RPM2 is a Saccharomyces cerevisiae nuclear gene that encodes the protein subunit of mitochondrial RNase P and has an unknown function essential for fermentative growth. Cells lacking mitochondrial RNase P cannot respire and accumulate lesions in their mitochondrial DNA. The effects of a new RPM2 allele, rpm2-100, reveal a novel function of RPM2 in mitochondrial biogenesis. Cells with rpm2-100 as their only source of Rpm2p have correctly processed mitochondrial tRNAs but are still respiratory deficient. Mitochondrial mRNA and rRNA levels are reduced in rpm2-100 cells compared to wild type. The general reduction in mRNA is not reflected in a similar reduction in mitochondrial protein synthesis. Incorporation of labeled precursors into mitochondrially encoded Atp6, Atp8, Atp9, and Cytb protein was enhanced in the mutant relative to wild type, while incorporation into Cox1p, Cox2p, Cox3p, and Var1p was reduced. Pulse-chase analysis of mitochondrial translation revealed decreased rates of translation of COX1, COX2, and COX3 mRNAs. This decrease leads to low steady-state levels of Cox1p, Cox2p, and Cox3p, loss of visible spectra of aa3 cytochromes, and low cytochrome c oxidase activity in mutant mitochondria. Thus, RPM2 has a previously unrecognized role in mitochondrial biogenesis, in addition to its role as a subunit of mitochondrial RNase P. Moreover, there is a synthetic lethal interaction between the disruption of this novel respiratory function and the loss of wild-type mtDNA. This synthetic interaction explains why a complete deletion of RPM2 is lethal.
MITOCHONDRIAL DNA (mtDNA) in the yeast Saccharomyces cerevisiae codes for components of complexes required in oxidative phosphorylation and electron transport as well as RNAs necessary for their expression by the mitochondrial translational machinery (Attardi and Schatz 1988; Pon and Schatz 1991). The vast majority of mitochondrial proteins, however, are encoded by nuclear genes, translated on cytoplasmic ribosomes, and delivered to mitochondria for function (Grivellet al. 1999).
S. cerevisiae has been a useful organism for studying many aspects of mitochondrial biogenesis because, as a facultative anaerobe, it can grow by either fermentation or respiration. Therefore, depending on the carbon source used, mutants with defects in genes required for respiration can be recovered and studied. A problem associated with using yeast to study aspects of mitochondrial gene expression is the link between translation of mitochondrial gene products and the maintenance of the mitochondrial genome. Mutations in nuclear genes that disrupt mitochondrial gene expression induce either the complete loss of the mitochondrial genome or large fragments thereof (Myerset al. 1985). Secondary effects associated with the loss of the mitochondrial genome can be particularly troublesome in the study of multifunctional proteins when one function is required for mitochondrial gene expression. A case in point is the nuclear gene, RPM2, which encodes a protein subunit of mitochondrial RNase P (Moraleset al. 1992; Dang and Martin 1993). The RNA subunit of mitochondrial RNase P, Rpm1r, is encoded in the organelle (Miller and Martin 1983; Underbrink-Lyonet al. 1983). Mitochondrial RNase P is an enzyme required for processing 5′ leader sequences from organelle tRNAs. Cells carrying an insertional disruption of RPM2 (rpm2::LEU2) produce a carboxyl-terminally truncated Rpm2p and accumulate mitochondrial tRNA precursors with extensions at their 5′ ends (Moraleset al. 1992). Like other nuclear and mitochondrial mutants defective in mitochondrial protein synthesis (Myerset al. 1985), these cells cannot maintain their mitochondrial DNA and either accumulate mitochondrial genomes with large deletions or lose their mitochondrial genomes entirely.
The unexpected observation that a complete deletion of the RPM2 open reading frame prevented growth on fermentable carbon sources revealed that Rpm2p has another function, in addition to its role in RNase P activity (Kassenbrocket al. 1995). This second function could be involved in mitochondrial biogenesis and yet still affect growth on fermentable carbon sources. For example, mutations in genes encoding components of the mitochondrial import machinery affect the localization of proteins required for a number of essential functions in addition to proteins required for respiration (Baker and Schatz 1991; Herrmann and Neupert 2000). Alternatively, it has been shown that certain proteins required for respiratory growth become essential for growth on fermentable carbon sources in the absence of a wild-type mitochondrial genome (for review see Chen and Clark-Walker 1999b; Contamine and Picard 2000). However, a mechanism for this phenomenon has not been established.
Although we have previously isolated alleles of RPM2 that lose mitochondrial RNase P activity, but allow cells to grow on fermentable carbon sources, we had not isolated any that retain RNase P activity but were otherwise compromised for growth. Here we describe a novel allele of RPM2, rpm2-100, which produces a protein missing amino acids 146-246. Cells with this allele as their sole source of Rpm2p grow on fermentable media, retain mitochondrial RNase P activity in vivo, and maintain wild-type mitochondrial genomes. However, even though mitochondrial tRNA processing appears normal, rpm2-100 cells grow poorly on respiratory carbon sources. Further analysis revealed that these cells have a specific defect in the synthesis of mitochondrially encoded cytochrome c oxidase subunits. This results in the loss of visible spectra of aa3 cytochromes and low cytochrome c oxidase activity. Therefore, Rpm2p has another mitochondrial function, in addition to its role in RNase P activity. Interestingly, cells defective in this second function possess a limited capacity to divide upon losing the wild-type mitochondrial genome. These data are consistent with the view that RPM2 has two mitochondrial functions and the loss of either affects growth on nonfermentable carbon sources. Moreover, mutations that disrupt both functions are synthetically lethal.
MATERIALS AND METHODS
Strains and media: Strains used in this study are listed in Table 1. Rich glucose media, YPD, included 1% Bacto-yeast extract, 2% Bacto-peptone, and 2% glucose. Rich glycerol/ethanol media, YPGE, contained 1% Bacto-yeast extract, 2% Bacto-peptone, 3% (v/v) glycerol, and 2% (v/v) ethanol. Synthetic complete (SC) media lacking specific amino acids contained 0.67% Bacto-yeast nitrogen base, 2% glucose. Solid media for plates included 2% Bacto-agar. Culture media reagents were Fisher Scientific (Pittsburgh) or Difco (Detroit) brand. Standard yeast methods (Shermanet al. 1986) were used. Yeast cells were transformed with plasmid DNA using the lithium acetate method (Chenet al. 1992). The plasmid-shuffle protocol was performed as described (Sikorski and Boeke 1991).
Construction of RPM2 mutants: Standard procedures were used for the preparation and ligation of DNA fragments and for transformation and recovery of plasmid DNA from Escherichia coli (Sambrooket al. 1989). Restriction and modification enzymes were used as recommended by the supplier (New England Biolabs, Beverly, MA). Plasmid DNA was isolated using QIAGEN columns, and DNA fragments were isolated from agarose gels using a QIAEX II extraction kit (QIAGEN, Chatsworth, CA). DNA was sequenced with a Sequenase Version 2.0 Kit (United States Biochemical, Cleveland). To construct plasmid pRS314/rpm2-100, plasmid pRS315/GAL-COX4-247.rpm2 (containing RPM2 sequence coding for amino acids 247-1202 fused to the COX4 mitochondrial targeting sequence under the transcriptional control of the GAL1 promoter; our laboratory, unpublished data) was cut with EcoRV, which cuts downstream of the COX4 sequence, and SacI, which cuts downstream of the RPM2 gene. Plasmid pRS314/RPM2 containing wild-type RPM2 was cut with BglII, filled in with Klenow, and subsequently cut with SacI. After gel electrophoresis, a 5780-bp fragment containing the plasmid sequence with 5′-end sequence (-570 to +432) of RPM2 was ligated with the EcoRV/SacI fragment. This pRS314/rpm2-100 construct resulted in a deletion of amino acids 146-246 coding region. To construct plasmids pRS425/RPM2 and pRS425/rpm2-100, XhoI/SacI fragments containing RPM2 and rpm2-100 gene were cloned into pRS425 cut with XhoI/SacI. To integrate the rpm2-100 allele at the RPM2 locus, pRS314/rpm2-100 was cut with XhoI and SacI and a 4.0-kb fragment containing rpm2-100 was cloned into pRS316. Integration of rpm2-100 at the RPM2 locus was performed as described (Rothstein 1991) and confirmed by PCR. To construct Δ735-1190, pRS314/RPM2 was cut with HpaI/PpuMI, filled in with Klenow, and ligated. To construct Δ715-1098, pRS314/RPM2 was cut with PstI, gel purified, and ligated. To construct Δ528-734, pRS314/RPM2 was cut with HincII, gel purified, and ligated.
DNA and RNA analysis: Total yeast RNA was isolated by hot phenol extraction (Kohrer and Domdey 1991). Total RNA (25-30 μg) was separated on a 4 or 6% polyacrylamide/8 m urea/TBE gel for the Rpm1r and tRNA analysis, respectively. For the mRNA and rRNA analysis total RNA was separated on a 1% formaldehyde agarose gel. Blotting, hybridization, and probing for and Rpm1r were performed as described previously (Stribinskiset al. 1996). Oligonucleotide probes (Table 2) were radiolabeled with [γ-32P]ATP as described (Sambrooket al. 1989). The signals were detected using phosphorimager scanning (PhosphorImager SF, Molecular Dynamics, Eugene, OR) or autoradiography. To quantify RNAs, the signals were normalized to that for U3 RNA.
Total yeast DNA was isolated as described (Philippsenet al. 1991). DNA was digested with HincII and TaqI, which released 1300 bp of RPM1 and 624 bp of ACT1. The products were separated on an agarose gel and Southern analysis was performed (Sambrooket al. 1989). The RPM1 riboprobe (above) was used to determine the amount of mitochondrial DNA; an ACT1 oligonucleotide (Table 2) was used to determine the amount of nuclear DNA. The signals were detected using phosphorimager scanning.
Labeling of mitochondrial translation products: In vivo pulse labeling of mitochondrial translation products with [35S]methionine (New England Nuclear, Boston) and mitochondrial isolation was essentially as described (Foxet al. 1991), except that log-phase cells were grown in galactose media. Cells (25-50 OD600) were used for the pulse labeling with 0.1 mCi of [35S]methionine, and 250-500 OD600 for pulse-chase labeling with 0.5 mCi [35S]methionine. Labeling was stopped by adding an excess of cold methionine. The radiolabeled proteins were fractionated by SDS-PAGE in a 16.5% gel, and analysis of the dried gel was performed using phosphorimager scanning.
Extraction and spectra of mitochondrial cytochromes: Mitochondria were isolated after conversion of cells to spheroplasts (Dieckmann and Tzagoloff 1983). The mitochondria were suspended in 50 mm Tris-HCl, pH 8.0, at a protein concentration of 10 mg/ml. Cytochromes were extracted and difference spectrum was obtained as described (Tzagoloff 1995).
Mitochondrial enzyme assays: The cytochrome c oxidase and the NADH-cytochrome c reductase activities were determined spectrophotometrically as described (Coruzziet al. 1979). Reduced cytochrome c was prepared by adding small amounts of sodium dithionite to a solution of horse heart cytochrome c (Sigma, St. Louis) followed by aeration to remove excess dithionite. The nonenzymatic rates were measured at 550 nm for 1 min after which 20 μg of mitochondria was added and the reaction was followed for an additional 2 min. The enzymatic rate was calculated from the difference in absorbance at 550 nm between 15 and 45 sec after addition of mitochondria. The specific activity is expressed as micromoles of cytochrome c oxidized or reduced per minute per milligram of mitochondrial protein at 23°.
Western analysis: Proteins were separated by 16.5% SDS-PAGE for the analysis of cytochrome c oxidase subunits and by 7.5% SDS-PAGE for the analysis of Rpm2 proteins using the buffer system of Laemmli, transferred to Immobilon-P membranes (Millipore, Bedford, MA), and treated with antibodies. The monoclonal antibodies against Cox1p, Cox2p, and Cox3p were used as recommended (Molecular Probes, Eugene, OR); antibodies that recognize nuclear-encoded subunits of cytochrome c oxidase (Glerumet al. 1995) and antibodies against Mdh1p and Cytb (Chacinskaet al. 2000) were also used. The anti-Rpm2p antibodies were made against a peptide encoding amino acids 306-323 (QCB Inc., Hopkinton, MA) and were used at 1:200 dilution.
Protein concentrations were determined using a Bio-Rad DC protein assay kit (Bio-Rad, Hercules, CA).
The rpm2-100 mutant is respiratory deficient but maintains wild-type mtDNA and grows on fermentable carbon sources: The RPM2 mutant allele, rpm2-100, has a deletion in the coding region of the RPM2 open reading frame downstream of the mitochondrial presequence such that the protein product does not contain amino acids 146-246. We introduced plasmids containing RPM2, rpm2-100, or vector alone into cells harboring a complete deletion of the RPM2 gene on a chromosome, but carrying a wild-type RPM2 gene on a URA3-containing plasmid, and then measured the ability of these cells to grow on media containing 5-fluoroorotic acid (5-FOA). Only cells that lose the URA3-containing plasmid and have another source of functional Rpm2p can grow under these conditions. Both RPM2 and rpm2-100 cells grew on plates containing 5-FOA (Figure 1A). The mutant cells grew at rates comparable to wild type on rich glucose medium (YPD; Figure 1B). Together, these results show that the mutant allele rpm2-100 supports the essential function of RPM2 and that amino acids 146-246 are dispensable for growth on glucose. In contrast, rpm2-100 cells do not form visible colonies after 4 days on plates containing the nonfermentable carbon sources glycerol/ethanol (YPGE; Figure 1B). The respiratory growth deficiency of the rpm2-100 mutant strain is leaky and colonies are observed upon prolonged incubation.
Since Rpm2p is required for the maintenance of the wild-type mitochondrial genome, defects in the integrity of mtDNA could explain the respiratory deficiency of rpm2-100 cells. To determine whether rpm2-100 cells maintain wild-type mtDNA, they were crossed to a wild-type strain devoid of mtDNA. The resulting diploids grow on nonfermentable carbon sources (data not shown), indicating that the rpm2-100 mutation is recessive and that rpm2-100 cells retain wild-type mtDNA. The amount of mtDNA relative to nuclear DNA was also examined in rpm2-100 cells, in which the chromosomal copy of RPM2 was replaced by rpm2-100, and wild-type cells. Cells were grown in glucose medium and shifted for 14-16 hr to glycerol/ethanol medium. A Southern blot with RPM1 as a probe for mtDNA and ACT1 as a probe specific for nuclear DNA revealed that RPM1 gene content was comparable between wild-type (lanes 1 and 3 in Figure 1C) and mutant cells (lanes 2 and 4 in Figure 1C). Both strains appeared to increase their mtDNA copy number to the same extent when grown under derepressing conditions. Although rpm2-100 cells maintain wild-type amounts of mtDNA, they respire poorly.
RNase P-related functions appear normal in rpm2-100 cells: We examined Rpm1r biosynthesis and RNase P activity to determine whether alterations in the known functions of Rpm2p could account for the respiratory defect in rpm2-100 cells. Total RNA was isolated from wild-type and rpm2-100 cells that were grown in glucose before a shift to glycerol/ethanol medium for 14-16 hr. Northern analysis was performed with probes specific for mitochondrial , tRNAPhe, tRNAGlu, and Rpm1r. These tRNAs represent three different mitochondrial transcription units and have either short ( , tRNAPhe) or long (tRNAGlu) 5′ leader sequences. The rpm2-100 cells make mature mitochondrial , tRNAPhe, and tRNAGlu (Figure 2, A and B). Levels of mature tRNAs are comparable to wild type under both fermentative and respiratory growth conditions. There is no evidence that tRNA precursors accumulate in either strain.
Synthesis of the RNA subunit of mitochondrial RNase P, Rpm1r, is also dependent on Rpm2p (Stribinskiset al. 1996). To determine if the rpm2-100 mutation affects Rpm1r synthesis, the RNA was blotted with a probe to Rpm1r (Figure 2C). Mature Rpm1r is made under all growth conditions, although it does appear that more precursors accumulate in rpm2-100 cells. Steady-state levels of mitochondrial RNA increase after cells are shifted to nonrepressing carbon sources such as glycerol/ethanol media (Uleryet al. 1994). It is clear from the data in Figure 2 that this response is not defective in the rpm2-100 cells. Since neither the amount nor the processing of mitochondrial tRNAs we examined were impaired, we decided to examine mitochondrial protein synthesis. It was possible that processing of a mitochondrial tRNA that we did not test might be defective in rpm2-100 cells and this could lead to a reduction of mitochondrial protein synthesis. We reasoned that if translation were reduced to the point that it no longer supported respiration, but not to the point where mtDNA stability was compromised, a respiratory phenotype such as seen in rpm2-100 cells could be observed.
Analysis of rpm2-100 mitochondrial translation products revealed defects in the synthesis of mitochondrially encoded Cox1p, Cox2p, and Cox3p: We compared the synthesis of mitochondrial gene products in RPM2 and rpm2-100 strains by pulse labeling for 30 min with [35S]methionine. These experiments were carried out in the presence of cycloheximide to inhibit cytoplasmic protein synthesis. Mitochondria were then isolated and proteins were analyzed by SDS-PAGE and phosphorimaging. Both strains incorporated radiolabeled methionine into mitochondrial translation products and the total amount of label incorporated was comparable (data not shown). However, while rpm2-100 cells synthesized all major mitochondrial gene products, the relative amount of label incorporated into each of the mitochondrially encoded proteins differed between rpm2-100 and wild-type cells (Figure 3A, lanes 1 and 2). Labeling of Cox1p, Cox2p, and Cox3p was reduced in the mutant relative to wild type. The incorporation of label into Var1p in the mutant was about twofold less than that in wild type. In contrast, incorporation of label into apocytochrome b and ATPase subunit 6 was elevated in the mutant relative to wild type. Thus, rpm2-100 cells have an altered pattern of mitochondrial protein synthesis. To determine if the labeling profiles were a consequence of differences in protein stability, cells were pulse labeled and chased for up to 6 hr in the presence of excess nonradioactive methionine. In this experiment, mitochondrial proteins were fractionated using a long gel, which allowed the separation of all major mitochondrial proteins. Figure 3B shows that the stability of radiolabeled mitochondrial proteins appeared to be comparable in both mutant (even lanes) and wild type (odd lanes). The incorporation of [35S]methionine into all three mitochondrially encoded subunits of ATPase was more efficient in the mutant relative to wild-type cells.
To address the possibility that a pulse-labeling period shorter than 30 min might be necessary to observe equivalent rates of protein synthesis in the two strains, pulse labeling for 5 min was performed. As shown in Figure 3C, decreased amounts of Cox1, Cox2, Cox3, and Var1 proteins were observed in rpm2-100 (lane 2) relative to wild type (lane 1). This confirms that protein synthesis, but not protein stability, is affected in rpm2-100 cells.
The Var1 protein, a component of the yeast mitochondrial small ribosomal subunit (Grootet al. 1979; Terpsta and Butow 1979), was reduced in rpm2-100 cells. However, this reduction is not limiting for the synthesis of other mitochondrially encoded proteins in rpm2-100 mutant, since Atp6p, Atp8p, Atp9p, and Cytbp were synthesized even more efficiently compared to wild type. The observed pattern of protein synthesis indicates that a reduction in cytochrome c oxidase levels is the most likely cause of the respiratory defect in rpm2-100 cells.
rpm2-100 cells lack visible spectra of aa3 cytochromes and have low cytochrome c oxidase activity: We performed spectral analysis of cytochromes, enzyme activity assays, and immunoblot analysis using mitochondria isolated from rpm2-100 and wild-type cells to determine the effects of the rpm2-100 mutation on cytochrome c oxidase activity and subunit accumulation. The cytochrome composition of mutant mitochondria was determined from the visible spectrum of extracts obtained under conditions known to quantitatively solubilize all the respiratory components of the organelle (Tzagoloff 1995). Room temperature cytochrome spectra revealed that the absorption band corresponding to cytochromes aa3, which reflects the amount of cytochrome c oxidase, was dramatically reduced in the mutant. In contrast, the level and absorption maxima for cytochromes c + c1 and b were unaffected in rpm2-100 cells (Figure 4A). To determine the biochemical consequence of the cytochromes aa3 defect, we measured cytochrome c oxidase and NADH-cytochrome c reductase activities in rpm2-100 mitochondria. Table 3 shows that rpm2-100 cells have only 10% of the wild-type cytochrome c oxidase activity, while their NADH-cytochrome c reductase activity is normal.
Western blot analysis of the mutant cell extracts revealed low steady-state levels of the Cox1, -2, and -3 proteins, but wild-type levels of mitochondrially encoded Cytb (Figure 4B). Immunoblots also revealed that the levels of nuclear-encoded subunits Cox4p, Cox5p, Cox6p, Cox7p, and Cox8p in rpm2-100 cells were comparable to wild type (Figure 4C). Thus, the respiratory deficient growth phenotype of rpm2-100 appears to be caused by decreased levels of Cox1p, Cox2p, and Cox3p.
Steady-state levels of mRNA and rRNA were reduced in rpm2-100 mitochondria: Differential regulation of yeast mitochondrial genes appears to take place via gene-specific controls of RNA processing, stability, and translation (Attardi and Schatz 1988; Costanzo and Fox 1990; Dieckmann and Staples 1994). To determine whether the decrease in mitochondrially encoded cytochrome c oxidase subunits resulted from a decrease in the steady-state levels of their mRNAs, total RNA was isolated from RPM2 and rpm2-100 cells, fractionated, blotted to a membrane, and hybridized with oligonucleotide probes specific for COX1, COX2, COX3, CYTb, ATP9 mRNA, as well as 15S and 21S rRNA (Table 2). Steady-state levels of all mature forms of mitochondrial mRNAs and rRNAs analyzed were reduced in rpm2-100 cells relative to wild type (Figure 5). The reduction varied from 2- to 8-fold for different RNAs and did not correlate with the presence or absence of introns. The ratio of precursor to mature RNA, when precursors were observed, was comparable to those in wild-type cells. Finally, there was no direct correlation between abundance of mRNA and the synthesis of the corresponding protein during protein labeling in vivo. For example, despite a 4-fold reduction in mRNA for CYTb and 8-fold for ATP9, the relative incorporation of [35S]methionine into apocytochrome b (Cytbp) and ATPase subunit 9 (Atp9p) in rpm2-100 cells was greater than that in wild type. These results indicate that some proteins can be synthesized efficiently in rpm2-100 mitochondria, even when the amount of their corresponding mRNA is reduced. Others have observed substantial decreases in mRNA levels without concomitant effects on the synthesis of protein products. For example, despite 2- to 5-fold reduction in CYTb and 20-fold reduction in COX1 mRNA levels in SUV3-1, the synthesis of Cox1 and Cytb polypeptides was about the same as that in wild-type cells (Conrad-Webbet al. 1990). Two other reports demonstrate that COX2 mRNA levels are not limiting for Cox2p synthesis, and 40-fold reduction still allowed growth by respiration (Pinkhamet al. 1994; Dunstanet al. 1997). Finally, Cliften et al. (1997) showed that mitochondrial transcription is reduced at higher temperatures and concluded that mitochondrial function can be maintained with only 10-20% of wild-type transcript levels. Therefore, the 2- to 8-fold reduction of mRNAs and the 4-fold reduction of rRNAs should not preclude appreciable growth on nonfermentable carbon sources. These considerations suggest that the major effect of the rpm2-100 mutation is at the translational level.
RPM2, but not rpm2-100, is a high-copy suppressor of tom40-3: RPM2 was isolated as a high-copy suppressor of tom40-3, a temperature-sensitive allele coding for a component of the mitochondrial protein import channel (Kassenbrocket al. 1995). It was clear in these previous experiments that RNase P activity was not required for high-copy suppression of tom40-3. However, all alleles that provided the essential function also suppressed the tom40-3 allele. Although the mechanism of suppression of tom40-3 is, as yet, unclear, it does suggest a model relating Rpm2p function to mitochondrial import, which is an essential feature of mitochondrial biogenesis. To determine whether rpm2-100 could also serve as a high-copy suppressor of tom40-3, we transformed the mutant with rpm2-100 on a high-copy vector. Figure 6 shows that rpm2-100 is not a high-copy suppressor of tom40-3.
RPM2 alleles that complement the rpm2-100 respiratory defect also provide the function essential for growth on glucose: The fermentative growth function of Rpm2p has been localized to the amino-terminal 734 amino acids of the RPM2 reading frame. The same portion of Rpm2p also suppressed the tom40-3 temperature-sensitive growth when provided on a high-copy vector (Kassenbrocket al. 1995). To determine if the novel function reported here can be uncoupled from the previously identified unknown function necessary for growth on all carbon sources, we asked whether mutant alleles of RPM2 that support the essential function also complement the respiratory growth defect of rpm2-100 cells. Deletions were introduced to remove increasing amounts of the carboxy terminus (Δ735-1190 and Δ715-1098) or 206 amino acids in the middle (Δ528-734) of Rpm2p. The ability of these alleles to support growth on the fermentable carbon source glucose was tested using plasmid shuffling (Figure 7A). rpm2.Δ735-1190 has the same phenotype as an insertional disruption of chromosomal RPM2 at the HpaI site (rpm2::LEU2; Moraleset al. 1992). Cells lose mtDNA at high frequency, but grow well on glucose. rpm2.Δ715-1098 and rpm2.Δ528-734 cells have phenotypes comparable to Δrpm2 and are unable to grow on glucose medium (Figure 7A). Each allele was transformed into yeast containing chromosomal rpm2-100 as its only source of Rpm2p. Figure 7B shows that wild-type RPM2 and rpm2.Δ735-1190, but not rpm2.Δ715-1098 or rpm2.Δ528-734, could complement the rpm2-100 respiratory growth defect. We used the same strains to determine whether stability or mitochondrial localization of Rpm2p was affected by the different mutations. Western analysis (Figure 7C) showed that alleles that do not provide function do produce detectable amounts of the mutant proteins that are localized to mitochondria. In fact, these alleles generally produced higher amounts of Rpm2 protein than the functional derivative rpm2.Δ735-1190p or wild-type Rpm2p. Therefore, because only alleles of RPM2 that provide the function essential for fermentative growth complement rpm2-100, we conclude that these two functions are related.
The rpm2-100 mutation converts S. cerevisiae to a petite negative yeast: Mitochondrial RNase P is not essential for growth on fermentable carbon sources in the yeast S. cerevisiae. Petite mutants completely lacking mtDNA are capable of growth on fermentable carbon sources. To determine the consequences of mtDNA depletion in the rpm2-100 background, we cultivated wild-type and rpm2-100 cells in glucose medium in the presence of ethidium bromide (EB). EB is a potent mitochondrial mutagen that causes deletions in mtDNA (Slonimskiet al. 1968). After 48 hr of cultivation with EB, aliquots of cells were plated on solid glucose medium without EB, and their ability to form colonies was determined. Figure 8 shows that rpm2-100 cells do not form visible colonies on YPD plates after 4 days, while RPM2 cells do. Only after prolonged incubation (over a week) did some cells give rise to colonies. Cells from these colonies grew at variable (slow) rates on glucose and were unable to grow on nonfermentable carbon sources (data not shown). This phenotype is reminiscent of Δrpm2 mutants, which display a significant rate of phenotypic reversion on glucose-containing medium (Kassenbrocket al. 1995; Lutzet al. 2000). Thus, rpm2-100 cells undergo a limited number of cell divisions and arrest growth after they lose mtDNA. This result indicates that the fermentative growth of rpm2-100 cells is dependent on the maintenance of the wild-type mitochondrial genome. Therefore, we conclude that the RNase P activity retained in rpm2-100 cells becomes essential for growth on glucose, through its effect on the maintenance of the wild-type mitochondrial genome.
Rpm2p is a multifunctional protein required for mitochondrial RNase P activity and for fermentative growth. Cells lacking mitochondrial RNase P activity lose wild-type mtDNA and thereby respiratory competence but grow by fermentation (Moraleset al. 1992). This dual requirement has prevented a detailed characterization of the role of RPM2 in mitochondrial biogenesis. We describe here an allele of RPM2, rpm2-100, that supports mitochondrial RNase P activity and yet is unable to support normal respiratory growth. Cells harboring the rpm2-100 allele are deficient in the synthesis of mitochondrially encoded cytochrome c oxidase subunits Cox1p, Cox2p, and Cox3p. The resultant decrease in cytochrome c oxidase activity provides an explanation for the respiratory deficiency observed in rpm2-100 mutant cells. Thus, RPM2 has at least two separable functions that contribute to respiratory growth.
The defect in mitochondrial protein synthesis in rpm2-100 cells is selective. Cox1p, Cox2p, and to a lesser extent Cox3p and Var1p are decreased in the mutant relative to wild type, whereas Cytbp and ATPase subunits 6, 8, and 9 are increased. There are a number of nuclear genes that affect the synthesis of specific mitochondrial gene products (Attardi and Schatz 1988; Tzagoloff and Dieckmann 1990; Dieckmann and Staples 1994; Pel and Grivell 1994; Fox 1996). Most, if not all, of the eight major mitochondrially encoded mRNAs are translated under the direction of mRNA-specific translational activator proteins specified by nuclear genes (Fox, 1996). Each mitochondrial mRNA appears to require its own specific activators for translation. The best studied, COX3 mRNA, is specifically activated by a complex containing the nuclear encoded gene products Pet54p, Pet122p, and Pet494p, (Mülleret al. 1984; Costanzo and Fox 1986; Costanzoet al. 1986; Foxet al. 1988; Brownet al. 1994). The Pet111p nuclear gene product (Poutre and Fox 1987; Mulero and Fox 1993) specifically activates translation of COX2 mRNA, whereas mutations in two genes, MSS51 (Decosteret al. 1990) and PET309 (Manthey and McEwen 1995) block translation of COX1 mRNA. Interestingly, Rpm2p has sequence similarity with Pet309p over a region spanning residues 189-302 of Rpm2p. This region, which contains 49% sequence similarity with Pet309p, partially overlaps with residues 146-246 deleted in rpm2-100. Thus, it is possible that in addition to its role as a component of mitochondrial RNase P, Rpm2p could act as a translational activator for the mitochondrially encoded cytochrome c oxidase genes.
RPM2 joins a number of nuclear genes involved in mitochondrial RNA processing that play a second role in mitochondrial biogenesis or function. NAM2 encodes the leucyl-tRNA synthetase and NAM2 is also required for the splicing of the COB bI4 and COX1 aI4 introns (Herbertet al. 1988). The splicing defect in PET54 mutants can be suppressed in intron-less strains, even though the remaining defect in the translation of COX3 does not allow respiratory competence (Valenciket al. 1989), indicating a dual mitochondrial function for PET54. The genetic evidence suggests that different functional domains of the Pet54 protein facilitate expression of the mitochondrial genes COX1 and COX3 (Valencik and McEwen 1991). The product of the MSS18 gene is required for the splicing of the COX1 aI5β intron by promoting the cleavage of the 5′ exon-intron junction (Seraphinet al. 1988). However, strains missing the aI5β intron and MSS18 grow only half as well on lactate as those with MSS18. This suggests that MSS18 has a second, as yet unidentified, respiratory function (Seraphinet al. 1988). The nuclear gene MSS116 has also been implicated in the splicing of several introns of both COX1 and CYTb, as well as having an additional role in the translation of mitochondrial encoded genes (Seraphinet al. 1989). For each of these genes, as well as for RPM2, the dual functions of their proteins can be at least partially separated by genetics. Some of these genes have a direct role in translation, while others, including RPM2, may act indirectly.
A role of Rpm2p in translation of mitochondrial COX mRNAs does not, however, readily explain the role of Rpm2p in fermentative growth, since growth on glucose is not dependent on the mitochondrial COX genes or their translational activators. Therefore, it is likely that the defect in the synthesis of mitochondrially encoded proteins is a reflection of some other process that, when disrupted, leads to mitochondrial dysfunction. In this context, a defect in the synthesis of mitochondrially encoded cytochrome c oxidase subunits was reported for mutants of the nuclear gene SSC1, which encodes mitochondrial Hsp70, a protein that plays an important role in the folding and assembly of proteins that are either newly imported or synthesized within the organelle (Kanget al. 1990; Manning-Krieget al. 1991; Herrmannet al. 1994; Westermannet al. 1996). SSC1, like several other components of the mitochondrial import apparatus, is an essential gene. When Hsp70 function is altered, the pattern of proteins synthesized in mitochondria changes. While mitochondrial translation continues in ssc1-2 and ssc1-3 mutants at the nonpermissive temperature, the amount of Cox1, -2, and -3 proteins is reduced relative to wild-type cells (Herrmannet al. 1994).
A link between Rpm2p and the mitochondrial import apparatus was established when RPM2 was isolated as a high-copy suppressor of tom40-3, which encodes a temperature-sensitive component of the mitochondrial protein import channel (Kassenbrocket al. 1995). Tom40 is an essential protein of the outer membrane translocase (Bakeret al. 1990) and forms the basic import core (Hillet al. 1998; van Wilpeet al. 1999). Several studies have shown that proteins destined for import into mitochondria can be unfolded once engaged by the import apparatus. This unfolding has been assigned to the outer membrane translocation apparatus (Mayeret al. 1995). Mitochondrial preproteins are in close contact with Tom40p and associate with the translocation machinery by interaction through both the presequence and the mature portion of the protein (Rapaport et al. 1997, 1998). This intimate contact maintains the preprotein in a translocation-competent form. It has also been shown that unfolding is associated with the protease-resistant part of the translocation machinery rather than with the surface receptors, which characterize the protein import complex as a membrane-integrated chaperone (Mayeret al. 1995). The observation that multiple copies of RPM2 can suppress a mutation in a key component of this complex, while rpm2-100 cannot, suggests that the same function of Rpm2p required for the synthesis of mitochondrially encoded subunits of cytochrome c oxidase within the organelle might be required for tom40-3 suppression. If the defect in cytochrome c oxidase synthesis in rpm2-100 cells is related to a chaperone function of Rpm2p within the organelle, this same activity could underlie the mechanism of suppression of the TOM40 mutant at the mitochondrial surface. Alternatively, Rpm2p may be involved in the synthesis of nuclear-encoded chaperones involved in mitochondrial biogenesis and function. A critical feature of either model infers an extramitochondrial localization of a fraction of Rpm2p.
The function of RPM2 compromised by the rpm2-100 mutation makes yeast cells dependent on the wild-type mitochondrial genome for fermentative growth. A small number of proteins required for respiratory growth have been shown to be essential for growth on fermentable carbon sources in the absence of wild-type mtDNA. These genes include AAC2 (Kováčet al. 1967), PGS1 (Subik 1974), YME1 (Weberet al. 1995), and genes encoding α- and β-subunits of F1-ATPase (Chen and Clark-Walker 1999a). Little is known regarding the mechanism linking the maintenance of an intact mitochondrial genome to the function of these genes, since a common primary defect is unknown (for review see Chen and Clark-Walker 1999b; Contamine and Picard 2000).
Nonetheless, each of these genes can be tied in, either directly or indirectly, to the mitochondrial import process. AAC2 encodes the major ADP/ATP carrier and defects in its activity could alter adenine nucleotide pools and thereby affect import, since the import of proteins into mitochondria needs energy available inside the organelle (Nelson and Schatz 1979). PGS1 catalyzes the first step in a biosynthetic pathway of phosphatidylglycerol and cardiolipin (Changet al. 1998), two anionic phospholipids that are confined mainly to mitochondrial membranes (Zinseret al. 1991). Anionic phospholipids participate in the formation of α-helices in the presequences of mitochondrial proteins (Wang and Weiner 1994; Chupinet al. 1996) and appear to facilitate the unfolding of proteins during import into mitochondria (Eilerset al. 1989; Endoet al. 1989). The α-subunit of F1-ATPase (encoded by ATP1) shares sequence similarity with molecular chaperones (Luiset al. 1990; Alconadaet al. 1994) and is required for the normal function of the inner membrane in promoting efficient protein import (Yuan and Douglas 1992). Finally, there is a genetic interaction between ATP1 and YME1, which suggests that there could be some functional overlap between F1-ATPase and Yme1p. YME1 encodes a putative ATP and zinc-dependent protease localized to the mitochondrial inner membrane (Weberet al. 1996) and has a potential role as a molecular chaperone (Nakaiet al. 1995). The inability of yme1 mutants to grow in the absence of wild-type mtDNA can be suppressed by mutations in the γ-subunit of F1-ATPase (Weberet al. 1995). These mutations are identical to those in the petite-negative yeast Kluyveromyces lactis, which convert it to petite-positive yeast (Chen and Clark-Walker 1995), and the authors have proposed that a novel property of the F1 complex other than ATP hydrolysis is responsible for the suppression (Chen and Clark-Walker 1999a). Thus, it is possible that mutations in these genes, as well as in RPM2, may decrease the efficiency of mitochondrial import. If the efficiency is further reduced by the loss of the mitochondrial genome and the concurrent reduction in membrane potential, import efficiency may be reduced below a point consistent with supporting growth on both nonfermentable and fermentable carbon sources.
The link between the function lost by the rpm2-100 mutation and the dependence on a wild-type mitochondrial genome provides an explanation for why a complete deletion of RPM2 prevents growth on fermentable carbon sources. The mitochondrial RNase P is required for the maintenance of the wild-type mitochondrial genome. Mitochondrial genomes in cells lacking mtRNase P activity accumulate deletions or are lost completely after a limited number of divisions. Cells compromised in the second Rpm2p function characterized here require the wild-type mitochondrial genome and are unable to grow in the absence of RNase P activity. Thus, a complete deletion of RPM2 causes lethality because mutations in the two functions of Rpm2p in combination are lethal, while either one can support growth on glucose.
We thank Marlene Steffen and Paul Weis for technical assistance. We thank Dr. Alex Tzagoloff for the generous donation of crude antisera against cytochrome c oxidase. National Institutes of Health grant GM-27597 to N.C.M. supported this work.
Communicating editor: M. Johnston
- Received December 27, 2000.
- Accepted March 5, 2001.
- Copyright © 2001 by the Genetics Society of America