210 was transformed by using this linear fragment. His ⁺ transformants were isolated and the deletion was confirmed by PCR (
Data not shown). Western immunoblotting Prepare whole cell protein extract as described (12), SDS-PAG
Separated by E and immunoblotted. For detection of secreted chitinase, the protein in the culture supernatant was precipitated by adding 10 volumes of ice-cold acetone.
Centrifuged at 2,000 Xg. Anti-chitinase antibody (from W. Tanner) was used at a 1: 1000 dilution. Culture supernatants containing the monoclonal anti-HA antibody 12CA5 were used at a 1:10 dilution. Chemiluminescence (ECL, Amersham) using a secondary anti-rabbit or anti-mouse antibody (Amersham) conjugated to horseradish peroxidase at a 1: 3000 dilution.
, Followed by autoradiography. Indirect immunofluorescence SEY6210 or pYRHL-HA3 or TiOCH-HA (15)
10 ml of a logarithmic culture of SEY6210 (1-3 × 10 ⁷ cells / ml) containing 3
. Fixed by the addition of up to 7% formaldehyde at room temperature for 30 minutes. Cells are harvested and 10 ml of 3.7% formaldehyde; 0.1 M KPO ₄ , pH
Resuspend at 6.8 and fix for an additional 1-2 hours at room temperature. Fixed cells were treated with 1.0 M sorbitol; 100 mM HEPES; pH 6.8; 5 mM Na
It was washed with N ₃ and made spheroplast by addition of 30 μg / ml zymolase 100T. Spheroplasts were treated with 0.1% Triton X-100 for 5 minutes at room temperature. After attachment to glass slides, cells were submerged in methanol at 20 ° C. for 6 minutes and then in acetone at −20 ° C. for 30 seconds. Slides were incubated overnight with primary antibody (12CA5 culture supernatant, diluted 1:10) and
Washed twice with PBS and then diluted 1-2 in anti-mouse IgG: FITC or anti-rabbit: IgG: FITC (Jackson ImmunoResearch, PA) diluted 1: 200.
Incubated for hours. After washing, cells were overlaid on an encapsulation medium containing 25 ng / ml DAPI. Radiolabeling and lipid analysis of cells Wickelham's minimal medium containing 2% glucose and lacking myo-inositol (
Cultures were grown in (WH-I) (18) to an OD ₆₀₀ of 1. Sign is 5
Started by the addition of μCi / ml [3H] -myo-inositol (American Radiolabeled Chemicals, St. Louis, Mo.). Cells are metabolically labeled for 10 minutes at 30 ° C. and WH containing 40 μg / ml unlabeled myo-inositol
Followed by addition of 4 volumes. The reaction was stopped by the addition of ice-cold NaN ₃ . The cells were washed once with NaN ₃ was suspended in NaN ₃ in 100 [mu] l, it was disrupted by vortexing with glass beads.

The lysate is removed from the glass beads and 600 μl of chloroform / methanol (1: 1) is added to 90 μl of cell extract to a final concentration of chloroform / methanol / aqueous solution (10: 10: 3). This extracted the liquid. After centrifugation, the precipitate was extracted with chloroform / methanol / water (10: 10: 3) for 45 minutes. The stored liquid fraction was dried under nitrogen gas and desalted by phase separation of n-butanol and water. The lipid was resuspended in 40 μl of a chloroform / methanol / aqueous solution (10: 10: 3) in thin layer chromatography.

HPTLC silica-60 gel plates (0.2 mm) (Merck, Darmstead, Germany) were dried (110 ° C.) for 2 hours and then cooled to room temperature before use. Samples were applied (150,000 cpm per lane) and ascending chromatography was performed using a solvent system of 0.22% KCl in chloroform / methanol / water (55:45:10) (1-2 h solvent). In an equilibrated tank). After chromatography, plates were air dried and EN ³ HANCE
(New England Nuclear) and a fluorograph was performed overnight. Preparation of Permeabilized Yeast Cells Permeabilized yeast cells (PYC) suitable for use in measuring Golgi nucleotide-sugar transporters are prepared with (20) modifications according to the method described. did. 100-200 ml of cells were placed in YPAD medium containing 0.5 M KCl for 3 times.
Grow at 0 ° C. to an OD ₆₀₀ of 1-2. After collection, the cells are
Tris-HCl, pH 9.4; 50 OD units / m in 10 mM DTT
and left at room temperature for 5 minutes. Centrifuge the cells to 0.75 XY
PA, 0.5% glucose, 0.7 M sorbitol, 10 mM Tris-HC
l, pH 7.5, suspended at 50 OD units / ml, and 10 U of lyticase / OD units of cells were added to form spheroplasts. After incubation at 30 ° C. for 20 minutes, more than 80% of the yeast cells turned into spheroplasts. The spheroplasts were centrifuged at 1,500 Xg for 3 minutes,
. Suspended in 0.75X YPA medium containing 7M sorbitol and 1% glucose. After a 20 minute incubation at 30 ° C. to allow recovery by metabolism, cells were buffered (400 mM sorbitol; 20 mM HEPES).
, PH 6.8; 150 mM potassium acetate; 2 mM magnesium acetate) and resuspended in buffer at 300 OD units / ml. For permeabilization, aliquots of PYCs were slowly frozen over liquid nitrogen for 1 hour and immediately transferred to -70 ° C. GDP-mannose transport assay using permeabilized yeast cells GDP-mannose transport was measured using permeabilized spheroplasts. Reactions were 20 mM HEPES (pH 6.8); 150 mM potassium acetate, 250
mM sorbitol, 5 mM magnesium acetate; 3 μM GDP mannose and 50 nCi GDP- [ ³ H] -mannose (15 Ci / mmole) in a final volume of 25 μl. The permeabilized cells were quickly thawed and thawed three times with 1 ml of ice-cold reaction buffer (buffer H) (20 mM HEPES, pH 6.8; 150 mM
Washing with potassium acetate, 250 mM sorbitol, 5 mM magnesium acetate) removed cytoplasm and endogenous GDP-mannose. The membrane was concentrated to half the initial volume using buffer H. The reaction was started by mixing 5 μl of the membrane (containing 10-20 μg of protein) with 20 μl of reaction buffer to a final protein concentration of 0.4-0.8 mg / ml. Protein concentration was measured with BCA reagent (Pierce Chemical, Rockford, IL).

After incubation at 30 ° C. for 6 minutes, the reaction was stopped by adding 0.5 ml of ice-cold buffer H and placing the sample on ice. Membranes were precipitated by centrifugation at 14,000 Xg or 100,000 Xg in an ultracentrifuge (Beckman Optica TL). Free radioactive solutes were removed by washing the membrane precipitate three times with 1.0 ml of ice-cold buffer H. 10 precipitates
Resuspended in 0 μl 0.1% Triton X-100, 50 μl sample was removed, added to 1 ml scintillation mix and radioactivity was counted in a scintillation counter. By subtracting the radioactivity of the membrane at zero time incubation the value of solute bound to the membrane measured was measured GDP- ^[3 H] mannose non-specifically bound to the outside of the membrane. The percent activity was calculated by dividing this value by the total cpm in the reaction [% transport activity = CPM in sediment for 6 minutes-CPM in sediment for 0 hours / total CPM]. When compared to PYC preparations from another strain, each value was normalized by dividing the percent transport activity by the total amount of protein in each reaction. Guanosine diphosphate assay GDPase was purified as described (7) using strain JPY256c (VRG4
)) Or in solubilized P100 fractions prepared from PYCs from JPY263d (vrg4-2). Inorganic phosphoric acid was measured by the method of Ames (
21). One unit of GDPase was defined as the activity of releasing 1 nmole of inorganic phosphate per minute. Subtracting the background value measured by assaying the reaction lacking substrate or protein gave the stated value.

Example 4 Vrg4 Variant Lacks Both N- and O-Linked Sugar Modifications VRG4 is Required for N-Linked Glycosylation (10-12) O-
To evaluate the effect on bound glycosylation, we examined the glycosylation site of chitinase. Chitinase is a secreted protein containing carbohydrates that are exclusively O-linked. Thus, any effect on O-linked glycosylation can be detected by electrophoretic mobility shift (22). Whole cell extracts were prepared from isogenic wild-type and vrg4 cells and assayed by immunoblotting using an anti-chitinase antiserum. As a control, chitinase mobility in cells containing the mnn10-2 mutation (23) lacking only N-linked glycosylation was also tested. The shift in mobility is v when compared to wild type cells.
It was detected with chitinase from rg4-2 but not in mnn10-2 cells (FIG. 1). This result indicates that the vrg4 mutation affects O-linked glycosylation and is therefore required for both classes of glycosylation.

Example 5 The vrg4 mutant lacks sphingolipid mannose formation When observed by electron microscopy, vrg4 cells show an abnormal morphology of intracellular membranes (12). In the vrg4 mutant, the membrane accumulates but stains only slightly with potassium permanganate. This observation suggested that the VRG4 gene product may be necessary to maintain the normal protein / lipid ratio of these Golgi membranes whose properties are altered by the vrg4 mutation. Synthesis of sphingolipids in yeast requires vesicle transport to the Golgi apparatus, suggesting that their synthesis occurs in this compartment (24). Thus, it was of interest to determine whether the vrg4 mutation affected sphingolipid biosynthesis. S. cerevi
In siae, there are three main classes of sphingolipids. These include inositol phosphoryl selemides (IPCs) and mannosyl inositol phosphoryl selemides (MIPC, and M (IP) ₂ C) (for review see ref. 5). To test the idea that VRG4 is required for sphingolipid biosynthesis, we examined the [ ³ H] in the genotype vrg4-2 mutant.
-Inositol-labeled lipids were compared to the wild type strain. 1 with [ ³ H] -inositol
Cells were labeled for 0 minutes and chased for 20 or 40 minutes. Lipids were extracted and analyzed by thin layer chromatography. The most significant difference between the wild type and vrg4-2 cells was that vrg4-2 strain could not accumulate MIPC and M (IP) ₂ C (Figure 2). These results demonstrate that VRG4 is required for sphingolipid biosynthesis, and suggest that this deficiency specifically affects the form of mannose formation.

Example 6 Development of In Vitro GDP-Mannose Transport Based on Permeabilizable Yeast Cells The effect of the vrg4 mutation on the biosynthesis of glycoproteins and sphingolipids was
This suggests that VRG4 is normally required for mannose formation in the Golgi apparatus. v
A simple model that can account for the pleiotropic phenotype of the rg4 mutant is that VRG4 is required for GDP-mannose accumulation or transport in the Golgi lumen.

To test this model, GDP-mannose transport activity was compared in isogenic wild type and vrg4-2 mutant strains. The transport of rumen GDP-mannose in yeast in vitro has been characterized using Golgi-rich vesicles (7). Using this system, we routinely observed a decrease in the activity of mutant membranes relative to wild type (data not shown). However, this method involves the preparation of large-scale cells, and the reaction typically requires milligram quantities of protein.
To allow the processing of more samples at the same time for comparative purposes, we
We sought to develop another system to measure GDP- [ ³ H] mannose transport on an analytical scale. Permeabilized cells (PYCs) were used for this purpose. P
YCs must be highly complementary to glycosylation in vitro when GDP-mannose is added, thus allowing efficient rumen GDP-mannose transport.

In a permeabilized yeast cell containing a dpm1 mutation that results in 90-95% of dolichol-phosphate-mannose synthase (Dpm1) activity in vitro,
GDP-mannose transport was characterized. The background of this mutant is E
Eliminates the competition reaction catalyzed by Dpm1p in GDP-mannose conferring mannose to form dolichol-phosphate-mannose (Dol-P-Man) acting as a mannose donor for glycosylation at R Needed to do so. This ER reaction was fairly efficient in vitro and may have masked or masked Golgi transport of GDP-mannose (7). Comparison of [ ³ H] -mannose incorporation into sealed membranes of wild-type isogenic strains (JPY256b) or the same genotype strain containing the dpm1 mutation (JPY256c) was compared to Dol-P-Man. Uptake of mannose,
It proved that the calculated [ ³ H] incorporation was more than 60% (
Data not shown). Thus, all experiments described below were performed with isogenic strains containing dpm1 mutations that did not otherwise affect the growth characteristics of these strains (
Data not shown).

[0102] GDP-To assay the incorporation of mannose, GDP-after incubating the PYCs in the presence of ^[3 H] mannose, bound to the washed vesicles ^[3 H] - supernatant the amount of mannose (S100). Vesicles are 100,000 × with extensive washing to remove bound radioactive solutes.
g (P100). Time course of ^[3 H] uptake, suggesting that GDP- mannose transporter was pretty good efficiency. Typically, [ ^3]
H] recovered in the P100 fraction, with GDP mannose about 25p.
It corresponded to the uptake of moles (FIG. 3A). The transport rate was up to the time of 6 minutes (FIG. 3A) and the protein concentration in the range of 0.4 to 1.2 mg / ml (FIG. 3A).
B) was a straight line. Transport was temperature dependent; maximal transport occurred at 30 ° C, decreased slightly at 25 and 42 ° C, and was inhibited at temperatures above 60 ° C (data not shown).

The uptake of GDP-mannose was determined with detergent (0.1% Triton X-100).
) Was completely inhibited and accompanied by a decrease in transport to less than 2%,
It has been demonstrated that accumulation of DP-mannose requires complete vesicles. Similarly, the stilbene derivative 4,4-diisothiocyanostilbene-2,2-disulfonic acid, which is known to inhibit nucleotide sugar transport in both mammalian (26) and human (7) systems The inclusion of 4 mM (DIDS) completely inhibited the activity. As previously demonstrated (7), transport was independent of energy or divalent cations, as ATP, Mg ⁺⁺ or EDTA did not affect the efficiency of transport (data not shown) ). However, we will be presumed that the elimination or inclusion of EDTA in Mg ⁺⁺ did not affect the activity of the endogenous acceptor glycosyltransferase utilizing labeled mannose, because transport of Mg ⁺⁺ It was stimulated about 2-fold in the presence (data not shown).

The physical properties of the rumen radiolabel were tested by analyzing the transport reaction products after phase separation. It separates lipid-binding oligosaccharides, but separates into an organic phase from protein-binding oligosaccharides and is insoluble in chloroform / methanol. After allowing the transport reaction to occur for 6 minutes, PYCs were extracted and lipids, proteins and water-insoluble products were separated as described in Waechter et al (27). According to this assay, most of the radiolabeled product bound to the membrane (87%) was either water soluble (GDP-mannose) or chloroform / methanol insoluble (protein). We conclude that GDP-mannose transport in PYCs appears to have all of the previously described (7) features for crude Golgi membranes.

Example 7 VRG4 Gene Product Is Required for Lumen Golgi GDP-Mannose To test the model that VRG4 is required for GDP-mannose transport,
PYCs were prepared in parallel from wild type and vrg4 mutant cells and their transport activity was compared. VRG4 (JPY256c) and vrg4-2 (JPY26
3d) The transport activity of the strain was tested as a function of time. In contrast to wild-type cells,
If more than 25% of the endogenous GDP- [ ³ H] -mannose is transported, v
The rg4-2 membrane showed a significant defect in GDP mannose uptake (<2
% Transport) (Figure 4). This deletion was caused by the plasmid containing the VRG4 gene.
It was partially complemented among the 4-2 mutants. This is consistent with the observation that this plasmid does not completely complement the glycosylation phenotype of the vrg4-2 mutant in vivo.

To determine whether the effect of the vrg4-2 mutation was specific for GDP-mannose incorporation, another Golgi protein in soluble P100 fractions prepared from wild-type and vrg4-2 PYCs Was assayed for activity. As shown in Table 2, GDPase in P100 fraction derived from wild type or vrg4-2
Were essentially indistinguishable. Vrg4p is therefore GDP
-Specifically required for mannose uptake.

[0106]

[Table 2]

Example 8 VRG4 is an Endogenous Golgi Protein The VRG4 gene product is required for many different Golgi functions (12). If these effects are due to their role in nucleotide sugar incorporation, Vrg
4p would be expected to be internal to the Golgi complex. To determine the subcellular localization of Vrg4p, three tandem copies of the HA epitope were added at the carboxyl terminus of the VRG4 gene (see Materials and Methods). Vrg4-HA3p in whole cell extracts was barely detectable by immunoassay when expressed as a single copy, even when three tandem copies of the HA epitope were added (FIG. 5A). The HA-added form of VRG4 did not complement the slowly growing form of the vrg4-2 mutant to the same extent as the wild-type VRG4 gene,
The sensitivity to hygromycin B as well as the lethality with the VRG4 deletion concentration could be complemented (data not shown). This suggests that the C-terminal addition of the HA-epitope does not significantly alter the normal function of the Vrg4 protein.

The intracellular location of Vrg4-HA3 was tested by indirect immunofluorescence using an antibody against the HA epitope. The fluorescent dot pattern characteristic of the Golgi complex is Vr
This was observed in cells expressing g4-HA3 (FIG. 5B). This staining pattern was similar to the early G1,6 mannosyltransferase, Och1p, another Golgi-localized protein (FIG. 5B). One difference in the staining patterns of these proteins is that, in general, more dot-like spots are Vrg4-HA3p
That was observed in the expressing cells. Most of the observed Vrg4-H
In A3p-expressing cells, the average number of HA-stained spots per cell observed by shifting the focal plane was 20-25. This indicates that analysis of 1 μ thick optical sections through individual cells showed an average number of 25 spots per cell, Z
-Confirmed by performing a series (data not shown). Och1-H
Cells expressing A3p contained between 7-10 spots / cell, and no qualitative differences were observed in cells expressing Och1p. From these results we conclude that Vrg4p is present in the Golgi complex. Taken together with immunoelectron microscopy studies suggesting that the yeast Golgi consist of about 30 spot-like structures (28), unlike Och1p, which is more spatially restricted, V
The rg4 protein appears to be widely distributed throughout the Golgi complex.

Example 9 Homology to VRG4 protein was determined by other putative S. VRG4 encodes a highly conserved protein that predicts the presence of cerevisiae nucleotide sugar transporters. Thirteen different members were identified, and Leishmania LPG2 and Kluyveromyces
lactis MNN2 gene product (12,29,30). In the case of Lpg2p and Mnn2p, both proteins were identified as nucleotide sugar transporters (2
9-31). S. Search of cerevisiae genomic database is Vr
The identification of several other yeast ORFs with sequence similarity to g4 suggests that these putative proteins may function in nucleotide sugar transport. These putative yeast proteins are listed in Table 3 under the ORF name. We have named HVG1 (H omologous to V R G 4), one of these ORFs (Yer039p) encodes a high similarity predicted protein against Vrg4p (80% identical). The other proteins listed in Table 3 are more distantly related to Vrg4 and Hvg1p (approximately 25% identical and 45% similar along their length), but near overlap in their respective hydropathic profiles (Data not shown), each of these proteins is the same size (35-45 kD) and has a similar predicted structure.

[0110]

[Table 3]

By using the sequence of the Vrg4 protein, the BLAST algorithm (42)
Using S. Search the cerevisiae genome database and search for Clust
Identical proteins were aligned using the DNASTAR MegAlign program with the al algorithm. The accession numbers for each are as follows: (hvg1 = Ye
m9 / yer039p accession number P40027); Yea4 / ye1004p accession number P40004; YMD8 accession number Q03697; Yor306c, accession number Q04835; Ym1018C accession number Z46659x21.

Because of the high degree of identity to Vrg4p, it was interesting to test the phenotype of the Hvg1 protein and null mutants. The predicted ORF and flanking sequences were cloned by PCR amplification of yeast genomic DNA (see Materials and Methods). To determine whether the HVG1 gene can complement the vrg4 mutation,
The VG1 gene was introduced into the vrg4-2 mutant (NDY5) in either the single copy expression plasmid or the high copy expression plasmid. Although the encoded gene products are very similar, the HVG1 gene does not complement the glycosylation of the vrg4-2 mutant and the slow growing phenotype or the nonviability of the null vrg4 allele. Unlike VRG4, which is required for survival, deficiency of HVG1 has no discernable effect on the growth characteristics of vegetatively growing cells. Similarly, vrg4-2
The hvg1 double mutant did not show any synthetic phenotype, and PYCs prepared from the hvg mutant had wild-type levels of GDP-mannose transport activity (data not shown). These results demonstrate that the Hvg1p and Vrg4 proteins do not have overlapping functions. They also suggest that Hvg1p fulfills an overlapping function with the other as an unidentified protein, or whether that function is completely essential for vegetative growth in yeast.

Example 10 We have used Vr as a model to understand nucleotide sugar transport in the Golgi.
We have begun functional analysis of g4p. We analyzed epitope-added alleles of VRG4 fused to either myc or HA epitope. The results from the co-immunoprecipitation experiments demonstrate that the Vrg4 protein multimerizes with specificity and high affinity both in vivo and in vitro. The molecular weight of the Vrg4p-containing complex calculated by gel filtration is twice that of the monomer, indicating that the enzyme is a dimer of the same subunit. In addition to the wild-type protein, we have also characterized the protein encoded by the mutant allele of VRG4. The mutein is catalytically inactive for nucleotide sugar-transport, but retains the ability to multimerize, is normally localized in the Golgi apparatus, and is as stable as its wild-type control. is there. Sequence analysis of the vrg4-2 allele revealed a single base pair change that changed from an alanine residue to an asparagine residue.
This alanine was buried in a region that was highly conserved in another GDP-mannose transporter but was different in other nucleotide sugar transporters. These results are consistent with a model that identifies this amino acid as a site involved in or binding to GDP-mannose transport. Materials and Methods Yeast Strains and Media Standard yeast media and genetic techniques were used (48). Hygromycin B sensitivity was determined using yeast extract / peptone / adenine sulfate / dextran plate (Y) supplemented with 50 μg / ml hygromycin B (Boehringer Mannheim).
(AD) (49) Tested as described above. The wild type strain used was SEY
6210 (MATα ura3-52 leu2-3,112 his3-Δ2
00-trp1-Δ901 lys2-801 suc2Δ9) NDY5 (M
ATα ura3-52 leu2-211 vrg4-2) (12) as vrg
Used as a source of genomic DNA for cloning the 4-2 allele. The same genotype parent strain is RSY255 (MATα ura3-52 leu2-211).
Met. Cloning of vrg4-2 Allele and DNA Sequence Analysis A 1.35 kB fragment containing the open reading frame of vrg4-2 and a 5 'flanking sequence of 237 base pairs and a 3' flanking sequence of 72 base pairs was obtained from vrg4- From genomic DNA isolated from two strains NDY5 (12),
By PCR using LA Taq thermophilic DNA polymerase (TaKaRa Shuzo, Japan), the following primers were used: 5′CGTAATGAATCGCAATAT
ACG 3 '(SEQ ID NO: 25) and 5' TTGCATTAGATAGCCCTCT
Amplification was performed using ATAA 3 '(SEQ ID NO: 26). LA Taq polymerase has as high fidelity as Pfu, but lacks 3 'to 5' proofreading exonuclease activity like Taq polymerase. This is directly related to pCRII-TA
PCRII by insertion into a cloning vector (Invitrogen)
This results in a PCR product containing the TA overhang producing vrg4-2. Plasmids from three individual clones were isolated, each of these sequences was compared by PCR to the VRG4 gene isolated from the isogenic parent strain, and PCR-based mutations were eliminated in the same manner. DNA sequencing was performed using the Thermo Sequence cycle sequencing kit (Amersham Pharmacia Biotech) by the dideoxy chain termination method (45) as described (50). DN
A sequence analysis was performed using an automated LI-COR 4000L DNA sequencer. Plasmid Construction Standard molecular biology techniques were used for all plasmid constructions (45). DNA
Sequence analysis identified a single C to A base pair change at nucleotide 857 within the vrg4-2 allele. VRG4 or v differ only in this mutation
To construct an equivalent series of expression plasmids containing any of the rg4-2 alleles, a 251 base pair HpaI from pCRIIvrg4-2 containing this point mutation was constructed.
The same region in the wild-type VRG4 gene was replaced by using the / MfeI fragment. Plasmid pRS316Vrg4-A286D contains CEN6 / URA3
It was created by replacing this fragment in pRHL (12) containing the VRG4 gene under its own promoter on the EcoRI / HindIII fragment in the vector pRS316 (46).

[0114] pRS316Vrg4-A286D-HA ₃ encodes Vrg4-A286D mutation protein which is added three copies of the HA epitope at the C- terminus in pRS316. It, HpaI / MfeI fragment pRHL-HA ₃ a (51),
It was constructed by replacing with the HpaI / MfeI fragment from pCRIIvrg4-2. To construct the YEp352-Vrg4-A286D-HA ₃ containing HA- additional vrg4-2 allele on 2.mu. / URA3 plasmids, full Vr
including g4-A286D-HA _3, it was subcloned into YEp352 (52) a HindIII / XbaI fragment from pRS316 Vrg4-A286D-HA _3.

[0115] In order to build a pRHL-myc _{3, 3} a VRG4 gene in-frame
A copy of the myc epitope was cloned. Lacks a stop codon and 5'H
A fragment containing the VRG4 ORF flanking the indIII and 3'NsiI sites was isolated by PCR. This fragment was digested with HindIII-PstI digested pS
K ^- P / X myc _3, Blue script SK ^- was cloned into a derivative (Stratagene). pSK ^- P / X myc ₃ is a myc epitope (EQKLISEEDL) cloned between the PstI and XbaI sites (SEQ ID NO: 27)
Has a 172 bp fragment containing a sequence that encodes three copies in tandem. That is, pS
K ^- P / X myc ₃ construct contains in-frame fusion of three copies of the myc epitope to the carboxyl terminus of Vrg4p. The use of VRG4-myc _3, VRG4-m on EcoRI / HindIII fragments in pRS316
to generate the pRHL-myc _3, including yc _3. vrg4-A2 with myc added
The 86D allele was constructed in several steps. First, MfeI / S
vrg4-A286 containing three HA tags but lacking a point mutation on the acl fragment
The fragment containing the 3 'end of the D-HA _3, MfeI corresponding from VRG4-myc ₃
/ SacI fragment. The resulting plasmid pRS316-VRG4-A
286D-myc ₃ is in pRS316, including vrg4-2 alleles added myc. The HindIII / XbaI fragment of 1.3kB containing ORF and promoter sequences of the complete _{vrg4-A286D-myc 3, LEU2} / 2μ
The vector was subcloned into YEplacl181 and YEplacl181 was subcloned.
To generate the -Vrg4-A286D-myc _3.

The HA-added GDA1 plasmid was subjected to PCR to 5 ′ of the GDA1 ORF.
And 3 'were created by introducing a SalI / EcoRI site. Sal
After digestion with I and EcoRI, the fragment pSK ^- pSK by ligating SalI / EcoRI sites of P / X HA3 plasmid (15) ^- GDA1
To produce a -HA _3. This results in an in-frame fusion of GDA1 with three HA epitope copies at the 3 ′ end followed by a sequence encoding a stop codon. pS
K ^- sequence of GDA1-HA ₃ was confirmed by DNA sequencing as described above. ^{Finally, pSK - GDA1-HA SalI /} Not including GDA1-HA ₃ from ₃
It was generated pYO323 GDA1-HA ₃ by subcloning into the I fragment HIS3 / 2.mu. expression vector pYO323 (53). Preparation of Cell-Free Lysate Exponentially growing yeast cells (A600: 1-3) were collected and converted to spheroplasts using lyticase (SIGMA) as described in (59) above. Spheroplasts are 400 μl containing 1% digitonin or 1% Triton X-100.
of ice-cold lysis buffer (150 mM NaCl, 10 mM HEPES-KO
The membrane proteins were solubilized by resuspension in H (pH 7.5), 5 mM MgCl ₂ , 1 mM PMSF) and the residue was removed by centrifugation at 14 kG for 5 minutes at 4 ° C. These detergent extracts were used for both FPLV analysis and co-immunoprecipitation assays, as described below. Whole cell extracts were prepared by TCA as described (59).

For preparation of the membrane fraction, 50 A ₆₀₀ units of cells were spheroplasted with lyticase (59). Spheroplast is a protease inhibitor cocktail (1 mM phenylmethylsulfonyl fluoride (PMSF), 1 μg / ml
Pepstatin, 50 μg / ml N-tosyl-L-lysine chloromethyl ketone (
TLCK), 1 ml containing 100 μg / ml N-tosyl-L-phenylalanine chloromethyl ketone (TPCK) and 100 μg / ml topsin inhibitor)
Ice-cold lysis buffer (0.1 M sorbitol, 50 mM potassium acetate, 2 mM
Resuspended in EDTA, 20 mM HEPES (pH 7.4), 1 mM DTT. Lysis is performed on ice with dounce homogenization (
(25 strokes) and unbroken cells were removed from the lysate by centrifugation in a microcentrifuge tube for 5 minutes. The membrane is Beckman Optima TL
Isolated by centrifugation at 100 kG for 30 minutes in an ultracentrifuge. The membrane precipitate was resuspended in 150 μl lysis buffer and used for protease protection assays (see below). Co-immunoprecipitation, Western immunoblotting and immunofluorescence 400 μl of detergent extract (above) was added to 200 μl of 12CA5 monomer anti-H
Incubation with the A antibody-containing hybridoma cell culture and 25 μl of protein A-Sepharose (Pharmacia) for 2 hours at room temperature resulted in H
The protein to which A was added was immunoprecipitated. Protein A-Sepharose beads and bound proteins were centrifuged and centrifuged in the same lysis buffer (1% digitonin or 1% Triton X-100; 150 mM NaCl, 50 mM HEPES).
-KOH (pH 7.5), washed three times with 5mM MgCl _2, 1mM PMSF). After resuspension in Laemmli's sample buffer and solubilization at 45 ° C. for 3 minutes. The immunoprecipitate was subjected to 10% SDS-PAGE.
, Transferred to Immobilon-PVDF membrane (Millipore) and anti-myc
Immunoblot was performed with an A-14 polyclonal antibody (Santa Cruz Biotechnology). Secondary anti-rabbit antibody conjugated to horseradish peroxidase (Amersham) was used at a 1: 3000 dilution and detected by chemiluminescence (ECL, Amersham) followed by autoradiography.

Indirect immunofluorescence of yeast cells expressing Vrg4-HAp or Vrg4A286D-HA was performed as described (51). The sample was observed with a Zeiss axiscope and photographed with a Sony DXC-9000 cooled CCD camera. Images were captured using NIH Image Software and all processing was performed on canvas version 5 (Deneba). Results Vrg4-protein multimerizes in vivo and in vitro To test whether the Vrg4 protein functions as a monomer or as a high-order structure, the Vrg4 protein interacts with itself using a co-immunoprecipitation assay first. It was measured whether it was possible. HA on high copy plasmid
Yeast strains were constructed that co-express the-and myc-added VRG4 alleles. Since both of these added alleles can complement the hygromycin B sensitivity of the vrg4 mutant, but not as much as the non-added allele,
It is shown that these epitopes do not significantly alter the normal function of Vrg4p. Membrane protein from this strain was solubilized with 1% digitonin to produce 12CA5 anti-
Immunoprecipitated with the HA monoclonal antibody. To determine the relative amount of Vrg4-myc protein bound to HA-added Vrg4p, the precipitate was subjected to SDS-PAGE.
And immunoprecipitated using a rabbit antiserum against the myc epitope (FIG. 10A). Vrg4-mycp was efficiently immunoprecipitated using Vrg4-HA, but could only be detected in the presence of an extract containing Vrg4-HA (FIG. 10A, compare lanes 1 and 2). Using anti-myc antibody for immunoprecipitation,
Similar results were obtained only when the anti-HA antibody was used for western blotting (not shown), indicating that co-precipitation is not antibody dependent. Vrg4-H
A strain that co-expresses Ap and Vrg4-mycp at high levels was used for the experiment shown in FIG. Vrg4p efficiently multimerized when expressed from the chromosomal allele that was found. These results suggest that Vrg4 efficiently multimerizes.

Vrg4 is extremely hydrophobic and contains 6 to 8 predicted membrane spatial domains. As a control for non-specific aggregation due to its hydrophobicity, we tested whether we could not detect the interaction of Vrg4p with other membrane proteins. Gdal-HAp, a Golgi-paired Gdal-HAp with a single transmembrane domain (FIG. 10B
Lane 4), oligomerization of Vrg4p as neither Yndl-HAp (not shown) nor Pmalp, a plasma membrane protein containing 10 predicted transmembrane domains (not shown), co-precipitated with Vrg4-mycp. Is not due to non-specific hydrophobic interactions.

To determine whether the observed Vrg4p-containing complexes assemble in vivo, we determined that digitonin extracts were prepared from strains that expressed either Vrg4-mycp or Vrg4-HAp. A mixing experiment was performed. These extracts were combined together prior to immunoprecipitation with anti-HA antibodies. After fractionation by SDS-PAGE, Vrg4-mycp could not be detected in the precipitate (FIG. 10A,
Lane 3) demonstrates that the complex was stably formed in vivo and did not degrade in vitro in the presence of digitonin.

In the process of determining the optimal conditions for extracting the Vrg4-containing complex, we noticed that the Vrg4-containing complex behaves differently in Triton X-100 than digitonin. As observed with digitonin, stable V
Although rg4p oligomers could be extracted from 1% Triton X-100 solubilized yeast, Vrg4-mycp efficiently co-precipitated with Vrg4HA to form Vrg4p.
This is because detection was possible only in the presence of the HAp-containing extract (FIG. 10B, lanes 1 and 2). However, when Triton extracts from strains expressing Vrg4-mycp or Vrg4-HAp were mixed prior to immunoprecipitation with anti-HA antibodies, significant amounts of Vrg4mycp were precipitated with Vrg4-HAp (FIG. 10B, lanes). 3)
. The strains used for these experiments contain Vrg4p, which is not endogenously added, in addition to the epitope-added version. Presumably, the added and unadded forms of Vrg4p complex with each other in vivo. Thus, any binding between Vrg4-mycp and Vrg4-HAp we observed in vitro required degradation of the assembled complex in vivo. Since the amount of Vrg4mycp co-precipitated with Vrg4-HAp after mixing was three-fold lower than that co-precipitated from extracts prepared from cells co-expressing these two proteins (FIG. 10B, lanes 2 and 3). The Vrg4-containing complex suggests less stability in Triton X-100 than in digitonin. Triton
Of Crg4 within is specific since no binding to other membrane proteins such as Gdal-mycp (FIG. 10B, lane 4) or Yndl-mycp (not shown) was observed. . This was true in extracts from strains co-expressing these control proteins (not shown) or in extracts containing each of these proteins mixed prior to immunoprecipitation (FIG. 10B, lane 4). . Taken together, these results demonstrate that Vrg4 has the ability to multimerize with high specificity and efficiency both in vivo and in vitro. The mutant protein encoded by the vrg4-2 allele load is stable and retains the ability to interact with the protein. Deletion of the VRG4 gene is lethal, but we have previously demonstrated significant glycosylation both in vivo and in vitro. A viable allele of vrg4 having the phenotype was isolated. The vrg4-2 strain exhibits about 25-fold reduced levels of nucleotide sugar transport activity in vitro than that of the wild-type strain in vitro. To analyze muteins in yeast extracts, the vrg4-2 allele was cloned by PCR amplification of genomic DNA from the vrg4-2 mutant to add an HA- or myc-epitope at the C-terminus (Material). And methods). To test its inability to complement the hygromycin B growth activity of the vrg4-2 mutant (FIG. 11A),
Alternatively, isolation of the mutant allele was confirmed by sequence analysis by Western blotting analysis (FIG. 11B).

Sequence analysis demonstrated that the vrg4-2 allele contained a single point mutation that changed alanine at position 286 to aspartic acid (see below). Therefore, this mutant protein is called Vrg4-A286Dp. To determine whether the A286D mutation altered the stability of the protein, Western blot analysis using anti-HA antibodies revealed that steady-state levels of the HA-added mutant protein were normal Vrg4-HA.
Comparison with protein (FIG. 11B). By this assay, we have determined that steady-state levels of mutant and wild-type Vrg4 proteins are low-copy CEN vectors (FIG. 1).
1B, comparison of lanes 2 and 3) or high copy 2μ vector (FIG. 11B, lane 4)
And 5) were found to be virtually indistinguishable when expressed from either of the above. These results demonstrate that the Vrg4 A286D-HA protein is as stable as its wild-type control.

The co-precipitation assay was used to test the multimerization of mutant Vrg4 A286D-HA. HA- and myc-added mutant and wild-type V, respectively.
A yeast strain co-expressing any of the rg4 proteins or a yeast strain co-expressing the HA- and myc-addition mutant Vrg4-A286D protein was constructed. The relative affinities of the mutant protein itself and the wild-type Vrg4 protein were compared by quantifying the amount of Vrg4 A286Dmycp precipitated with anti-HA antibody in the detergent extract. Although the Vrg4A286D mutant protein could be multimerized in the same manner as the wild-type, the same amount of vrg4A286D-mycp was Vrg-HAcp even in Vrg4-HAp.
This is because 4-A286D-HAp was also precipitated (FIG. 12, lanes 2 and 3). This resembled, but not identical to, the wild type Vrg4 protein precipitated by itself, which was typically observed within a range of 1.5 to 3 times greater (FIG. 12, lanes 3 and 4).
Vrg4-A286D protein is collectively localized in the Golgi apparatus The vrg4-2 mutation causes severe low-grade glycosylation of both proteins and lipids. In addition, this mutation is a background level of in vitro G
4 shows the level of DP-mannose transport activity. This raises the question of what is the biochemical basis for loss of nucleotide transport activity, as the Vrg4-A286D mutein is stable and also retains the ability to homo-dimerize. GDP
-Mannose transport activity is associated with the Golgi membrane, and the Vrg4-HA protein is localized in the Golgi of yeast. One possible explanation for its inactivity is Vr
g4 A286D is unable to exit the ER. To test whether the mutant Vrg4-A286D protein was in the wrong place, we compared its subcellular location to that of the normal Vrg4 protein. VRG4-HA or vrg4-A
Cells expressing 286D-HA were fixed with formaldehyde and HA-added proteins were detected by indirect immunofluorescence using an antibody directed against the HA-epitope. This assay showed that the mutant protein exhibited the same punctate pattern features as the yeast Golgi observed for the wild-type Vrg4 protein, which was identified for perinuclear ER staining (FIGS. 13A-13D). This suggests that the protein is in the correct location in the Golgi. Thus, the inactivity of the mutant Vrg4 A286D protein is not due to its incorrect localization. Sequence Analysis of Mutant vrg4-2 Alleles The above results indicate that the vrg4-2 allele contains mutations that affect nucleotide sugar transport but do not affect homooligomerization, Golgi localization or protein stability. showed that. To identify the molecular basis for the phenotype of this mutant,
The sequence of the rg4-2 mutant allele was determined. The mutant gene was cloned using a PCR approach (see Materials and Methods). Comparison of the DNA sequence from three separate clones containing the mutant allele with the wild-type VRG4 gene revealed a single C to A base pair change at nucleotide position 857. This point mutation results in the replacement of alanine by aspartic acid at position 286, whose position in the protein is graphically represented by the hydropathy plot in FIG. 14A. This amino acid is Vrg4 specifically conserved in Vrg4-related proteins.
It is located in the region of p (FIG. 14B).

Example 11 FIGS. 15A and 15B show S. cerevisiae by Candida VRG4 gene. cerev
2 shows the complementation of the vrae-4 mutation of isiae. This figure demonstrates that the isolated genes are not exactly structurally homologous, but functionally homologous, ie, the isolated C. elegans. albicans VRG4 gene proves to be a true GDP-mannose transporter. A second important aspect of this data is that the Candida gene is an S. aureus. cerevisiae, which is a Candid
a means that the targeted method of inhibiting the protein can be performed in this non-pathogenic strain more than in Candida.

[0125]

[Table 4]

[0126]

[Table 5]

[0127]

[Table 6]

[Sequence list]

[Brief description of the drawings]

FIG. 1. Immunoblot analysis of chitinase in vrg4 mutation and wild-type extracts. RSY255 (VRG4, lane 1), N as described in Materials and Methods.
Protein was extracted from the culture supernatant of DY5 (vrg4-2, lane 2) or HTY10 (mnn10-2, lane 3) by acetone precipitation, and 8% SDS-PA was extracted.
Fractionated by GE and subjected to immunoblot analysis with anti-chitinase antiserum. Arrows indicate the mobility of wild-type chitinase.

FIG. 2. Analysis of sphingolipids in vrg4-2 and wild type cells. RSY255 (VRG4) or NDY5 (vr) as described in Materials and Methods.
G4-2) sphingolipid in and pulsed with ^[3 H] myo-inositol, and Chase 20-40 minutes using unlabeled inositol, extracted and separated by thin layer chromatography. PI, IPCs, M indicated by arrows
The evaluation of IPC and M (IP) ₂ C is based on comparing their mobilities on TLC to those reported in the literature and many of their links.

FIG. 3. GDP-mannose transporter in permeabilized yeast cells as a function of time and protein concentration. PYCs (strain JPY25) as described in Materials and Methods.
3cM GDP-mannose and 50nCi GDP
Incubated in a final volume of 25 μl in reaction buffer containing-[ ³ H] -mannose (15 Ci / mmole). FIG. 3A shows the transport of GDP- [ ³ H] -mannose as a function of time when the reaction was performed at a final protein concentration of 0.5 μg / μl. FIG. 3B shows the protein dependence of the reaction performed at 30 ° C. for 6 minutes under the above conditions. A GDP mannose transport of 20 pmoles corresponds to a 27% lumen uptake of GDP-mannose in the reaction. Typical values of absolute cpms transferred to vesicles prepared from 25 μl reactions range from 6000-12,000 cpm over 10 separate experiments.

FIG. 4. GDP-mannose transporter activity in vrg4-2 and wild type cells.
PYCs were prepared in parallel from JPY26 3d (vrg4-2), JPY26 3d containing pRHL carrying the wild-type VRG4 gene or wild-type strain JPY256c of the same genotype as described in Materials and Methods. PYCs are standard GD
The time of incubation was varied for use in the P-mannose transporter assay. The percentage of GDP-mannose transported was determined as described in Materials and Methods.

FIG. 5. Western blot and cytology analysis of Vrg4 protein. FIG.
Whole cell protein extracts were purified from CEN plasmid (as described in Materials and Methods).
pRHL-HA3), prepared from yeast cells (SEY6210) with or without a plasmid expressing Vrg4-HA3p on a 2μ plasmid (pYRHL-HA3) or vector alone (pYEp352) and 1
Fractionated by 0% SDS-PAGE and subjected to Western blot analysis using anti-HA antibody. Figures 5B and 5C. SEY6210 cells or Vrg4-
SEY62 expressing HA3p (FIG. 5B) or Och1-HA3p (FIG. 5C)
Indirect immunofluorescence of 10 cells. The fixed cells are treated with anti-HA antibody and then FI
Treated with TC-conjugated anti-mouse IgG and viewed by confocal microscopy.

FIG. 6: VRG4 derived from Saccharomyces cerevisiae
Gene nucleotide sequence (SEQ ID NO: 1) and amino acid sequence (SEQ ID NO: 2)
). Five potential glycosylation sites at amino acid positions 81, 119, 242, 246, and 249 are indicated by asterisks. Domains consisting of at least 20 uncharged residues and spanning four dominant membranes with charged residues around are underlined. This sequence was found to be identical to the sequence of VAN2 gene (GenBank ^TM accession number U15599 (11)).

FIG. 7 shows the nucleotide sequence (SEQ ID NO: 3) and amino acid sequence (SEQ ID NO: 4) of the VRG4 gene derived from Candida albicans.

FIG. cerevisiae and C.I. albicans VRG4 protein alignment. Shown here are C.I. albicans VRG4 analog (
SEQ ID NO: 5) and S. cerevisiae VRG4 protein (SEQ ID NO: 6) is its conserved region. The alignment is based on the gapped BLAST algorithm (Altschul, S. et al 1997, Gapped B).
LAST and PSI-BLAST: A New Generation
of Protein Database Programs, Nucleic
Acids Research 25: 3389-3401). The two proteins show 65% homology and 78% similarity along their full length.

FIG. 9 is a physical map of the pSK ^- CaVRG4 plasmid containing a portion of the VRG4 gene from Candida albicans.

FIG. 10. Co-immunoprecipitation of stable Vrg4p multimers in detergent extract. The epitope-added Vrg4p-containing complex was transformed with 1% digitonin (PANEL 1
Extracted from yeast cells by treatment with OA) or 1% Triton X-100 (PANEL 10B) and immunoprecipitated with anti-HA antibody. After loading an equal amount of protein in each lane and fractionating by SDS-PAGE, the precipitate was subjected to anti-myc
The antibodies were immunoblotted and detected by chemiluminescence. Extracts were prepared using VRG
_{4-myc (YEplac81-Vrg4myc 3} ) only whether expression (lane 1), VRG4-myc (YEplac181 -Vrg4myc 3) and VRG4
-HA (YEp352-Vrg4-HA ₃ ) (lane 2
), Or _{VRG4-myc (YEplac181-Vrg4myc 3} ) or VRG4-HA (YEp352-Vrg4- HA 3) expressing either,
Prepared from yeast cells (SEY6210) and mixed after extraction and before immunoprecipitation (lane 3). The extract was purified using VRG4-myc (YEplacl181-Vr).
g4myc ₃ ) and GDA1-HA (pY023-GDA1-HA ₃ ) were prepared from SEY6210 co-expressing (PANEL 10A, lane 4) or from strains expressing any of these genes and mixed prior to immunoprecipitation. (PANEL 1
OB, lane 4).

FIG. 11. Expression of cloned vrg4-2 (A286D) mutant allele. The vrg4-2 mutant allele was cloned to add an epitope, as described in Materials and Methods. PANEL 11A. VRG4-HA (YEp-
RHL-HA ₃₎ or vrg4-A286D-HA (YEpVrg4- A28
6D-HA ₃ ) was transformed with a plasmid containing any of the same genotype wild type (PSY255) or vrg4-2 mutant cell (NDY5) at 50 μg /
Streaked on medium plus or minus ml of hygromycin B. PANEL 11B. Mutant Vrg4-A286D-HA and wild-type Vrg4
-Western blot of HA protein. CEN plasmid (pRHL-HA ₃₎ (
Lane 2) or V on the 2μ plasmid (YEpRHL-HA3) (lane 5).
RG4-HA or CEN plasmid (pRS316-Vrg4-A286D
-HA ₃₎ (lane 3) or 2μ plasmid (YEp352-Vrg4-A2
86D-HA ₃₎ (extracted from the wild type yeast expressing vrg4-A286D-HA on lane 4) (SEY6210). After loading an equal amount of protein per well, proteins were fractionated by SDS-PAGE, subjected to Western blot analysis with anti-HA antibody, and detected by chemiluminescence.

FIG. 12. Vrg4-A286D mutein is itself as well as wild-type Vrg4
Interacts stably with proteins. The extract was treated with VRG4-myc only (YEplac181-Vrg4-myc)._Three
) Is expressed (lane 1) or VRG4-HA (YEpRHHLHA)._Three) And vrg
4-A286D-myc (YEplac181-Vrg4-A286D-myc _Three ) Is co-expressed (lane 2) or vrg4A286D-HA and vrg4-A
286D-myc (YEp352-Vrg4-A2826-HA_ThreeAnd YEpla
c181Vrg4-A286D-myc_Three) Is co-expressed (lane 3) or
Or VRG4-HA and VRG4-myc (YEpRHL-HA_ThreeAnd YEplac
181-Vrg4-myc_Three) (Lane 4), a yeast strain (SEY6)
210). Vrg4p-containing complex was treated with 1% digitonin
Extracted from yeast cells and immunoprecipitated with anti-HA antibody. In each lane
And after fractionation by SDS-PAGE with loading of an equal amount of protein, the precipitate was anti-m
Immunoblot with the yc antibody was detected by chemiluminescence.

FIG. 13. Vrg4-A286D mutein is localized in the Golgi apparatus. _{Vrg4-HAp (pRHL-HA 3} ) (13C), mutant Vrg4-A286
D-HAp (pRS316-Vrg4- A286D-HA 3) (13D), Oc
Indirect immunofluorescence of SEY6210 expressing h1-HAp (Golgi marker) (13B) and Ost4-HAp (ER marker) (13A). Fixed cells were treated with anti-HA antibody and then FITA-conjugated anti-mouse I
Treated with gG.

FIG. 14. The vrg4-2 allele contains a single mutation (A286D) within a region of the protein that is highly conserved among other NSTs. PANEL 14A. The hydropathy profile of the Vrg4 protein is shown with the location of the A286D mutation shown in circles. PANEL 14B. Alignment of the region surrounding the A286D mutation (indicated by an asterisk) in other nucleotide sugar transporters.
Group I proteins include the Leishmania donovani Lpg2 protein and S. aureus. cerevisiae is closely related to the GDP-mannose transporter defined by the Vrg4 protein. Group II proteins are most closely related to those that transport UDP-sugars. Indicates the accession number for the related but unidentified ORFs. The identity of these proteins was selected using BLAST version 2.0s [Altschul, 1997 # 242]. The alignment is D
Performed using the Clustal algorithm, using the NASSTAR MegAlign program. The consensus was strictly defined as 5 out of 6 identical residues for Group I proteins (black shade) and 5 out of 8 identical residues for Group II (gray shade). . Group II residues identical to Group I are shaded in black.

FIGS. 15A and 15B show S. cerevisiae by Candida VRG4 gene.
3 shows the complementation of the cerevisiae vrg-4 mutation. This figure demonstrates that the isolated genes are not exactly structurally homologous, but functionally homologous, ie, the isolated C. elegans. albicans VRG4 gene proves to be a true GDP-mannose transporter.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C12N 1/19 C12N 1/21 4C084 1/21 C12P 21/02 C 4H045 5/10 21/08 C12P 21 / 02 C12Q 1/02 21/08 1/18 C12Q 1/02 1/68 A 1/18 G01N 33/15 Z 1/68 33/50 Z G01N 33/15 33/53 D 33/50 M 33/53 33 / 566 33/577 B 33/566 C12N 15/00 ZNAA 33/577 5/00 B (81) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SL SZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY , CA, CH, CN, CU, CZ, DE, DK, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG , SI, SK, SL, TJ, TM, TR, TT, UA, UG, US, UZ, VN, YU, ZA, ZWF terms (reference) 2G045 AA40 DA12 DA13 DA36 FB02 FB03 4B024 AA01 AA11 BA80 CA03 CA09 CA20 DA02 DA05 DA12 EA04 GA11 GA19 GA27 HA13 HA14 4B063 QA01 QA05 QA13 QA17 QQ07 QQ43 QQ53 QQ61 QQ98 QR08 QR 32 QR42 QR55 QR62 QR72 QR80 QS16 QS25 QS28 QS34 QX02 QX07 QX10 4B064 AG01 AG27 CA06 CA10 CA19 CA20 CC01 CC24 DA01 DA13 4B065 AA01X AA72X AA72Y AA90X AB01 AC14 BA02 BA10 BA25 BA30 BB01 4 DA76 EA20 EA29 EA50 FA72 FA74

Claims (85)

[Claims] 1. A method for measuring the activity of a yeast-derived nucleotide-sugar transporter, comprising the steps of: A) converting a nucleotide-sugar into a permeabilizable yeast spheroplast comprising a nucleotide-sugar transporter and yeast Golgi; B) measuring the amount of nucleotide-sugar bound to the Golgi apparatus as an indicator of nucleotide-sugar transporter activity. 2. The method of claim 1, wherein the source is permeabilized using liquid nitrogen. 3. A method for measuring the activity of a yeast-derived nucleotide-sugar transporter, comprising: A) supplying a nucleotide-sugar or a derivative thereof to a mammalian cell, provided that the mammalian cell is a yeast-derived nucleotide-sugar. B) determining the amount of nucleotide-sugar bound to the Golgi, provided that the amount is indicative of the activity of the nucleotide-sugar transporter. . 4. The method according to claim 1, wherein the nucleotide-sugar is GDP-mannose or a derivative thereof. 5. The method according to claim 1, wherein the yeast is Saccharomyces specialty or Ca.
4. The method according to claim 1 or 3, wherein the method is ndida specialty. 6. The method according to claim 6, wherein the yeast is Candida albicans, Candida.
4. The method according to claim 1 or 3, wherein the method is tropicalis or Torulopsis glabrata. 7. The method according to claim 1, wherein the yeast is Saccharomyces cerevisiae. 8. The method according to claim 1, wherein the yeast is Cryptococcus neoformans. 9. The method according to claim 1, wherein the yeast is Aspergillus species. 10. The method of claim 1, wherein the source is a yeast cell containing a dolichol phosphate-mannose synthase mutation. 11. The method of claim 1, wherein the yeast cell contains a foreign gene encoding a nucleotide transporter. 12. The method of claim 1, wherein the yeast cell contains a foreign gene encoding a GDP-mannose binding domain. 13. The method of claim 1, wherein the source is yeast strain JPY263D deposited at the ATCC under accession number ATCC74461. 14. The method according to claim 1, wherein the nucleotide-sugar transporter is a VRG4 protein or a homolog thereof. 15. The method according to claim 14, wherein the VRG4 protein is encoded by SEQ ID NO: 1 or SEQ ID NO: 3, or is a functional protein thereof. 16. The method according to claim 14, wherein the VRG4 protein has SEQ ID NO: 2 or SEQ ID NO: 4 or a functional protein thereof. 17. The VRG4 protein is SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO:
15. The method of claim 14, comprising 11 or SEQ ID NO: 23 or a homolog thereof. 18. The method according to claim 18, wherein the VRG4 protein is Saccharomyces special.
The method of claim 14, wherein the method is derived from e or Candida specie. 19. The VRG4 protein may be Cryptococcus neofor.
15. It is derived from C. mans or Aspergillus species.
The described method. 20. A method for screening an inhibitor of Golgi nucleotide-sugar transporter activity in yeast, comprising: A) prior to or simultaneously with the nucleotide-sugar, the candidate inhibitor is added to the nucleotide-sugar transporter and yeast Golgi. And B) measuring the amount of nucleotide-sugar bound to the Golgi, provided that the amount of Golgi-bound nucleotide-sugar in the absence of the inhibitor is measured. A method wherein the decrease in the amount of Golgi-bound nucleotide-sugar in the presence of the inhibitor relative to the amount is indicative of an inhibitor of transporter activity. 21. A method of screening for inhibitors of Golgi nucleotide-sugar transporter activity in yeast, comprising: A) providing a candidate inhibitor to a mammalian cell prior to or simultaneously with the nucleotide-sugar, The mammalian cell contains the yeast nucleotide-sugar transporter and B) measures the amount of nucleotide-sugar bound to the Golgi, provided that the amount of Golgi-bound nucleotide-sugar in the absence of the candidate inhibitor is measured. A method wherein the decrease in the amount of Golgi-bound nucleotide-sugar in the presence of the compared inhibitor is indicative of an inhibitor of transporter activity. 22. The method according to claim 20, wherein the nucleotide-sugar transporter is a GDP-mannose transporter. 23. The GDP-mannose transporter is Saccharomyces
specification, Candida specification, Cryptococcus s
pesie, Torulopsis species, or Aspergill
23. The method of claim 22, wherein said method is derived from a U.S. species. 24. The GDP-mannose transporter comprises SEQ ID NO: 2, SEQ ID NO: 4,
23. The method of claim 22, comprising SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 23, or a functional portion thereof. 25. The method of claim 22, wherein the GDP-mannose transporter is encoded by SEQ ID NO: 1, SEQ ID NO: 3, or a functional portion thereof. 26. The method of claim 20, wherein the inhibitor is a nucleotide-sugar analog. 27. The method of claim 20, wherein the inhibitor is a competitive or non-competitive inhibitor. 28. The method according to claim 20, wherein the inhibitor is stilbene or a derivative thereof. 29. The inhibitor of claim 4, wherein the inhibitor is a derivative of 4,4-diisothiocyanostilbene-2,2-disulfonic acid that specifically inhibits a GDP-mannose derivative and is non-toxic to mammalian cells. 22. The method according to 20 or 21. 30. The method of claim 20 or 21, wherein the inhibitor is an antibody or fragment thereof immunoreactive with VRG4 or an epitope thereof. 31. The method of claim 20, wherein the inhibitor is part of a VRG4 protein.
The method of claim 1. 32. The inhibitor of claim 2, wherein the inhibitor is a GDP-mannose binding domain.
22. The method according to 0 or 21. 33. A kit comprising permeabilized yeast spheroplast and optionally a nucleotide-sugar for use in the method of claim 1, 14, 20 or 21. 34. The permeabilizable yeast is derived from Saccharomyces.
A kit according to claim 33. 35. The nucleotide-sugar is GDP-mannose.
The kit as described. 36. Golgi G measured by the method according to claim 20 or 21.
An antifungal compound having DP-mannose transporter inhibitor activity. 37. A pharmaceutical composition for inhibiting the growth of yeast in a patient, comprising the compound of claim 36 as an active ingredient and a pharmaceutically acceptable vehicle. 38. A method for inhibiting yeast growth in a patient, comprising administering to the patient an amount of an antifungal compound effective to inhibit the activity of a Golgi GDP-mannose transporter. 39. An isolated nucleotide sequence comprising VRG4 from pathogenic yeast or a part thereof. 40. The nucleotide sequence consisting of SEQ ID NO: 3 or a portion thereof,
40. The isolated nucleotide sequence of claim 39. 41. The isolated nucleotide sequence of claim 39, wherein the pathogenic yeast is Candida albicans. 42. The pathogenic yeast is Cryptococcus neoforma.
40. The isolated nucleotide sequence of claim 39, which is ns or Aspergillus species. 43. The isolated nucleotide sequence of claim 39, which encodes a GDP-mannose transporter activity. 44. The gene encoding a GDP-mannose binding domain.
An isolated nucleotide sequence as described. 45. The isolated nucleotide sequence of claim 39, which encodes SEQ ID NO: 4 or a homolog thereof. 46. SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 1
An isolated nucleotide sequence encoding one or a homolog thereof. 47. An isolated nucleotide sequence encoding a consensus sequence comprising SEQ ID NO: 23. 48. A recombinant protein encoded by the nucleotide sequence according to claim 39 or part thereof. 49. SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9
, SEQ ID NO: 11, SEQ ID NO: 13, a protein or peptide comprising an amino acid sequence comprising a substantially homologous sequence thereof, or a portion thereof. 50. A recombinant expression vector comprising the nucleotide sequence of SEQ ID NO: 3 or a part thereof. 51. The S deposited at the ATCC under the accession number ATCC 203137.
51. The recombinant expression vector according to claim 50, designated as K ^- CaVRG4. 52. A recombinant expression vector encoding a GDP-mannose binding domain. 53. A recombinant expression vector wherein the GDP-mannose binding domain comprises the amino acid sequence of SEQ ID NO: 7, SEQ ID NO: 11 or SEQ ID NO: 23. 54. A host cell transformed or transfected with the recombinant expression vector according to any one of claims 50-53. 55. The host cell of claim 54, wherein the host cell is selected from the group consisting of a yeast cell, a mammalian cell, a bacterial cell, and an insect cell. 56. The method of claim 54 or 5 which expresses a GDP-mannose transporter.
6. The host cell according to 5. 57. The cell is Saccharomyces cerevisiae.
The host cell according to any one of claims 54 to 46, wherein the host cell is: 58. A dolichol phosphate-mannose synthase mutation,
58. The host cell of claim 57. 59. The host cell according to claim 57, which is the yeast strain JPY263D deposited at the ATCC under accession number ATCC74461. 60. A lacking endogenous VRG4 gene and a non-functional endogenous VRG
60. The host cell according to any one of claims 54 to 59, having four genes. 61. The host cell of any one of claims 54-56 or 58-60, which is a mammalian cell. 62. A method for producing a recombinant VRG4 protein or a part thereof, comprising: A) inserting a nucleotide sequence encoding a VRG4 protein into an expression vector; B) transferring the expression vector into a host cell; C) A method comprising culturing a host cell under conditions that allow expression of the VRG4 protein or a portion thereof. 63. The method of claim 62, wherein the expression vector is selected from the group consisting of a yeast expression vector and a mammalian expression vector. 64. An isolated antibody immunoreactive with the VRG4 protein or an epitope thereof. 65. The isolated antibody of claim 64, wherein said antibody is a monoclonal antibody or a recombinantly produced antibody. 66. The isolated antibody of claim 65, wherein said recombinantly produced antibody is a single chain antibody. 67. A method for detecting a VRG4 genomic nucleic acid sequence in a biological sample, comprising: a) a VRG gene under conditions that form a complex between the VRG4 nucleic acid sequence and the nucleic acid sequence of the sample. Contacting all or a portion of the nucleic acid sequence of the above with a genomic nucleic acid isolated from a biological sample; and b) detecting a VRG4 genomic sequence complex. 68. The method of claim 67, wherein the VRG4 mutated genomic DNA sequence is detected. 69. A VRG4 gene detection kit comprising at least one oligonucleotide primer specific for the VRG4 gene. 70. A method for determining the therapeutic effect of an antifungal compound in a human patient, comprising: a) obtaining a tissue sample from a patient who has been treated for yeast infection; b) a VRG4 gene in the tissue sample. The coding sequence or VRG4 mRNA molecule is compared to the wild-type VRG4 gene coding sequence or VRG4 mRNA molecule, provided that the observed change in the VRG4 gene coding sequence or mRNA molecule in the tissue sample when compared to the wild type is indicative of a therapeutic effect. A method consisting of showing. 71. A VRG4 mRNA alteration is detected in mRNA isolated from a tissue sample.
71. The method of claim 70, wherein said method is detected by hybridization of RNA to a VRG4 gene probe. 72. The yeast according to claim 7, wherein the yeast is Candida albicans.
0. The method of claim 0. 73. The method of claim 70, wherein the yeast is Cryptococcus neoformans or Aspergillus species. 74. The method of claim 70, wherein the tissue sample is selected from the group consisting of cells, tissue, blood, serum, feces, urine, amniotic fluid, mucus secretions or sputum. 75. A method for detecting VRG4 protein or a portion thereof in a biological sample, comprising: a) contacting with a reagent specifically reacting with VRG4 protein in the sample; and b) protein and reagent Detecting the formation of a complex during the process. 76. The method of claim 75, wherein the sample is a membrane selected from the group consisting of yeast cells, mammalian tissues, mammalian cells, Golgi samples, and artificial membranes. 77. The method of claim 75, wherein said reagent is an antibody or a fragment thereof. 78. The method of claim 75, wherein said reagent is a monoclonal antibody. 79. The method of claim 75, wherein said reagent is a polyclonal antibody. 80. The method of claim 75, wherein the biological sample is an individual infected with Candida albicans. 81. The biological sample is a Cryptococcus neof
77. The method of claim 75, wherein the individual is infected with ormans or Aspergillus species. 82. The reagent of claim 7, wherein the reagent is a nucleotide-sugar or an analog thereof.
5. The method according to 5. 83. The method of claim 75, wherein said nucleotide-sugar is GDP-mannose or an analog thereof. 84. The method of claim 75, wherein the nucleotide-sugar is labeled for detection. 85. The sample is permeabilized yeast spheroplast.
78. The method of claim 75.