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Vol. 18, Issue 2, 617-626, February 2007
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*Department of Biological Sciences, Columbia University, New York, NY 10027; and Adolf-Butenandt-Institut für Physiologische Chemie, Ludwig-Maximilians-Universität München, München 81377, Germany
Submitted September 19, 2006;
Revised November 17, 2006;
Accepted November 21, 2006
Monitoring Editor: Thomas Fox
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Senior et al., 2002). It also promotes a transfer of protons from the matrix to the intermembrane space when ATP is hydrolyzed (Boyer, 1997; Senior et al., 2002). FO is a hydrophobic protein consisting of upward of nine different subunits in fungal and mammalian mitochondria (Velours and Arselin, 2000; Ackerman and Tzagoloff, 2005). Proton transfer occurs at an interface between subunit 6 (subunit a) and subunit 9 (subunit c), the latter being present in 10–11 copies forming a ring structure that rotates with respect to the single subunit 6 (Nakamoto et al., 1999).
Three subunits of the ATPase complex of yeast mitochondria are encoded by mitochondrial DNA (mtDNA) (Hensgens et al., 1979; Macino and Tzagoloff, 1979, 1980; Macreadie et al., 1983). They are the already-mentioned subunits 6 and 9 of FO, and subunit 8, another component of FO, the function of which is still unclear at present. The remaining subunits of FO as well as the five subunits of F1 ATPase are products of nuclear genes that are imported into different compartments of the organelle where they assemble with their mitochondrial partners to form the holoenzyme. In addition to the structural and catalytic subunits of the F1-FO complex, the nuclear genome also codes for proteins that are not part of the complex but are essential for its assembly. Three such factors have been shown to be necessary for oligomerization of the F1 ATPase (Ackerman and Tzagoloff, 1990a; Lefebvre-Legendre et al., 2001). Other factors have been implicated in expression of subunits 6 and 9 of FO (Ackerman and Tzagoloff, 1990b; Payne et al., 1991; Helfenbein et al., 2003; Ellis et al., 2004).
Subunit 6 is synthesized with an N-terminal extension that is proteolytically removed after insertion/assembly of the precursor (Michon et al., 1988). Until now, the enzyme responsible for the maturation of the subunit 6 precursor has not been identified. As part of an effort to catalogue and functionally characterize nuclear gene products involved in assembly of the respiratory pathway, we have screened respiratory-deficient mutants of Saccharomyces cerevisiae for defects in the ATPase complex. In the present communication we report that the ATPase subunit 6 precursor is processed to the mature protein by the metallopeptidase encoded by the nuclear gene ATP23 (reading frame YNR020C on chromosome XIV). Our results also indicate that the efficiency of processing of the precursor depends on the presence of Atp10p, a mitochondrial inner membrane protein previously shown to be required for biogenesis FO (Ackerman and Tzagoloff, 1990b). In addition to their roles in processing of subunit 6, Atp23p and Atp10p are also essential for assembly of this subunit into a functional FO.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
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except that Zymolyase 20,000 instead of Glusulase was used to convert cells to spheroplasts. For the localization of Atp23p, mitochondria were obtained by the method of Glick (1985). ATPase activity was assayed by measuring release of Pi from ATP (King, 1932) at 37°C in the presence or absence of oligomycin.
Cloning and Sequencing of ATP23
ATP23 was cloned by transformation of the respiratory-deficient mutant aE884/UL1 (MATa leu2-3,11, ura3-1 atp23-1) with a yeast genomic library consisting of partial Sau3A fragments of nuclear DNA cloned in YEp24 (Botstein and Davis, 1982). This plasmid library was kindly provided by Dr. Marian Carlson (Department of Genetics and Development, Columbia University). Transformation of aE884/UL1 (108 cells) with 10 µg of library DNA yielded a single uracil-independent and respiratory-competent clone (aE884/UL/T1). The plasmid pG200/T1 conferring respiratory competence to the mutant was amplified in Escherichia coli RR1 and was used to subclone the gene. The sequences at the junctions of the 8.2-kb insert in pG200/T1 were sequenced and matched to the region of chromosome IX between nucleotides 666087 and 674355.
Disruption of ATP23
The following strategy was used to delete most of the ATP23 coding sequence. The 2.3-kb EcoRI-HindIII fragment containing the ATP23 reading frame and flanking sequences was transferred to pUC18. The resultant plasmid (pG200/ST5) was used as a template for PCR amplification of the entire plasmid and insert except for the internal 608 nucleotides coding for residues 58–260 of ATP23. The bidirectional primers used for the amplification were 5'-ggcgcggatccgtctcc-accactcaa and 5'-ggcgcggatccgatacgagaccgtttg. The resultant product was digested with BamHI and ligated to the yeast HIS3 gene on a 1-kb BamHI fragment yielding pG200/ST7. The deleted atp23::HIS3 allele, isolated from pG200/ST7 as a 2.8-kb linear XbaI-XmnI fragment, was substituted for the wild-type gene by homologous recombination (Rothstein, 1983).
Construction of the E
Q Mutant and of a Hybrid Gene Expressing Atp23p with a C-terminal Hemagglutinin Tag
ATP23 with a E168Q mutation was made by amplification of two separate fragments. The first containing 177 nucleotides of 5' sequence plus 507 nucleotides of coding sequence with two nucleotide changes to create a unique MfeI site and the glutamine codon, was amplified with primers 5'-ggcggatccgggccaaatattgaactag and 5'-ggccaattgat-gcgaaagcgtatcctc and was digested with a combination of BamHI and MfeI. The remainder of the gene starting with the glutamine codon and containing 155 nucleotides of 3' sequence was amplified with primers 5'-ggccaattgattcattatt-tcgatgatct and 5'-ggcaagcttgacattctaaggcatcc. This product was digested with MfeI and HindIII. The two PCR fragments were ligated to YEp352 and to YIp352 (Hill et al., 1986) linearized with BamHI and HindIII to yield pG200/ST14 and pG200/ST15, respectively.
To express Atp23p tagged with hemagglutinin (HA; Atp23p-HA), ATP23 in pG200/T1 was amplified with the primers 5'-acacacctagagctcacaattcaa and 5'-ggcgagctcaagcgtagtctgggacgtcgtatgggtatctgtaaatctcatcaaacgg. The product, consisting of 308 nucleotides of 5' sequence and the entire ATP23 gene fused in frame at its 3' end to a short sequence coding for the HA tag, was digested with SacI and cloned in YEp352 and YIp352 (Hill et al., 1986), yielding pG200/ST12 and pG200/ST13, respectively.
Miscellaneous Procedures
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). Proteins were separated on SDS-PAGE in the buffer system of Laemmli (1970). Protein concentrations were determined by the method of Lowry et al. (1951).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). The respiratory-deficient growth phenotype of the two mutants is complemented by a 
o tester confirming the presence of a recessive mutation in nuclear DNA. When grown under nonselective conditions on rich glucose medium, the mutant produces 70–80% –/o derivatives, indicating that the mutation has a secondary effect on the stability of mtDNA. This is also reflected in the visible spectrum of mitochondria, which shows a deficiency of cytochromes a, a3, and b (Figure 1A). The pleiotropic reduction of respiratory chain components is commonly found in mutants with a defective ATPase in which either the synthesis of F1 or FO is impaired (Paul et al., 1989; Arselin et al., 1996; Helfenbein et al., 2003; Ellis et al., 2004).
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, 2000). The decrease in subunit 4, which does not turnover in FO mutants as rapidly as subunit 6, was less pronounced. This was also true of the 
subunit of F1, which can assemble with its four partner subunits to form the active oligomer even in the absence of FO (Tzagoloff, 1969; Schatz, 1968). The presence of F1 was confirmed by assays of ATPase activity. Although the wild-type enzyme was more than 70% inhibited by oligomycin, the ATPase activity of mitochondria from E884 or aE884/UL1 was completely insensitive to the inhibitor even though the cultures consisted of 35 and 20% + cells, respectively (Table 2). Sedimentation of mitochondrial extracts in sucrose gradients also indicated that the ATPase in the mutant had properties similar to those of the F1 oligomer (Figure 1B).
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ATP23 and aW303ATP23) were respiratory deficient, did not complement E884, and displayed a biochemical phenotype similar to that of the point mutant. The atp23 null mutants had severely depressed levels of cytochromes a, a3, and b (Figure 1A) and were grossly deficient in subunit 6 but not in subunit 4 or the -subunit of F1 (Figure 1C). The ATPase activity of the null mutant, like that of E884, was also insensitive to oligomycin (Table 2).
Null Mutants in ATP23 Express an Aberrant Form of Subunit 6
Even though there is no immunologically detectable subunit 6 in the atp23 null mutant, it is able to synthesize a novel form of this protein. Pulse-labeling of whole cells with [35S]methionine in the presence of cycloheximide disclosed the presence of a novel mitochondrial translation product with a migration that is slightly retarded relative to mature subunit 6 (Figure 3, A and B). Because of its proximity to subunit 3 (Cox3p) of cytochrome oxidase in SDS-PAGE, the in vivo translation assays were also done with the atp23 mutant carrying a second mutation in PET494, which codes for a COX3-specific translation factor (Costanzo and Fox, 1986). The results obtained with the double mutant lacking Cox3p confirmed the slower migration of subunit 6 (Figure 3C).
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). The two conserved histidine and the glutamic acid residues of this motif are essential for protease activity (Becker and Roth, 1992). The importance of this sequence for the function of Atp23p was assessed by substitution of the glutamic acid by glutamine. The mutant gene was introduced into an atp23 null background either on a multicopy plasmid (W303ATP23/ST14) or by insertion of the gene in an integrative plasmid at the ura3 locus of nuclear DNA (W303ATP23/ST15). In both cases, the gene expressing the E168Q mutant protein restored wild-type growth of the atp23 null strain on glycerol/ethanol (Figure 5A).1
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Mutation(s) in Mitochondrial DNA Suppress the atp23 Null Mutation
The atp23 mutant gives rise to spontaneous revertants capable of slow growth on glycerol/ethanol (Figure 5C). Two such revertants (W303ATP23/R1 and R2) had spectra intermediate between that of wild type and the mutant, with partial restoration of cytochromes a, a3, and b (Figure 1A). The ATPase activity of mutant mitochondria was completely insensitive to oligomcyin, whereas the activity measured in the two revertants was partially inhibited (15 and 22%) by oligomcyin (Table 2). The fact that most of ATPase in the revertants remained insensitive to oligomcyin is consistent with the sedimentation properties of the ATPase in the mutant and the R1 revertant (Figure 1B). Unlike the F1-FO complex of wild-type mitochondria, the position of the ATPase in these strains relative to the 
-galactosidase marker was similar to that previously reported for F1 (Tzagoloff and Meagher, 1971). The subunit 6 precursor was also found to be more stable in the revertant than in the mutant. Almost none of the precursor was detected after 90 min of chase in the mutant (Figure 5E). In contrast 
25% was still present in both wild type and the revertant.
Only low amounts of the subunit 6 precursor and no mature protein were detected by Western analysis of the revertant mitochondria (Figure 1C). The subunit 6 precursor was also evident in the pattern of the mitochondrial translation products synthesized by the revertants (Figure 5D). Because suppression does not depend on cleavage of the precursor these results confirm that cleavage of the N-terminal 10 residues is not an essential step for expression of functional FO in yeast.
The suppressors in two revertants were ascertained to have dominant mutations. Diploid cells issued from crosses of the revertants to the atp23 mutant had a growth phenotype similar to that of the haploid revertant. This was not true of diploid cells obtained from crosses of o derivatives of the revertants to the atp23 mutant, which indicated that the suppressors were in mtDNA. Attempts to localize the mutations in the mitochondrial genome by deletion mapping with a – library generated from the revertants or by direct sequencing ATP6, ATP8, and ATP9 genes were unsuccessful. Introduction of the suppressor mutation in an atp10 revertant reported previously (Paul et al., 2000) also failed to rescue the atp23 null mutant. Our inability to find mutations in any of the three mitochondrial ATPase genes suggests that the revertants are likely to have informational suppressors either in mitochondrially encoded tRNA or rRNA genes. No further attempts were made to identify the suppressor(s).
Suppression of atp10 Mutants by One or More Copies of ATP23
ATP10 codes for an inner membrane protein that was shown to target subunit 6 and to be necessary for the interaction of this FO constituent with the subunit 9 ring (Tzagoloff et al., 2004). Like other mutants blocked in FO assembly, atp10 mutants have severely reduced levels of subunit 6 (Paul et al., 2000).
Transformation of atp10 null mutant with an extra copy of ATP23 integrated into nuclear DNA or with the gene in a high-copy plasmid conferred substantial growth on glycerol/ethanol (Figure 6A). Approximately 30% of subunit 6 in the atp10 null mutant was unprocessed (Figure 6B). In contrast only mature subunit 6 was detected in the atp10 mutant harboring an extra copy of wild-type ATP23. Only weak suppression was observed when the atp23 mutant was transformed with ATP10 on a high-copy plasmid (data not shown). To determine if proteolytically inactive EQ Atp23p also has suppressor activity, the mutant ATP23 gene was introduced into the atp10 mutant on an integrative or a high-copy episomal plasmid. Neither the high-copy or chromosomally integrated mutant gene restored respiration in the atp10 null strain (data not shown).
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ATP23/ST13, an atp23 null mutant with a chromosomally integrated ATP23 fusion gene expressing the protein with a C-terminal tag (Atp23p-HA). A monoclonal antibody against the HA tag detected a protein of 30 kDa, consistent with the expected size of Atp23p (Figure 7A). Atp23p-HA was not present in the cytosolic fraction or in the comparable fractions of wild-type yeast (not shown). It was recovered in the membrane fraction (SMP) obtained from sonically disrupted mitochondria (Figure 7B) and was partially extracted from the membrane vesicles in the presence of salt and deoxycholate (Figure 7C).
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), processing of the precursor by Atp23p is likely to occur in this compartment. This location of Atp23p is consistent with the sensitivity of the HA-tagged protein to proteinase K in mitoplasts (Figure 7D). Treatment of mitoplasts but not mitochondria resulted in a decrease of Atp23p-HA to about the same extent as of Sco1p, an inner membrane protein facing the intermembrane space (Beers et al., 2002). Cytochrome b2, a soluble intermembrane constituent, was largely depleted in the mitoplast fraction. In contrast, -ketoglutarate dehydrogenase, a component of the soluble matrix -ketoglutarate dehydrogenase complex, was protected against proteinase K in both mitochondria and mitoplasts, indicating that the inner membrane remained intact after lysis of mitochondria under hypotonic conditions (Figure 7D).
Sizing of Atp23p
The native size of Atp23p was assessed by sucrose gradient sedimentation. The HA-tagged protein was extracted from mitochondria of W303ATP23/ST13 with deoxycholate and centrifuged through a 7–25% sucrose gradient containing 0.1% Triton X-100. The peak of Atp23p-HA sedimented at a position midway between those of lactate dehydrogenase and hemoglobin, indicating an Mr of 100,000 (Figure 8). This suggests that the native protein is homo-oligomeric or is part of a hetero-oligomeric complex.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In this study we present evidence that Atp23p is a mitochondrial protease that removes the 10-residue-long N-terminal prepeptide of the subunit 6 precursor (Michon et al., 1988). The function of Atp23p was gleaned from the phenotype of a respiratory-deficient mutant previously assigned to complementation group G200 of our mutant collection (Tzagoloff and Dieckmann, 1990). In vivo labeling of the mitochondrial translation products in atp23 point and null mutants and in partial revertants revealed the presence of a novel form of subunit 6, which migrates as a slightly larger protein than mature subunit 6. The retarded electrophoretic migration of subunit 6 was also observed in Western blots of total mitochondrial proteins in atp23 revertants.
Atp23p is associated with the inner membrane in an orientation such that the C-terminus faces the intermembrane space. It is conserved among eukaryotic organisms from yeast to humans. It has an HEXXH motif previously shown to be part of the active sites of metalloproteases including zinc proteases (Jongeneel et al., 1989; Becker and Roth, 1992). The two histidine and the glutamic residues of this motif participate in zinc binding and in the catalytic mechanism, respectively, and are essential for enzymatic activity. Substitution of the essential glutamic acid by glutamine prevents cleavage of the N-terminal prepeptide, confirming that Atp23p processes the subunit 6 precursor. Surprisingly, the EQ mutant protein is able to restore normal growth of the atp23 mutant on respiratory substrates, even though all the subunit 6 remained in the unprocessed form. The ability the subunit 6 precursor to assemble into a functional ATPase excludes removal of the 10 N-terminal residues as a necessary condition for the function of this FO subunit.
The ATPase deficiency of atp23 mutants implies that in addition to catalyzing processing of the subunit 6 precursor, Atp23p has still another function in assembly of the ATPase complex. Clues about the second function of Atp23p have emerged from the observation that ATP23 is able to suppress the ATPase defect of atp10 mutants. Atp10p was previously shown to form a complex with and to confer stability on newly synthesized but unassembled subunit 6 (Tzagoloff et al., 2004). It was also inferred to be required for the association of subunit 6 with the subunit 9 ring (Tzagoloff et al., 2004). This interaction may be a rate-limiting step in FO assembly as Atp10p appears to minimize turnover of unassembled subunit 6 (Tzagoloff et al., 2004). It is significant that 5–15% of F1-FO ATPase is assembled in the atp10 null mutant (Ackerman and Tzagoloff, 1990b). This probably represents the small fraction of subunit 6 that escapes degradation. The presence of an extra copy of ATP23 is able to partially compensate for the absence of Atp10p suggesting that, when overexpressed, Atp23p helps to stabilize subunit 6. A possible explanation is that there is a cooperative interaction of Atp10p and Atp23p with the subunit 6 precursor. A physical interaction of the two proteins, if it occurs, is likely to be transient and unstable as the two proteins do not cosediment in sucrose gradients (data not shown). In the absence of either Atp10p or Atp23p the increased instability of the precursor would be expected to result in its more extensive turnover. The biochemical cooperativity of Atp23p and Atp10p is also indicated by the less efficient processing of subunit 6 precursor in the atp10 mutant. Proteolytically inactive Atp23p can promote assembly of a functional ATPase complex, indicating that the subunit 6 precursor is functional. Unlike the wild-type Atp23p, the EQ protein does not suppress the atp10 mutant. This may be the result of a greater susceptibility of the subunit 6 precursor to degradation in the absence of Atp10p, even when there is excess mutant Atp23p present. These observations are integrated in the model shown in Figure 9.
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Subunit 6 of the ATPase and subunit 2 (Cox2p) of cytochrome oxidase (COX) are the only mitochondrially encoded proteins of yeast synthesized as precursors. The amino terminal presequence of the Cox2p precursor (pCox2p) is cleaved by the IMP complex located in the intermembrane space of mitochondria (Jan et al., 2000). Mutations affecting the proteolytic activity of the IMP complex block maturation of pCox2p and elicit a COX deficiency. A similar phenotype is observed in cox20 mutants (Hell et al., 2000). Cox20p is not a protease but forms a stable complex with pCox2p, a prerequisite for processing by the IMP complex (Hell et al., 2000). Homologues of the yeast COX20 gene have recently been reported in animals and other organisms that synthesize Cox2p without a presequence (Hell et al., 1997). On the basis of these observations, Herrmann and Funes (2005) have inferred that the Cox20p acts both as a chaperone for proteolytic maturation of pCox2p by IMP and additionally promotes assembly of the mature subunit. This is also supported by observations that processing of pCox2p is not essential for its assembly and electron carrier function. Translocation of the N-terminal catalytic domain of pCox2p across the inner membrane depends on Oxa1p (He and Fox, 1997; Hell et al., 1997). The respiratory deficiency of a yeast mutant with a temperature-sensitive allele of oxa1 has been shown to be suppressed by a mutation in COX2 (Meyer et al., 1997). The mutant with the suppressor mutation in Cox2p assembles functional COX even though it does not process pCox2p (Meyer et al., 1997). The dual role of Cox20p in Cox2p biogenesis is in some ways similar to the results described here except that unlike Cox20p, which is not a protease, Atp23p functions both as the protease and assembly factor for subunit 6.
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ACKNOWLEDGMENTS |
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Q Atp23p. We also thank Dr. Jean Velours (Institut de Biochimie et Genetique Cellulaires du CNRS, Bordeaux, France) for his generous gift of the polyclonal antibody against subunit 6 of the yeast ATPase. This research was supported by National Institutes of Health Research Grant HL2274 and an Alexander Humboldt Award (A.T.). |
Footnotes |
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1 While these studies were in progress, we learned that Osman et al. (2007) obtained similar data. 
Address correspondence to: Alexander Tzagoloff (spud{at}cubpet.bio.columbia.edu)
Abbreviations used: o mutant, respiratory-deficient mutant lacking mitochondrial DNA; – mutant, respiratory-deficient mutant with a partially deleted mitochondrial genome; pet mutant, respiratory-deficient mutant of yeast with a mutation in a nuclear gene; DOC, potassium deoxycholate; PMSF, phenylmethylsulfonyl fluoride; SMP, submitochondrial particles.
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REFERENCES |
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