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Vol. 19, Issue 4, 1366-1377, April 2008

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ATP25, a New Nuclear Gene of Saccharomyces cerevisiae Required for Expression and Assembly of the Atp9p Subunit of Mitochondrial ATPase

Department of Biological Sciences, Columbia University, New York, NY 10027

Submitted August 3, 2007; Revised January 3, 2008; Accepted January 15, 2008
Monitoring Editor: Janet Shaw

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
We report a new nuclear gene, designated ATP25 (reading frame YMR098C on chromosome XIII), required for expression of Atp9p (subunit 9) of the Saccharomyces cerevisiae mitochondrial proton translocating ATPase. Mutations in ATP25 elicit a deficit of ATP9 mRNA and of its translation product, thereby preventing assembly of functional F₀. Unlike Atp9p, the other mitochondrial gene products, including ATPase subunits Atp6p and Atp8p, are synthesized normally in atp25 mutants. Northern analysis of mitochondrial RNAs in an atp25 temperature-sensitive mutant confirmed that Atp25p is required for stability of the ATP9 mRNA. Atp25p is a mitochondrial inner membrane protein with a predicted mass of 70 kDa. The primary translation product of ATP25 is cleaved in vivo after residue 292 to yield a 35-kDa C-terminal polypeptide. The C-terminal half of Atp25p is sufficient to stabilize the ATP9 mRNA and restore synthesis of Atp9p. Growth on respiratory substrates, however, depends on both halves of Atp25p, indicating that the N-terminal half has another function, which we propose to be oligomerization of Atp9p into a proper size ring structure.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Expression of the mitochondrial gene products of Saccharomyces cerevisiae depends on messenger-specific stabilization and translation factors, some of which have been shown to interact with sequences in the 5' untranslated leaders of their respective RNAs. Such factors have been described for the three mitochondrially encoded subunits of cytochrome oxidase, cytochrome b and subunits of the proton translocating ATPase or F₁–F₀ complex (reviewed in Costanzo and Fox, 1990; Dieckmann and Staples, 1994). Two nuclear genes are known to be required for expression of Atp9p (subunit 9 or subunit c) of F₀, the proton transclocating sector of the ATPase complex. AEP1/NCA1 has been shown to facilitate translation of Atp9p (Payne et al., 1991; Payne et al., 1993), whereas AEP2/ATP13 acts at an earlier step by promoting stability and processing of the ATP9 mRNA (Ackerman et al., 1991; Ellis et al., 1999). Similar factors have been described for Atp6p, another subunit of the F₀ sector (Zeng et al., 2007a). Nuclear genes have also been described for proteins that promote posttranslational steps in the F₀ assembly pathway. These include Atp10p and Oxa1p both of which have been shown to chaperone the interaction of Atp6p with the Atp9p ring (Tzagoloff et al., 2004; Jia et al., 2007) and Atp23p that cleaves the Atp6p precursor but in addition is required for some still unknown step of F₀ assembly (Zeng et al., 2007b; Osman et al., 2007).

Most of the currently known mRNA-specific translational and stabilization factors of yeast mitochondria have been identified through biochemical characterizations of respiratory-deficient mutants (pet) (Ebner et al., 1973; Michaelis et al., 1982; Tzagoloff and Dieckmann, 1990). As part of continuing efforts to identify and functionally characterize nuclear genes involved in mitochondrial biogenesis, mutants representative of different complementation groups in our collection of pet strains have been screened for defective ATPase. Here, we present evidence that the ATPase lesion of mutants in complementation group G29 stems from a deficit of Atp9p. The gene responsible for this phenotype, designated ATP25, corresponds to reading frame YMR098C on yeast chromosome XIII. Like Aep1p and Aep2p/Atp13p (Payne et al., 1991; Payne et al., 1993), the ATP25 product seems to target solely Atp9p. We present evidence that the Atp9p deficit in atp25 mutants is a consequence of increased turnover of ATP9 mRNA, and we conclude that Atp25p is a new stabilization factor specific for the ATP9 mRNA. In addition to stabilizing the ATP9 mRNA, Atp25p acts posttranslationally by promoting assembly of Atp9p into the oligomeric ring structure. Our results indicate that RNA stability and oligomerization of Atp9p are catalyzed by two separate domains corresponding to the C- and N-terminal halves of Atp25p, respectively.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Strains and Growth Media
The genotypes and sources of the S. cerevisiae strains used in this study are listed in Table 1. The compositions of the solid and liquid growth media have been described previously (Myers et al., 1985).

Cloning of ATP25
ATP25 was cloned by transformation of C279/U1 with a yeast genomic plasmid library consisting of partial Sau3A fragments of yeast nuclear DNA averaging 12 kb in YEp24 (Botstein and Davis, 1982). This library was kindly provided by Marian Carlson (Department of Human Genetics, Columbia University, NY). Transformation of 10⁸ cells with 10 µg of library DNA yielded three uracil-independent and respiratory-competent clones, of which one clone (C279/U1/T2) was used to subclone the gene.

Construction of Clones Expressing the Entire and Partial Atp25p Tagged with hemagglutinin (HA)
A short sequence coding for the HA tag was introduced immediately after the termination codon of ATP25 by polymerase chain reaction (PCR) amplification of the region of ATP25 starting from the unique BstX1 site with primers 5'-cattttccaaacatttggaga and 5'-ggcaagctttcaagcgtagtctgggacgtc-gtatgggtattgattgtttgttccacggactag. The PCR fragment was digested with BstX1 and HindIII, and then it was substituted for the native sequence in pG29/ST8 (see Figure 3). The resultant plasmid pG29/ST17 contained ATP25 with an in-frame 27-nucleotide-long sequence coding for nine extra C-terminal amino acids constituting the HA tag. The fusion gene was also transferred as a SacI–HindIII fragment from pG29/ST17 to the CEN plasmid pRS316 (Sikorski and Hieter, 1989). This construct was designated as pG29/ST18.

The sequence coding for the N-terminal half of Atp25p was PCR amplified with primers 5'-ggcggatccgaactatcgtaactttag and 5'-ggcctgcagtcaagcgtagtctgggacgtcgtatggg-taataccttctcctttgc. The fragment containing 300 base pairs of 5' untranslated sequence followed by the ATP25 sequence coding for the first 292 residues and ending with a short sequence coding for the HA tag was digested with BamH1 and PstI, and cloned into YEp351 (Hill et al., 1986). The resultant plasmid pG29/ST32 was used to transform aW303ATP25(H) to obtain aW303ATP25/ST32. Similarly, the sequence coding for the C-terminal half of Atp25p was amplified with primers 5'-ggcggatccatgtcaactatcaacccaaacggg and 5'-ggcaagctttcaagcgtagtctgggacgtcgtatgggtattgattgtttgttccacggactag. This PCR product consisting of the C-terminal 332 codons of ATP25 preceded by a methionine codon and terminating with the sequence coding for the HA tag was digested with BamH1 and HindIII, and cloned into YEp352 and YEp351 (Hill et al., 1986), yielding pG29/ST23 and pG29/ST40. Both plasmids were introduced into W303ATP25(H).

Expression of trpE-ATP25 Fusion Proteins in Escherichia coli
A trpE/ATP25 fusion protein was obtained by PCR amplification of the sequence from the 313th codon to the end of the gene with primers 5'-caaaaggaagagctcaaaggagcc and 5'-tggtgtgaaggatccacgttgga. This fragment was digested with SacI and BamH1 and cloned in pATH20 (Koerner et al., 1991). E. coli transformed with this construct expressed a protein of 70 kDa, corresponding to the N-terminal half of anthranylate synthase component 1 fused to the C-terminal sequence of ATP25 starting from residue 313. The insoluble fusion protein was isolated from E. coli, dissociated in 1% SDS, and size fractionated on a Biogel A-5 column. The highly enriched protein was used to immunize rabbits (Koerner et al., 1991).

Construction of a Temperature-sensitive (ts) Allele of atp25 in W303
ATP25 was mutagenized by PCR amplification of the gene under conditions causing misincorporation of deoxynucleotides (Barros et al., 2002). The gene was amplified from pG29/T2 with primers 5'-ggcggatccgaactatcgtaactttag and 5'-ggcggatccgagattaagatattaatatacagg in four separate reactions containing 0.03 mM MnCl₂, 1.25 mM MgCl₂, and 0.2 mM dNTPs but having a threefold lower concentration of one of the four deoxynucleotides. The products of the four reactions were pooled digested with BamH1 and cloned into YCplac22, a centromeric plasmid containing the TRP1 marker (Gietz and Sugino, 1988). The resultant library was used to transform the atp25 null mutant W303ATP25(H). Tryptophan prototrophic transformants were obtained at 30°C and checked for growth at 37°C. The ts mutants W303/ATP25ts-1 was selected based on its clear ts growth phenotype on YEPG (rich glycerol/ethanol).

Miscellaneous Procedure
Standard methods were used for the preparation and ligation of DNA fragments and for transformation and recovery of plasmid DNA from E. coli (Sambrook et al., 1989). The conditions for Northern hybridizations have been described previously (Barros et al., 2006). The ATP25 coding sequence plus 290 nucleotides of 5' and 500 nucleotides of 3' untranslated regions were sequenced by the method of Maxam and Gilbert (1977). The sequence obtained was identical to a region of chromosome XIII containing reading frame YMR098C. Proteins were separated by SDS-polyacrylamide gel electrophoresis (PAGE) (Laemmli, 1970). Where indicated the composition of the polyacrylamide gel were modified to permit better resolution of ATPase subunits. Native proteins were extracted with 2% final concentration of digitonin and separated by native blue (BN)-PAGE on a 4–13% polyacrylamide gel (Wittig et al., 2006). Western blots were treated with a rabbit polyclonal antibody against yeast Atp25p followed by a second reaction with anti-rabbit immunoglobulin G (IgG) coupled to peroxidase. The antibody complexes were visualized with the SuperSignal chemiluminescent substrate kit (Pierce Chemical, Rockford, IL). Protein concentrations were determined by the method of Lowry et al. (1951).

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Properties of Mutants in Complementation Groups G29
This complementation group consists of seven independent respiratory-deficient pet mutants with recessive point mutations in nuclear DNA. The mutations confer a secondary instability in mitochondrial DNA, which depending on the mutant strain causes the production of 30–90% ^–/o clones during vegetative growth. The high lability of mitochondrial DNA (mtDNA) is a hallmark of several different classes of nuclear mutants (Tzagoloff and Dieckmann, 1990). Most commonly, it occurs in strains with mutations in F₁-F₀ ATPase (Paul et al., 1989; Helfenbein et al., 2003) or in components of the mitochondrial translation system (Myers et al., 1985). Mitochondrial translation mutants display multiple deficiencies in cytochromes a, a₃, and b. Because such cytoplasmic petite mutants are able to synthesize F₁ but not F₀, their ATPase is oligomycin insensitive. ATPase mutants have a similar pleiotropic phenotype with deficiencies in the bc1 and cytochrome oxidase complexes, both of which contain subunits translated on mitochondrial ribosomes. ATPase mutants either lack ATPase activity altogether when F₁ biosynthesis is impaired or the ATPase is oligomycin insensitive if the mutations prevent F₀ assembly (reviewed in Ackerman and Tzagoloff, 2005). The latter phenotype, however, is also encountered among mitochondrial translation mutants.

Assays of mitochondrial cytochromes in mutants from complementation group G29 revealed severe reductions in all but c-type cytochromes (Figure 1A). Although the mutant mitochondria had ATPase activity, the enzyme was not inhibited by oligomycin, indicative of a lesion in F₀ (data not shown). Because the absence of oligomycin sensitivity was observed even in strains that underwent <50% conversion to ^–/o mutants, this suggested that the F₀ deficiency was not secondary to a translation defect but rather resulted from a mutation in an F₀ subunit or in a factor necessary for F₀ assembly. This was confirmed by analysis of mitochondrial translation products labeled in vivo in the presence of cycloheximide to inhibit cytoplasmic protein synthesis. Because of the high percentage of ^–/o in the cultures of the mutants used for labeling, they incorporated less [³⁵S]methionine than the wild type (Figure 1B). With the exception of Atp9p, the three mutants analyzed synthesized all the mitochondrial gene products including Atp6p and Atp8p, the other two mitochondrially derived subunits of F₀ (Figure 1B). The absence of Atp9p in the mutants was unlikely to be due to turnover of the protein, because there was no evidence of labeling even at very short pulse times with [³⁵S]methionine (Figure 1C). The normal synthesis of the other mitochondrial gene products suggested that the pleiotropic phenotype of the mutants stemmed from an ATPase deficiency and that the mutation was in a nuclear gene essential for proper expression of Atp9p.

View larger version (31K): [in this window] [in a new window]   Figure 1. Phenotype of G29 mutants. (A) Mitochondria of the wild-type W303-1A and of the G29 mutant aC279/U1 were extracted with 1% potassium deoxycholate at a final protein concentration of 5 mg/ml (Tzagoloff et al., 1975). Half of the extract was reduced with sodium dithionite, and the other half was oxidized with potassium ferricyanide. The difference spectrum was recorded at room temperature. The absorption maxima of the -bands of cytochromes a, a3, b and c, c1 are indicated. (B) In vivo labeling of mitochondria gene products. The parental wild-type strain D273-10B/A1 and C279, N124, and N210, three independent mutants from complementation group G29, were labeled with [35S]methionine (1,000 Ci/mmol; GE Healthcare, Piscataway, NJ) in the presence of cycloheximide as described previously (Zeng et al., 2007a). Total cellular proteins were separated by SDS-PAGE on three differently prepared polyacrylamide gels. The 17.5% polyacrylamide gel on the extreme left was prepared from a 30:0.8 acrylamide:bis-acrylamide stock, and it was run in the normal buffer system of Laemmli (1970). The middle gel was prepared from a stock solution of 30:0.2 acrylamide:bis-acrylamide, and it was run with a buffer adjusted to pH 8.3. The 12% polyacrylamide gel on the right was prepared from a 30:0.8 acrylamide:bis-acrylamide stock, and it contained 4 M urea and 25% glycerol. The relative migration of some proteins differs depending on the gel system used. Cox1p, Cox2p, and Cox3p are subunit of cytochrome oxidase; cytochrome b (Cyt. b) is a subunit of the bc1 complex; and Atp6p, Atp8p, and Atp9p are subunits of F0. (C) The wild-type parent D273-10B/A1 and the G29 mutant N210 were labeled in vivo for the indicated times with [35S]methionine in the presence of cycloheximide, and the labeled products were separated by SDS-PAGE on a 17.5% polyacrylamide gel with a 30:0.8 ratio of acrylamide:bis-acrylamide as in B.

View larger version (54K): [in this window] [in a new window]   Figure 2. Northern analysis of ATP9, tRNAser, and ATP6 transcripts in wild-type and G29 mutants. Mitochondria were isolated from the parental respiratory competent strains D273-10B/A1 and from the G29 mutants C279 and N210. The C279 and the N210 cultures consisted of 2 and 20% + cells, respectively. Total RNAs were extracted from mitochondria representing 1 mg of protein as described previously (Barros et al., 2006). After precipitation with alcohol, the RNA extracts were dried and dissolved in 20 µl of sterile water. The extracts (3 µl) were separated on a 1% agarose gel under nondenaturing conditions, and were stained with ethidium bromide (A). After transfer to a Nytran membrane, the blot was hybridized as described in Barros et al. (2006) with ATP9 (B), tRNAser (C), and ATP6 (D) probes that had been labeled with [-32P]dCTP by random priming (Feinberg and Vogelstein, 1983). The blots were exposed to x-ray film for 30 min. The sizes of some of the DNA standards and the migration of the mitochondrial 15S and 21S rRNA are indicated in the margins of the stained gel. The mature seryl-tRNA; the 1.5-, 2.2-, and 0.95-kb ATP9 transcripts; and the two ATP6 mRNAs are identified by the arrows. Because the gel was run under nondenaturing conditions the migration of the RNAs is slightly retarded. (E) Diagram of the ATP9/tRNAser/VAR1 region of mtDNA showing the ATP9 transcripts detected in the Northern hybridizations. The transcripts shown are based on Zassenhaus et al. (1984).

To verify that C279/U1 had a mutation in ATP25, nuclear DNA obtained from the mutant was digested with a combination of PstI and SphI and fragments of 2.1 kb were used to construct a library in the integrative vector YIp352 (Hill et al., 1986). The library was screened by colony hybridization with a probe consisting of the BglII fragment internal to ATP25. The sequence of atp25 in a clone isolated from the library disclosed the presence of a C-to-T transition at nucleotide 1231 of the gene creating a TAA stop at codon 410. The mutation confirmed that restoration of respiratory growth in the mutant by pG29/T2 (Figure 3A) was the result of complementation rather than suppression.

View larger version (18K): [in this window] [in a new window]   Figure 3. Phenotype of the atp25 temperature-sensitive mutant W303/ATP25ts-1. (A) Dilutions of the wild-type W303-1B and of W303/ATP25ts-1, grown to early stationary phase in liquid YPD, were spotted on two YPD and YEPG plates and incubated at 24 and 37°C for 2–3 d. (B) Spectra of mitochondrial cytochromes in W303-1B and W303/ATP25ts-1 grown at 24 and 37°C in YPGal to early stationary phase. Mitochondria were prepared and extracted, and spectra were recorded as described in Figure 1A.

Isolation of Temperature-sensitive Alleles of ATP25
The absence of ATP9 mRNA in atp25 mutants could be consequent to a block in transcription, RNA processing, or instability of the mRNA. Studies aimed at distinguishing among these possible functions of the ATP25 product were hindered by the mtDNA instability of the null and most atp25 point mutants. To circumvent this problem, temperature-sensitive alleles of the gene were obtained by low-stringency PCR amplification of the gene. Several such mutants were isolated and one mutant, W303/ATP25ts-1, was used to probe the biochemical lesion causing the Atp9p deficiency.

Growth of the ts mutant on ethanol/glycerol at 24°C was 2 times slower than that of wild type, and it was almost completely inhibited at 37°C (Figure 3A). Spectra of mitochondrial cytochromes showed decreases in cytochrome oxidase and to a lesser extent of cytochrome b in the mutant grown at 37°C (Figure 3B). Although the wild-type strain also sustained some loss of "a" and "b" type cytochromes when grown at 37°C, the decrease was less pronounced than in the mutant. The presence of cytochromes a, a₃, and b in the ts mutant grown at the nonpermissive temperature made it highly unlikely that Atp25p is involved in transcription, because translation of these respiratory carriers depends on both the seryl-tRNA and the ribosomal protein Var1p.

The ts mutant corroborated the results obtained with the point mutants, indicating that the product of ATP25 is required for expression of Atp9p and oligomycin-sensitive ATPase. The ATPase activity of mitochondria was inhibited by >60% in the presence of oligomycin when the ts mutant was grown at 24°C, but it was essentially unaffected by oligomycin when the cells were grown at 37°C, the nonpermissive temperature (Table 2). In vivo labeling of mitochondrial products in cells grown at 24°C and switched to 37°C for different times showed an almost complete arrest of Atp9p synthesis after incubation at 37°C for 4 h (Figure 4A). A temperature shift experiment was also done to assess the effect of the ts mutation on ATP9 transcripts. Mitochondria were prepared from cells that were first grown to early stationary phase at 24°C and subsequently incubated at 37°C for up to 7 h. Northern analysis of mitochondrial RNAs revealed a time-dependent loss of the Atp9p mRNA (Figure 4, B and C). The decrease of Atp9p mRNA and of in vivo synthesis of the protein both occurred after 4-h incubation at the restrictive temperature. Interestingly, the 1.5-kb ATP9 precursor normally seen in wild type was not affected at 37°C.

View larger version (59K): [in this window] [in a new window]   Figure 4. Northern analysis of ATP9 mRNA and of its translation product in the atp25 ts mutant after shift to the nonpermissive temperature. (A) The wild-type haploid parent W303-1B and the ts mutant W303/ATP25ts-1 were grown at 24°C overnight in YPGal, transferred to minimal galactose medium, and incubated at 24°C for 7 h or at 37°C for the indicated times. Samples of the cultures were labeled with [35S]methionine in the presence of cycloheximide to inhibit cytoplasmic protein synthesis (Zeng et al., 2007a). Total cellular proteins were separated by SDS-PAGE on a 17.5% polyacrylamide gel prepared with a 30:0.8 solution of acrylamide:bis-acrylamide. After transfer to nitrocellulose, the blot was exposed to x-ray film. (B) The wild-type and ts mutant were grown in YPGal to early stationary phase at 24°C. The culture was transferred to 37°C, and growth was continued. Equal samples of the culture was removed at the indicated times and used to prepare mitochondria. Only a 20% increase in mass occurred after 7 h at 37°C. Total mitochondrial RNAs were extracted as described in Figure 2, and then they were separated on a 1% nondenaturing gel. The amount of RNA loaded per lane was adjusted based on the ethidium bromide stain associated with the 21S and 15s rRNAs (marked in the margin). More of the mutant RNAs was loaded to compensate for the presence of 40% –/o in the culture. (C) The gel shown in B was blotted to Nytran and hybridized to a radioactively labeled ATP9 probe as described in Figure 2. The ATP9 precursor and mRNA are identified in the margin.

View larger version (37K): [in this window] [in a new window]   Figure 5. Detection of and localization of Atp25p. (A) Mitochondria were prepared from the wild-type strain W303-1B and from W303ATP25/ST18 and C279/U1/ST18, the atp25 null and point mutant, respectively, with ATP25-HA in a CEN plasmid. W303ATP25/ST17 and C279/U1/ST17 contain ATP25-HA on a multicopy plasmid. Mitochondrial proteins (40 µg) were separated by SDS-PAGE on a 12% polyacrylamide gel, and were transferred to nitrocellulose. The protein blot was probed with a monoclonal antibody against the HA tag followed by a peroxidase-coupled rabbit antibody against mouse IgG. The antibody–antigen complexes were visualized with the SuperSignal kit (Pierce Chemical). (B) Mitochondria were prepared from the wild-type strain W303-1B and from W303ATP25/ST18 and W303ATP25/ST17 (see above). Mitochondrial proteins (40 µg) were separated and transferred to nitrocellulose as described in A. The blot on the left was processed as described in A. The blot on the right was first reacted with a rabbit polyclonal antibody against the C-terminal half of Atp25p followed by a peroxidase-coupled goat antibody against rabbit IgG. Antibody–antigen complexes were visualized as described in A. The slower migrating bands above the Atp25p and Atp25p-HA are cross-reacting protein of unidentified nature. (C) Mitochondria from W303ATP25/ST18 were suspended in 0.6 M sorbitol, 20 mM HEPES, pH 7.5, at a protein concentration of 10 mg/ml. The mitochondria (0.4 ml) were disrupted with a Branson microprobe by irradiation for 5 s at half-maximal output. The suspension was centrifuged at 90,000 x gav for 20 min. The supernatant was collected and the pellet consisting of submitochondrial membrane vesicles was resuspended in the starting volume of 0.6 M sorbitol, 20 mM HEPES, pH 7.5. The mitochondria were also extracted with an equal volume of 0.2 M sodium carbonate. After incubation on ice for 20 min, the mixture was centrifuged at 90,000 x gav. The supernatant was collected and the extracted mitochondria were suspended in the starting volume of 0.6 M sorbitol, 20 mM HEPES, pH 7.5. A 40-µg sample of mitochondria (Mito) and equivalent volumes of the submitochondrial vesicles (SMP), sonic extract (supernatant), carbonate extracted mitochondria (Carb.-P), and carbonate extract (Carb.-E) were separated by SDS-PAGE on a 12% polyacrylamide gel. Proteins were transferred to nitrocellulose, and then they were probed with a mAb against the hemagglutinin tag and polyclonal antibodies against Atp6p and the β subunit of F1. (D) Mitochondria of the wild-type W303-1B and W303ATP25/ST18 at a protein concentration of 10 mg/ml were diluted with 6 volumes of either 0.6 M sorbitol, 20 mM HEPES, pH 7.5, or 20 mM HEPES, pH 7.5, in the presence and absence of 100 µg/ml proteinase K. After incubation at 4°C for 30 min the mitochondria were treated with PMSF at a final concentration of 0.5 mM. The mitochondria and mitoplasts formed under these hypotonic conditions were centrifuged, suspended in 0.6 M sorbitol, 20 mM HEPES, pH 7.5, and treated with 0.1 volumes of 50% trichloroacetic acid. The denatured proteins were centrifuged at 14,000 rpm for 5 min in a microcentrifuge, rinsed with water, and dissolved in Laemmli sample buffer (Laemmli, 1970). Proteins were separated by SDS-PAGE on a 12% polyacrylamide gel, transferred to nitrocellulose, and reacted with the rabbit polyclonal antibodies against the C-terminal half of Atp25p and with a monoclonal antibody against the HA tag as described in A. (E) W303ATP25/ST18 mitochondria were incubated as described in D in the absence or presence of proteinase K and the indicated concentrations of Triton X-100. Proteins were precipitated with 5% final concentration of trichloroacetic acid, rinsed in water before depolymerization in Laemmli sample buffer, separated by SDS-PAGE on a 12% polyacrylamide, and probed with a monoclonal antibody against the HA tag as described in A.

To confirm that the 35-kDa protein is a product of Atp25p and to identify the proteolytic cleavage site, six consecutive histidines codons were added to the 3' end of the of ATP25. When expressed from a multicopy plasmid, the hybrid gene expressed a protein with an electrophoretic migration similar to that of the 35-kDa HA-tagged protein. The polyhistidine-tagged protein was affinity purified on a nickel-NTA acid column and was further purified by SDS-PAGE electrophoresis. The N-terminal five residues of the purified protein had a sequence identical to that of Atp25p starting at serine 293.

Localization of the 35-kDa C-Terminal Fragment of Atp25p
The 35-kDa product of Atp25p cofractionated with submitochondrial membranes after disruption of mitochondria by ultrasound (Figure 5C). Extraction of mitochondria at alkaline pH with sodium carbonate released 30% of the HA-tagged Atp25p, indicating that it is probably a peripherally associated membrane protein. No Atp6p, an integral membrane protein was present in the carbonate extract, indicating that the Atp25p in this fraction was not due to contamination by membrane fragments. Some F₁, detected by the β-subunit antibody, was sheared from the membrane after sonic disruption of mitochondria. An additional 60% of the β-subunit in the submitochondrial membrane particles was extracted by carbonate (Figure 5C).

The carboxy-terminal 35-kDa protein is a constituent of the inner membrane facing the matrix side based on its resistance to digestion by externally added proteinase K in mitochondria and mitoplasts (Figure 5D). This was true both of wild-type mitochondria probed with the polyclonal antibody against Atp25p and of mitochondria from the transformant expressing the Atp25p-HA from a low copy plasmid probed with a monoclonal antibody against the HA tag. The Western blots of the mitochondria were also reacted with antibodies against cytochrome b₂ (a soluble intermembrane protein), against Sco1p (an inner membrane protein facing the intermembrane space), and against -ketoglutarate dehydrogenase (a subunit of the matrix localized -ketoglutarate dehydrogenase complex). The depletion of cytochrome b₂ in the mitoplasts and the sensitivity of Sco1p but not the -ketoglutarate dehydrogenase to proteinase K confirmed that most of the mitochondria had been converted to mitoplasts by the hypotonic treatment (Figure 5D). Treatment of mitochondria with proteinase K in the presence of 0.05 or 0.1% Triton X-100 resulted in complete digestion of HA-tagged Atp25p (Figure 5E).

Both the N-Terminal and C-Terminal Halves of Atp25p Are Required for Restoration of Respiratory Growth
The Atp25p antibody was obtained against the sequence starting from residue 313. This antibody does not recognize the N-terminal sequence of Atp25p preceding the cleavage site. Complementation tests, however, indicated that both halves of the protein are necessary for respiratory growth. Transformants harboring only the N- or C-terminal half of the gene were either not complemented for their respiratory defect (ATP25/ST32 in Figure 6A), or they showed only a very marginal restoration of growth on ethanol/glycerol (ATP25/ST23 in Figure 6A). Growth of the null mutant on glycerol/ethanol, however, was restored when plasmids coding for the N- or C-terminal half of Atp25p were present jointly in the same cell (ATP25/ST23, ST32).

View larger version (34K): [in this window] [in a new window]   Figure 6. Growth and biochemical phenotype of atp25 null mutants transformed with the N- and C-terminal halves of ATP25 singly and in combination. (A) The wild-type W303-1A, the atp25 null mutant W303ATP25 (ATP25), and the mutant transformed with plasmids coding either the N-terminal (ST32), C-terminal (ST23), or both halves of the protein were grown overnight in liquid YPD. Serial dilution were spotted on YPD and YEPG plates and incubated at 30°C for 2–3 d. (B) The null mutant expressing either the N-terminal half (ATP25/ST32), C-terminal half (ATP25/ST23), or both halves of Atp25p were grown in YPGal to early stationary phase. Mitochondria were prepared and their spectra recorded as in Figure 1A. (C) The point mutant C279 and the strains used for the spectral analysis in B were labeled in vivo in the presence of cycloheximide, as described in Figure 1B. Total cellular proteins were separated by SDS-PAGE on a 17.5% polyacrylamide gel. The radioactively labeled Atp8p and Atp9p are identified in the margin.

Detection of the N and C Half of Atp25p Expressed from Constructs Containing the Partial Genes
The N- and C-terminal halves of Atp25p tagged at their C termini with HA were cloned in multicopy plasmids. Expression of the two halves of Atp25p in transformants harboring either or both partial genes was confirmed by Western blot analysis of mitochondria (Figure 7A).

View larger version (23K): [in this window] [in a new window]   Figure 7. Detection and localization of HA-tagged N- and C-terminal halves of Atp25p expressed from the respective partial genes. (A) Mitochondria prepared from the wild-type W303-1B, the atp25 null mutant (ATP25), and the atp25 null mutant expressing either C-terminal half (ATP25/ST23) or N-terminal (ATP25/ST32) of Atp25p from multicopy plasmids were separated by SDS-PAGE on a 12% polyacrylamide gel. The proteins were transferred to nitrocellulose, and then they were probed with a monoclonal antibody against the HA tag as described in Figure 5A. (B) Mitochondria of the atp25 null mutant transformed with both halves of Atp25p (ATP25/ST23, ST32) were diluted with 6 volumes of either 0.6 M sorbitol, 20 mM HEPES, pH 7.5, or 20 mM HEPES, pH 7.5, and incubated at 4°C for 30 min in the absence or presence of 100 µg/ml proteinase K and further processed as described in Figure 5D. The HA antibody does not always detect the N-terminal fragment either in the single or double transformant, probably because of its lower concentration relative to the C-terminal fragment.

In contrast, all of the N-terminal polypeptide of Atp25p was resistant to proteinase K in mitochondria, indicating that the presence of the mitochondrial targeting sequence facilitated transport of all of the protein into mitochondria. The N-terminal fragment was partially sensitive to proteinase K in mitoplasts as was the intermembrane Sco1p maker, suggesting that like the latter fragment, this fragment is associated with the inner membrane facing the intermembrane compartment.

Function of the N-Terminal Half of Atp25p
The requirement of both halves of Atp25p for restoration of ATPase suggested that the N-terminal half of Atp25p catalyzes a function distinct from stabilization of the ATP9 mRNA. A reasonable hypothesis was that this function might be related to some aspect of Atp9p biogenesis. To test this possibility, the status of the ATPase in cells expressing only the C-terminal domain of Atp25p was probed immunochemically with antibodies against different subunits of F₁ or F₀ and by labeling mitochondrial translation products in vivo.

The ATPase complex of wild type and different mutants was extracted from mitochondria with digitonin and separated by BN-PAGE. Antibodies against the β-subunit of F₁ revealed that most of the ATPase extracted from wild-type mitochondria migrated as the monomer F₁–F₀ complex (Figure 8A). This was also true of RKY48-1, a mutant expressing Atp6p without its N-terminal presequence (Zeng et al., 2007c). No ATPase complex was detected in W303ATP25/ST40, a transformant expressing the C-terminal domain of ATP25 or in the atp10 mutant, which synthesizes the Atp9p ring but is blocked in its interaction with Atp6p (Tzagoloff et al., 2004).

View larger version (45K): [in this window] [in a new window]   Figure 8. Assembly of Atp9p in wild-type and mutant mitochondria. (A) Mitochondria from wild-type W303-1B, the atp25 null mutant expressing only C-terminal half of ATP25 (ATP25/ST40), the atp6 leaderless mutant (RKY48-1), and the atp10 null mutant (ATP10) were solubilized with 2 mg digitonin/mg mitochondrial protein, and were analyzed by BN-PAGE on a 4–13% polyacrylamide gel. The proteins were transferred to a polyvinylidene difluoride (PVDF) membrane and probed with polyclonal antibodies against the β subunit of F1 (left) and Atp9p (right). (B) The strains used in A (except RKY48-1) were labeled in vivo with [35S]methionione in the presence of cycloheximide as in Figure 1B. The labeled cells were converted to spheroplasts by incubation at 30°C for 10 min in the presence of 14 mg/ml Zymolyase 20,000. Mitochondria were prepared, mixed with unlabeled wild-type mitochondria, and extracted with digitonin as described in A. The soluble fraction obtained after centrifugation at 70,000 x gav for 15 min was separated either by SDS-PAGE on a 17.5% polyacrylamide gel (left) or by BN-PAGE (right), transferred to a PVDF membrane, and exposed to x-ray film. The band identified as the Atp9p ring was confirmed to contain Atp9p by SDS-PAGE in a second dimension (data not shown).

The absence of the Atp9p ring in mitochondria of W303ATP25/ST40 could be the consequence of turnover, resulting in a steady-state concentration of Atp9p too low for immunochemical detection. Although turnover of Atp9p cannot be excluded, it is unlikely in view of experiments showing that there is no difference in the stability of newly translated Atp9p in wild type and in the mutant harboring only the C-terminal half of Atp25p (data not shown). Alternatively, assembly of Atp9p into the ring may be blocked in the absence of the N-terminal domain. This explanation is consistent with in vivo pulse-labeling results. In the experiment of Figure 8B, newly translated mitochondrial gene products were extracted with digitonin and displayed by BN-PAGE. The pulse-labeling conditions used in this experiment preempted the newly translated Atp9p from assembling into the ATPase complex, and as a result, it was present as a ring in both wild type and the atp10 mutant (Figure 8B). No newly translated Atp9p, however, was incorporated into the ring structure in the transformant expressing only the C-terminal half of Atp25p (Figure 8B).

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Atp9p is a low-molecular-weight hydrophobic subunit of mitochondrial and bacterial proton-translocating ATPases. It exists as a 10- to 11-membered ring structure (Arechaga et al., 2002), which together with Atp6p defines the channel through which protons are translocated during ATP formation or hydrolysis (Fillingame and Dmitriev, 2002). In S. cerevisiae, expression of the mitochondrial gene coding for Atp9p has been shown to depend on several nuclear gene products (Payne et al., 1991, 1993). Although our understanding of the functions of these factors is still fragmentary, there is good evidence that they are required for processing/stability of the ATP9 transcript and for translation of the mRNA (Ackerman et al., 1991; Payne et al., 1993; Ellis et al., 1999). The product of the nuclear ATP25 gene reported here is a new member of this class of proteins.

The atp25 mutants have a phenotype similar to aep1 and aep2/atp13 mutants reported previously (Ackerman et al., 1991; Ellis et al., 1999). They contain catalytically active F₁ ATPase, but they are unable to incorporate it into the larger proton-translocating complex because of a block in assembly of F₀, the membrane sector of this inner membrane complex. In the current study, the absence of functional F₀ in atp25 mutants has been correlated with a severe deficit of the Atp9p subunit. In vivo labeling revealed that atp25 mutants are capable of translating all the mitochondrial gene products except Atp9p. This failure to synthesize Atp9p was unlikely to be due to a defect in transcription of the gene or processing of the primary transcript for the following reason. ATP9 is part of a polycistronic transcript that includes tRNA^ser and VAR1 (Zassenhaus et al., 1984). The ability of atp25 mutants to express all the normal mitochondrial gene products except Atp9p attests to the presence of both functional ribosomes and the seryl-tRNA. This in turn suggested that the defect was likely to be related to translation of Atp9p or stability of its mRNA. To further probe the biochemical basis of the Atp9p deficit, two of the more stable atp25 mutants were used to analyze ATP9 transcripts in mitochondria. Northern analysis of mitochondrial RNA confirmed the presence in the mutants of seryl-tRNA (the VAR1 transcript was not analyzed) and of the low abundance 1.5-kb ATP9 precursor transcript. The mutants, however, were grossly deficient in the 0.95-kb ATP9 mRNA.

The Northern results are most consistent with an aberrantly high rate of ATP9 mRNA turnover in the mutants. This was corroborated by Northern analysis of ATP9 mRNA stability in an atp25 temperature-sensitive mutant. The level of ATP9 mRNA in mitochondria of the mutant grown at the permissive temperature was estimated to be comparable with the wild type when corrected for the percentage of ^– and ° cells in the culture, but there was an almost total depletion of this transcript between 1.5 and 4 h after shift to the nonpermissive temperature. The ATP9 mRNA of wild-type cells grown and incubated under similar conditions was completely stable. As expected, the ts mutant showed a similar time-dependent loss of Atp9p translation after shift to the restrictive temperature. Based on these results, we propose Atp25p to be a novel RNA stabilization factor specific for the 0.95-kb ATP9 mRNA. Because the abundance of the 1.5-kb precursor, normally also seen in wild type, does not seem to be affected either in apt25 point mutants or the ts mutant grown under nonpermissive conditions, the function of Atp25p must be restricted to the fully processed mRNA. Several other mitochondrial RNAs are known to require protein factors for their stability. Cbp1p has been shown to affect the stability of the cytochrome b mRNA (Mittelmeier and Dieckmann, 1990), but in addition it also functions during translation of apocytochrome b (Islas-Osuna et al., 2002). Aep3p is another recently described factor that stabilizes the ATP8-ATP6 bicistronic mRNA (Ellis et al., 2004). Although the evidence that Atp25p is involved in stability of the 0.95-kb ATP9 mRNA is compelling, we cannot exclude the possibility that this factor, not unlike Cbp1p, may also play a role in translation of Atp9p.

Based on the sequence of ATP25, the primary translation product is predicted to have a mass of 70 kDa. Unexpectedly, an antibody directed against the C-terminal half of Atp25p detected a 35-kDa protein. Because the antibody cross reacted with proteins in the 60- to 70-kDa region it was not possible to decide whether the full size protein was also present. This question was resolved with an atp25 null mutant expressing Atp25p C-terminally tagged with an HA epitope. Mitochondria of strains expressing the HA-tagged protein from a low copy plasmid contained only the 35-kDa protein. The full-size product was not detected even when Western blots were overexposed. A 60 kDa HA-tagged protein was present in mitochondria of the atp25 null mutant transformed with the gene on a high copy plasmid. The 60-kDa band, however, represented only a small fraction of the signal associated with the 35-kDa protein. No increase in the 60-kDa product was observed by inclusion of PMSF at all stages of the mitochondrial isolation procedure or by purification of mitochondria on a gradient to remove possible lysozomal contamination. The 35-kDa Atp25p product resulting from cleavage after Tyr292 is, therefore, unlikely to be an artifact of the mitochondrial isolation procedure, but rather it seems to be a physiological and functional unit of the protein. The C-terminal half of Atp25p is associated with the inner membrane and it is protected from external protease, indicating that it faces the matrix side of the membrane, a location consistent with its proposed role.

The evidence that the N-terminal half of Atp25p is also required for respiratory competence is unambiguous. Transformation of the null mutant with constructs coding for either the N- or C-terminal half of Atp25p is not sufficient to produce respiration. Nearly wild-type growth on nonfermentable carbon sources is elicited, however, when both constructs are present in the mutant simultaneously. Transformants containing the gene coding for the C-terminal half of Atp25p are only very partially complemented for their growth defect, they display the same pleiotropic phenotype as the mutant, and they are deficient in oligomcyin-sensitive ATPase. Nonetheless, this domain of Atp25p is sufficient to promote expression of Atp9p. This observation strongly implies that Atp25p is a bifunctional protein with the C-terminal half affecting the stability of the ATP9 mRNA and the N-terminal half serving some other function.

The function of the N-terminal half of Atp25p was studied by assessing the status of Atp9p in the atp25 null mutant expressing only the C-terminal half of Atp25p (W303ATP25/ST40). Atp9p was present as the oligomeric ring in an atp10 mutant completely blocked in assembly of F₀ (Ackerman and Tzagoloff, 1990) or in an atp6 leaderless mutant, which assembles only 50% of the normal amount of F₀ (Zeng et al., 2007c). No ring, however, was detected in the atp25 mutant expressing only the C-terminal half of Atp25p. This was true both when Atp9p was analyzed under steady-state condition with an antibody or when it was assayed radiochemically after in vivo pulse labeling. Under the latter conditions, Atp9p is synthesized in all three strains, but it is assembled into the ring only in wild type and the atp10 mutant. These results indicate that the inability of the C-terminal domain to restore growth of the atp25 mutant on respiratory substrates stems from its failure to assemble Atp9p ring. The absence of the ring in the atp25 mutant lacking N-terminal half of Atp25p implies that this domain either promotes the interaction of the Atp9p monomer or it prevents the monomer from forming polymers of diverse size. It is of interest to note that the N-terminal half of Atp25p has homologues in fungi and contains the DUF143 domain found in a group of bacterial proteins of unknown function. In contrast the homology of the C-terminal half is confined to proteins found only in some budding and filamentous yeast such as Ashbya that are closely related to S. cerevisiae.

	ACKNOWLEDGMENTS

 
We thank Dr. Jean Velours (Institut de Biochimie et Genetique Cellulaire du Centre National de la Recherche, Bordeaux, France) for the generous gift of antibody against yeast ATPase Atp6p and Atp9p. This research was supported by National Institutes of Health Research grant HL-22174 (to A.T.) and a Fundação de Amparo à Pesquisa do Estado de São Paulo grant (to M.H.B.).

	Footnotes

 
This article was published online ahead of print in MBC in Press (http://www.molbiolcell.org/cgi/doi/10.1091/mbc.E07-08-0746) on January 23, 2008.

* Present address: Department of Microbiology, University of São Paulo, São Paulo, Brazil.

Address correspondence to: Alexander Tzagoloff (spud{at}cubpet.bio.columbia.edu)

Abbreviations used: BN, blue native; mtDNA, mitochondrial DNA; ° mutant, respiratory-deficient mutant lacking mitochondrial DNA; ^–, mutant, respiratory-deficient mutant with a partially deleted mitochondrial genome; PAGE, polyacrylamide gel electrophoresis; pet mutant, respiratory-deficient mutant of yeast with a mutation in a nuclear gene; PCR, polymerase chain reaction; PMSF, phenylmethylsulfonyl fluoride.

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