WO2004007724A1

WO2004007724A1 - Method of transporting protein to mitochondria

Info

Publication number: WO2004007724A1
Application number: PCT/JP2003/007252
Authority: WO
Inventors: Shinya Yoshikawa; Hideo Shimada; Kunitoshi Shimokata
Original assignee: Japan Science And Technology Agency
Priority date: 2002-07-11
Filing date: 2003-06-09
Publication date: 2004-01-22
Also published as: JP2004041067A

Abstract

It is intended to provide a means of transporting a protein to mitochondria without restraint of molecular weight, thereby making it possible to arbitrarily modify a protein synthesized in mitochondria and arbitrarily modify its function. Paying attention to the intracellular alignment (topology) of subunits, it is found out that a desired protein can be transported to mitochondria by using a transport signal inherent to a specific subunit. Such a subunit fulfills the following requirements: 1) being a subunit of a mitochondrial membrane protein encoded by nuclear DNA; and 2) in case of existing in a mitochondrial membrane, the N-end (preferably both of the N-end and the C-end) of the protein of the subunit existing in the mitochondrial matrix side.

Description

Description Method for transporting proteins to mitochondria

The present invention relates to a method for transporting a specific protein from the cytoplasm to mitochondria, and more particularly, to transport a protein encoded by mitochondrial DNA from the cytoplasm to mitochondria, and to modify the function of the protein encoded by mitochondrial DNA. About the method. Conventional technology

Intracellular mitochondria play a role in converting the chemical energy of carbohydrates, lipids, proteins, etc. into the chemical energy of ATP, and are essential for cell survival, but mitochondria contain DNA and contain 20 amino acids. Encodes the genetic information for tRNA and ribosome RNA corresponding to In other words, mitochondria have their own protein synthesis systems. In addition, mitochondrial DNA (mt DNA) encodes a partial subunit gene of a membrane enzyme that catalyzes the above-mentioned energy conversion reaction (respiration).

Methods for modifying mitochondrial DNA include (A) a method in which the gene is applied to metal microparticles and the gene is injected into mitochondria to introduce the gene (Methods in Enzymology, 264, 265-278 (1996), etc.). (B) A method of establishing a cell line in which mt DNA has been eliminated by using a ligature or by using an enzyme and introducing new mt DNA there (Proc. Natl. Acad. Sci. 85, 7288-7292 ( 1988)). (C) Furthermore, a method of modifying the function of mitochondria by incorporating the gene into the nucleus and transporting the protein expressed in the cytoplasm to mitochondria (Cell, 46, 837-844 (1986), etc.) is already known. Have been. The transport of membrane proteins related to this technology involves the transport of mitochondria by subunit 8 (SVIII) of ATP synthase encoded by yeast mtDNA consisting of 48 amino acid residues. Recovery Successful review (Proc. Natl. Acad. Sci. USA, 85, 2091-2095 (1988)) o

An object of the present invention is to develop a method for modifying the function of a multi-transmembrane protein encoded by mt DNA of a higher animal. In the conventional method that has been adopted to modify the function of the protein encoded by mt DNA, in the case of (A), the frequency at which the shot DNA is stably retained is low, and the mitochondria that are numerous in the cell Is not introduced into the gene. Therefore, this method is unsuitable for altering mitochondrial function, that is, a technique for treating mitochondrial disease. In the case of (B), the method for modifying the gene is correct in principle. However, it is necessary to establish a cell line in which mtDNA has been deleted. However, if DNA is lost, respiratory deficiency may occur, which may lead to problems such as slow cell growth, which is also technically difficult.

On the other hand, the method (C) requires an operation to change the unique codon used in the mitochondrial protein synthesis system to the cytoplasmic type, but a series of operations is relatively easy and can be achieved in a short time. In addition, protein transport is expected to occur equally in all mitochondria, which is desirable as a technique for modifying the function of mitochondria. In order to restore the function of mitochondria that has been lost, the method (C) transports a water-soluble protein encoded by mtDNA responsible for the function, and partially recovers the defective function. (Cell, 6, 837-844 (1986)).

In the case of the membrane protein encoded by mt DNA, SVIII, a single transmembrane yeast ATP synthase, was similarly transported to mitochondria and successfully expressed its function. However, eight transmembrane cytochromes 6 (subunits of the bc 1 complex in the respiratory system) and twelve transmembrane cytochrome oxidase subunits I (SI) cannot be transported to mitochondria. It has been reported (Eur. J. Biochem. 228, 762-771 (1995)). Here, the transport signal of the ATP synthase Subunit 9 is used as the transport signal, and both the N and C termini of the ATP synthase subunit 9 are located in the transmembrane space of mitochondria. (Science 286, 1700 (1999), subunit 9 above corresponds to subunit c in this paper.) The reason that high-molecular-weight proteins could not be transported may be due to the selection of this transport signal. Problems the invention is trying to solve

As described above, in the prior art, large-molecular-weight proteins such as eight-transmembrane cytochrome and SI of 12-transmembrane cytochrome oxidase cannot be transported to mitochondria. There has been a need for a technique that enables such proteins to be transported to mitochondria without being restricted by molecular weight. If such a technology becomes possible, it becomes possible to freely modify proteins synthesized in mitochondria, and as a result, it is possible to freely modify the functions of mitochondria. It can provide a means to treat defects in certain functions of tochondria. Means for solving the problem

Of the proteins synthesized in mitochondria, some are encoded by mitochondrial DNA, and others are encoded by nuclear DNA. The protein encoded by this nuclear DNA links a unique transport signal (also called the Targeting signal ^ Sorting signal) to cross the outer and inner membranes of the mitochondrial lipid bilayer, Transported to mitochondria. After transport, the transport signal is disconnected.

The present inventors paid attention to the orientation (topology) of subunits in the membrane, and found that a desired protein can be transported to mitochondria by using a transport signal specific to a specific subunit. The present invention has been completed.

Such a subunit satisfies the following conditions. 1) It is a subunit of the mitochondrial membrane protein encoded by nuclear DNA. Thus, this subunit is expressed in the cytoplasm and transported to mitochondria. 2) When this subunit is transported to the mitochondria and is present on the mitochondrial membrane, the N-terminus of the protein of this subunit, preferably both the N-terminus and the C-terminus, is present on the matrix side of the mitochondria. Subunit It is considered that a transmembrane type of two or more even times is suitable as such a subunit. By using a transport signal unique to such a substance, the above-mentioned problem can be solved. I was able to.

That is, the present invention relates to a method for binding a transport signal to the N-terminus of a specific protein to transport the protein from the cytoplasm to mitochondria, wherein the transport signal is a mitochondrial encoded by nuclear DNA. A subunit of a membrane protein of which the N-terminus, preferably both the N-terminus and the C-terminus, are present on the mitochondrial matrix side when the subunit is present in the mitochondrial membrane. A method characterized by being a transport signal.

The present effort also provides for mitochondrial membrane proteins having mitochondrial membrane proteins so produced, since subunits of membrane proteins can be transported to mitochondria in this manner to produce mitochondrial membrane proteins. And mitoplast in which the outer membrane has been removed from the mitochondria, and cells containing the mitochondria. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the mitochondrial SI gene prepared from the muscle of the mouse heart. TGA (single underline) and AT A (double underline) were modified to TGG and ATG, respectively.

FIG. 2 shows a process for preparing a subunit I expression vector of cytochrome _c oxidase having a histidine tag at the C-terminus and a transport signal at the N-terminus. FIG. 3 shows SDS-PAGE of purified ゥ cytochrome c oxidase at the same mobility as SI. (1) HeLa cells (mock cells) containing plasmid pRcZC MV without SI gene, mitoplasts prepared, and (2) Hela cells infected with plasmid (SI wt) (3) shows mitoplast (WT) obtained from cells, and (3) shows mitoplast (D51N) obtained from Hela cells infected with plasmid (pSI51n).

FIG. 4 shows an absorption spectrum of cytochrome c. Reduced cytochrome c shows a sharp absorption at 550 nm, but disappears in the oxidized form.

FIG. 5 shows cytochrome c oxidation activity. The vertical axis indicates the absorbance at 550 nm, and a decrease (below) indicates that oxidation has occurred. The horizontal axis indicates time. Steep The higher the activity, the higher the activity. Open arrows indicate the addition of reduced cytochrome c. Left (WT) expresses mitoplasts prepared from cells expressing wild-type SI, right (D51N) expresses mutant SI (subunit I with amino acid 51 mutated to asparagine) It shows the mitoplasts that do.

FIG. 6 shows the active transport activity of hydrogen ions. The vertical axis indicates the hydrogen ion concentration, and the lower the lower, the higher the hydrogen ion concentration. The horizontal axis indicates time. Open arrows indicate the addition of reduced cytochrome c. (A) shows WT, (B) shows WT plus uncoupler, and (C) shows D51N. Embodiment of the Invention

In the present invention, a specific transport signal is first bound to the N-terminal of the protein. Although there is no particular limitation on such a binding method, nuclear DNA is ligated by linking a nucleotide sequence encoding a transport signal to a gene encoding the protein so as to bind to an end corresponding to the N-terminus of the protein. In this case, a fusion protein in which a transport signal is linked to the N-terminal of the protein can be expressed in the cytoplasm. In this case, since the mitochondria and the nucleus have different genetic codes, it is necessary to appropriately modify the genetic code so that the mitochondrial gene brings about the same protein in the nucleus. In addition, the protein to be transported in the present invention may be such a protein, but one of the features of the present invention is that it can be applied to a protein encoded by mitochondrial DNA.

Such a protein has the same orientation as the subunit originally having the transport signal employed in the present invention, that is, when the subunit is present in the mitochondrial membrane, both its N-terminal and C-terminal are mitochondrial. Subunits that are present on the side of the matrix are most preferred. Such proteins include the subunits of mitochondrial membrane proteins such as SI of cytochrome b cytochrome oxidase.

Such a method can also be applied to proteins encoded by nuclear DNA, particularly membrane proteins.

Also, the method of the present invention is not limited by molecular weight, One of the features of the present invention is that the present invention can be applied to a protein whose amount is as large as 100,000 to 600,000.

As described above, the transport signal bound to the protein transported to the mitochondria is cleaved and removed, and the protein remaining as a result of this cleavage may function as it is in the mitochondria. If, for example, one of the subunits of the mitochondrial membrane protein, a mitochondrial membrane protein is formed from this protein and other subunits.

By this method, the function of mitochondrial proteins can be altered, and if such proteins are defective, they can be restored to normal ones. The invention's effect

The method of the present invention makes it possible to artificially alter various functions dependent on the multi-transmembrane protein encoded by mitochondrial DNA. 1 The success of SI with a transmembrane cytochrome oxidase was demonstrated by the fact that this protein was also used for the 8-transmembrane cytochrome 6, which was previously said to be unable to be transported to mitochondria. Since the protein and the orientation in the membrane are the same, the function can be changed by this method. Both proteins play a central role in mitochondrial respiration, thus enabling the treatment of severe genetic disorders caused by protein mutations. This method is effective not only for treating genetic diseases but also for studying the function of mitochondrial membrane proteins and the relationship between function and structure. In addition, membrane proteins encoded by nuclear DNA can be newly transported to mitochondria, and new functions can be imparted to mitochondria. Hereinafter, the present invention will be illustrated by way of examples, but these are not intended to limit the present invention.

The cytochrome oxidase targeted in this example is composed of three subunits derived from mitochondria (SI-III) and 10 subunits encoded in the nucleus and transported from the cytoplasm to mitochondria. SI is responsible for the active center of this enzyme It is an important subunit. In this example, in order to transport this SI to mitochondria and achieve function expression, the orientation (topology) in the membrane was the same, and the S-terminal (one-transmembrane type) with the N-terminus on the matrix side was used. Notably, the mitochondrial transport signal (SEQ ID NO: 6) bound to the N-terminus was fused to the N-terminus of SI and transported from the cytoplasm to mitochondria.

Human cultured cells were selected in consideration of human gene therapy. No protein has been transported from the cytoplasm to the mitochondria using this mitochondrial transport signal of SIV. However, a report using the transport signal of yeast cytochrome oxidase SIV (The EMBO J. 4, 2061-2068, 1985) Power S Power The yeast SIV corresponds to the mammalian cytochrome oxidase SV, There are no parts and the transport signals are different.

In this example, S I of wild-type cytochrome oxidase and two genes obtained by mutating the 51st aspartic acid from N of S I to asparagine were each integrated into the chromosome of a human HeLa cell. To detect SI transported to mitochondria, a histidine tag (amino acid sequence consisting of six consecutive histidines) was added to the C-terminus of SI. To examine the function, cultured cells in which the SI gene had been integrated into the chromosome were grown, mitochondria were prepared therefrom, and the mitochondrial cytochrome c oxidation activity and the hydrogen ion active transport activity were measured. Cloning of S I transgene

Mitochondria were prepared from the heart muscle of a fresh mouse, and mitochondrial DNA (mt DNA) was obtained by a conventional method. Since the gene sequence has already been determined for the mtDNA of the heart muscle (J. Mol. Biol. 156, 683-717 (1982)), the synthetic DNA shown in SEQ ID NOs: 2 and 3 was utilized using that sequence. Using this method, only the SI gene (FIG. 1, SEQ ID NO: 1) was amplified by PCR to clone the gene and inserted into the multicloning site of plasmid pUC18 (FIG. 2 (A)). This plasmid was named pSIm. The fourth to ninth bases of the synthetic DNA of SEQ ID NO: 2 are restriction enzymes cI, and the fourth to ninth bases of the synthetic DNA of SEQ ID NO: 3 are cleavage sites of PstI. Amplified subunit I gene at both ends The SacI-PstI DNA fragment obtained by cleaving the above-mentioned restriction enzyme cleavage site was inserted into the above plasmid. Base modification

In the mitochondrial gene, TGA and ATA encode tributophan and methionine, respectively. When they are incorporated directly into the nucleus, they correspond to translation termination and isoleucine. TGG and ATG were modified to be translated into tryptophan and methionine in the cytoplasm. The modification used the usual site-specific mutagenesis method. The plasmid carrying the modified gene was named psI.

In addition, the base of pSI was modified so that the first asppartic acid became asparagine. The nucleotide sequence and its modification were confirmed by DNA Sequencer. Addition of histidine tag and preparation of expression vector

Using the above plasmid pSI as PCR type II, the SI gene having a histidine tag at the C-terminal was amplified using the synthetic DNA of SEQ ID NO: 4 and the reverse primer for Takara Biomedical's pUC (SEQ ID NO: 5). (Fig. 2 (B) top). The 3rd to 8th bases of the above primer shown in SEQ ID NO: 4 indicate a restriction enzyme 5 zoII cleavage site, and the 12th to 29th bases indicate a portion complementary to the histidine tag-encoding sequence. The resulting DNA fragment containing the SI gene was cleaved with cI-Bg1II (Fig. 2 (B)) and inserted into the SacI and BamHI sites of pUC18 (projection after cleavage). Since the site is complementary to that of ^ ZoII, binding is possible), and plasmid pSI-ht was obtained (Fig. 2 (B), bottom). Addition of transport signal

The amino acid sequence serving as a transport signal (SEQ ID NO: 6) was linked to the SI gene by the following method.

Two oligonucleotides (SEQ ID NOS: 7 and 8, 3 to 8 of SEQ ID NO: 7 represent $$ acI, 2 to 7 of SEQ ID NO: 8 represent SjDzoI cleavage sites, SEQ ID NO: 3 The 5th to 55th positions and the 20th to 40th positions of SEQ ID NO: 8 show complementary sequences. ) Were synthesized, the two DNAs were ligated with these complementary sequences, and the complete double-stranded DNA was prepared by PCR.

It was subsequently cleaved with S _a c I, the other end was inserted into the left pUC 1 9 of c I and Sma i iL blunt {produce blunt ends with Sma I cleavage) (FIG. 2 (C) above) . The ^ col-Sp1 IDNA fragment was excised from here (arrow in FIG. 2 (C)).

On the other hand, the SI gene of pSI-ht was amplified with the primer of SEQ ID NO: 9 having a zoI site and the universal primer for pUC of Takara Biomedical (SEQ ID NO: 10) (FIG. 2 (C)). The obtained DNA fragment was digested with ゾ and Pst I (arrows in FIG. 2 (C)), and inserted into pBluescript KS together with the ^ col-Sp II DNA fragment described above, and plasmid pB, SIW was obtained (Fig. 2 (C), bottom). Preparation of expression vector

The SI gene was excised from plasmid p BZS IW at // ^ 11 1 and \ / "0 I and inserted into Invitrogen's plasmid pRc, CMV to obtain an expression vector pSI wt (wild type). .

5 Subunit I (mutant SI) in which the first amino acid was mutated to asparagine was treated in the same manner as above to obtain pSI51n. Incorporation into cultured cells

Each of the above two plasmids (SI wt, pSI51n) was infected with HeLa cells using Qiagen's Effectene transfection reagent, and Dulbecco's modified Eagle containing 10% fetal bovine serum. The cells were cultured in a medium, and then colonies that grew in the presence of the antibiotic geneticin were selected. Mitochondrial and activity ϋ≤ These two types of cultured cells were cultured in the above-mentioned medium containing geneticin, and when the cells became 100% confluent in the dish, the culture was stopped and the cells were collected. The obtained cells were disrupted in a 10 mM HEP ES-KOH (pH 7.4) buffer containing 0.2 M sucrose and 0.5 mM EDTA using a potter-type glass stirrer (fluorine resin whistle). Mitochondria were obtained by a conventional method. Furthermore, mitochondria are treated with hypotonic solution as usual and suspended in lmM HE PES-KOH (pH 7.0) containing 0.2M sucrose and 0.02% bovine serum albumin, and the outer membrane of mitochondria is treated. Were prepared. Mitoplasts obtained from cells that expressed wild-type SI are denoted by “WTJ”, and mutant SI mitoplasts are denoted by “D51N”. Detection of Subunit I by Western blotting

The mitoplasts prepared as described above were dissolved in 0.1% SDS, subjected to SDS-PAGE electrophoresis by a conventional method, transferred to a PVDF membrane, and then reacted with a histidine-tagged antibody. Figure 3 shows the results. Fig. 3 (2) shows that of WT, and Fig. 3 (3) shows that of D51N. In both cases, the presence of the protein was confirmed at the same mobility as that of S I of purified cytochrome c oxidase.

On the other hand, the above proteins were not observed in mitoplasts prepared from HeLa cells (moCK cells) into which plasmid pRcZCMV without the SI gene had been incorporated (Fig. 3 (1)). Measurement of cytochrome c oxidation activity

Reduced cytochrome c was prepared and reacted with the above mitoplast at 25 ° C. Reduced cytochrome c shows a sharp absorption at 550 nm, but disappears when oxidized to oxidized form (Fig. 4). Therefore, the oxidation activity of reduced cytochrome c was tracked at a wavelength of 55 Onm.

The reaction solution (660 μ 1) is as follows. 1 OmM HEPE S-KOH (pH 7.0) buffer solution, 5 OmM KC 1, 0.2 M sucrose, 40 nmolZmg N-ethylmaleimide, 0.2 nmolZmg Antimycin A, 45 pmo \ / mg valinomycin, 2.0 μΜ rotenone, 11.3 nmol / ml oligomycin, and 1.0 // M myxothiazole. About 5 μg of mitoplast was added.

The reaction was started by adding 8.4 nmol of reduced cytochrome c. When electrons are sent to cytochrome c oxidase by cytochrome c, cytochrome c becomes an oxidized form and the absorption at 550 nm is reduced. Fig. 5 shows the situation.

Mitoplasts prepared from cells expressing wild-type SI (WT) show a rapid cytochrome c inhibition activity inhibited by cyanide (Fig. 5, left), and the activity is almost the same as that of mitoplasts obtained from mock cells. It was the same.

On the other hand, mitoplasts expressing mutant SI (D51N: subunit I in which the 51st amino acid was mutated to asparagine) also showed rapid cytochrome c oxidation activity (Fig. 5, right). When compared in terms of specific activity, it was about 30% higher than that of mitoplast containing wild-type SI, but it is considered to be almost the same. Measurement of hydrogen ion active transport activity

The active transport activity of hydrogen ions was measured for the same two mitoplasts as described above. The active activity for hydrogen ion transport was the same as above, but the buffer concentration was reduced to 0.5 mM. The change in hydrogen ion concentration in the reaction solution was measured using a Beckman pH meter model ψ660 and a composite electrode (BECKMAN, 51082). In the measurement, the change in hydrogen ion concentration of the reaction solution caused by the addition of reduced cytochrome c was tracked. Fig. 6 shows the results. The lower the lower, the higher the hydrogen ion concentration. After the addition of reduced cytochrome c, mitoplasts (WT) expressing wild-type SI showed significant ^ 4 formation of the reaction solution (Fig. 6 (A), the area indicated by a). However, the reaction solution became more viscous over time. This is a result of oxygen reduction to water at the same time as reduced cytochrome c is oxidized, and hydrogen ions are consumed in mitoplasts. The mitoplast membrane is difficult to pass hydrogen ions but is not perfect, so non-enzymatically hydrogen ions pass through the membrane. This is a phenomenon well known as backf 1 ow. The difficulty after the start of the reaction (the area indicated by a in Fig. 6 (A)) is the result of the active transport of hydrogen ions by this enzyme. Is observed.

When the uncoupling agent (p-trifluoromethoxy carboxyl-cyanide phenylhydrazone) is added at 10 / xg before the reaction opening stage to allow hydrogen ions to freely enter and exit the membrane, the first ^ The tetracation disappeared (Fig. 6 (B)).

In the case of the mutant enzyme (D51N), the acidification caused by the addition of reduced cytochrome c did not occur (Fig. 6 (C)). This indicates that active transport of hydrogen ions was significantly reduced or eliminated.

This result indicates that the mutation had little effect on the cytochrome c oxidation activity of the cultured cells, but markedly reduced the other function, the active hydrogen ion transport activity. This experimental fact indicates that the function of mitochondria has been modified by Escherichia coli SI transported from the cytoplasm to mitochondria.

Claims

The scope of the claims

1. A method of transporting a protein from the cytoplasm to mitochondria by binding a transport signal to the N-terminus of a specific protein, wherein the transport signal is a subunit of the mitochondrial membrane protein encoded by nuclear DNA. A method comprising: a unit, wherein when the subunit is present in the mitochondrial membrane, the N-terminus is a subunit transport signal present on the matrix side of the mitochondria.

2. The method according to claim 1, wherein both the N-terminus and the C-terminus are present on the matrix side of the mitochondria when the subunit is present on the mitochondrial membrane.

3. The method according to claim 1 or 2, wherein the specific protein has a molecular weight of 100,000 to 60,000.

4. The method according to any one of claims 1 to 3, wherein the specific protein is a protein encoded by mitochondrial DNA.

5. The method according to claim 4, wherein the protein encoded by the mitochondrial DNA is a subunit of a mitochondrial membrane protein.

6. The method according to claim 4, wherein the protein has been modified.

7. The transport signal is cleaved and removed from the protein transported to the mitochondria by the method according to claim 5 or 6, and the mitochondrial membrane protein is removed from the remaining proteins and other subunits as a result of the cleavage. A method for producing a mitochondrial membrane protein, which comprises being formed.

8. A method for altering the function of a protein encoded by mitochondrial DNA by the method according to claim 7.

9. A mitochondria having a mitochondrial membrane protein produced by the method according to claim 7.

10. A mitoplast from which the outer membrane has been removed from the mitochondria according to claim 9.

11. A cell containing the mitochondria according to claim 9.