Expression of the Saccharomyces cerevisiae Gene YME1 in the Petite-Negative Yeast Schizosaccharomyces pombe Converts It to Petite-Positive

Corresponding author: Peter E. Thorsness, Department of Molecular Biology, University of Wyoming, Laramie, WY 82071-3944., thorsnes{at}uwyo.edu (E-mail)

Communicating editor: M. LICHTEN

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Organisms that can grow without mitochondrial DNA are referred to as "petite-positive" and those that are inviable in the absence of mitochondrial DNA are termed "petite-negative." The petite-positive yeast Saccharomyces cerevisiae can be converted to a petite-negative yeast by inactivation of Yme1p, an ATP- and metal-dependent protease associated with the inner mitochondrial membrane. Suppression of this yme1 phenotype can occur by virtue of dominant mutations in the - and -subunits of mitochondrial ATP synthase. These mutations are similar or identical to those occurring in the same subunits of the same enzyme that converts the petite-negative yeast Kluyveromyces lactis to petite-positive. Expression of YME1 in the petite-negative yeast Schizosaccharomyces pombe converts this yeast to petite-positive. No sequence closely related to YME1 was found by DNA-blot hybridization to S. pombe or K. lactis genomic DNA, and no antigenically related proteins were found in mitochondrial extracts of S. pombe probed with antisera directed against Yme1p. Mutations that block the formation of the F₁ component of mitochondrial ATP synthase are also petite-negative. Thus, the F₁ complex has an essential activity in cells lacking mitochondrial DNA and Yme1p can mediate that activity, even in heterologous systems.

MITOCHONDRIAL biogenesis requires the coordinated expression of genes encoded by mitochondrial and nuclear genomes, as well as the regulated assembly of a number of multicomponent protein complexes. Recent work in the yeast Saccharomyces cerevisiae has revealed the importance of a related set of mitochondrial proteases in the assembly of energy transduction complexes. Located in the inner mitochondrial membrane is a hetero-oligomer, composed of the homologous proteins Yta10p and Yta12p, that is necessary for the assembly of cytochrome oxidase and ATP synthase (PAUL and TZAGOLOFF 1995 ; ARLT et al. 1996 ; GUELIN et al. 1996 ). The yeast homolog of the Escherichia coli lon protease has also been implicated in the assembly of inner membrane protein complexes (REP et al. 1996 ). These proteases have an ATP requirement, and mutational analysis has led to a proposal that these proteins function largely as assembly factors with editing capability. Misfolded and damaged proteins or proteins present in excess that cannot be assembled into a higher-order complex are degraded by these proteases.

Yme1p forms another inner mitochondrial membrane complex with protease activity (LEONHARD et al. 1996 ). Yme1p determines the stability of cytochrome oxidase subunit II that is present in excess or is not assembled into the multicomponent cytochrome oxidase complex (NAKAI et al. 1995 ; PEARCE and SHERMAN 1995 ; WEBER et al. 1996 ). While Yme1p is envisioned to have a role as an assembly/editing factor similar to Yta10p/Yta12p and mitochondrial Lon, this has not yet been demonstrated experimentally. YME1 was originally identified in a screen for mutations that increase the rate at which mitochondrial DNA (mtDNA) is transferred to the nucleus (THORSNESS and FOX 1993 ). yme1 strains are also heat sensitive for growth on nonfermentable carbon sources (pet-ts) and cold-sensitive for growth on rich-glucose media and have altered mitochondrial morphology (THORSNESS et al. 1993 ; CAMPBELL et al. 1994 ). S. cerevisiae strains in which YME1 has been inactivated are petite-negative; that is, they are incapable of growth in the absence of mtDNA (THORSNESS et al. 1993 ). Wild-type S. cerevisiae is a petite-positive strain, capable of growing on fermentable carbon sources in the absence of mtDNA. As a consequence of dysfunctional mitochondrial structure and function, yme1 strains have an increased rate of mitochondrial compartment turnover by the vacuole (CAMPBELL and THORSNESS 1998 ). Clearly, Yme1p is involved in a number of important mitochondrial processes that must extend beyond the turnover of unassembled Cox2p.

In an effort to understand the physiological role of Yme1p in mitochondrial biogenesis and function, a number of suppressors of yme1 phenotypes have been characterized. A bypass suppressor of the yme1 null allele that suppresses all yme1 phenotypes was identified as YNT1/RPT3, a gene that encodes a regulatory subunit of the 26S protease (CAMPBELL et al. 1994 ). Inactivation of YNT20, which encodes a putative 3'-5' exonuclease located in mitochondria, suppresses the high rate of mtDNA escape in yme1 strains and also suppresses the synthetic nonrespiring phenotype of yme1 yme2 strains (HANEKAMP and THORSNESS 1998 ). Specific dominant mutations in the gene encoding the -subunit of the mitochondrial ATP synthase, ATP3, suppress the petite-negative phenotype of yme1 yeast (WEBER et al. 1995 ). For none of these suppressors has it been possible to demonstrate a substrate/product relationship between the identified gene products and the Yme1p protease. In the work presented here, a number of additional mutations that result in the suppression of various yme1 phenotypes are described, and another suppressor of the petite-negative phenotype of yme1 yeast is fully described. Additionally, the heterologous expression of Yme1p in the petite-negative yeast Schizosaccharomyces pombe was used to define the genetic basis of yeast strains that require mtDNA for viability.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Strains:
The E. coli strains used for preparation and manipulation of DNA were DH5 [F^- end, hsdR17 (r_k^- m_k⁺), supE44, thi-1, recA, gyrA96, relA1, (argF-lacZYA) U169, 80, lacZM15] and XL1 Blue [recA1, endA1, gyrA96, thi-1, rsdR17, supE44, relA1, lac (F' proAB, lacI^qZDM15, Tn10 (tet^r))]. The E. coli strains ES1301 [lacZ53, mutS201::Tn5, thyA36, rha-5, metB1, deoC, IN(rrnD-rrnE)] and JM109 [endA1, recA1, gyrA96, thi, hsdR17(r_k^- m_k⁺), relA1, supE44, -, (lac-proAB), (F', traD36, proA+B+, lacI^qZM15)] were used for in vitro mutagenesis and were obtained from Promega (Madison, WI). The genotypes of S. cerevisiae strains used in this study are listed in Table 1. Standard genetic techniques were used to construct and analyze the various yeast strains (SHERMAN et al. 1986 ; see below). The S. pombe strain PNY10 was a gift of Dr. Paul Nurse. The Kluveromyces lactis strain Y11401 was a gift of Dr. Claudia Abeijon. The genotypes of both strains are listed in Table 1.

Media:
E. coli strains containing plasmids were grown in Luria-Bertani (LB) medium (10 g bactotryptone, 10 g NaCl, 5 g yeast extract per liter; MANIATIS et al. 1982 ) supplemented with 125 µg/ml of ampicillin. Yeast strains were grown in complete glucose medium (YPD) containing 2% glucose, 2% Bacto-peptone, 1% yeast extract; complete ethanol-glycerol medium (YPEG) containing 3% glycerol, 3% ethanol, 2% Bacto-peptone, 1% yeast extract; or minimal glucose medium (SD) containing 2% glucose, 6.7 g/liter yeast nitrogen base without amino acids (Difco, Detroit) and supplemented with the appropriate nutrients. Nutrients were uracil at 40 mg/liter, adenine at 40 mg/liter, tryptophan at 40 mg/liter, lysine at 60 mg/liter, and leucine at 100 mg/liter. For agar plates, Bacto-agar was added at 20 g/liter. Where indicated, ethidium bromide was added at 25 µg/ml (WEBER et al. 1995 ).

yme11::URA3 suppressor isolation and analysis:
Bypass suppressors of yme1-1::URA3 were isolated by screening for spontaneous revertants of the pet-ts phenotype of PTY52. Overnight cultures grown in YPD at 30° were plated on YPEG plates at ~2000 cells/plate and incubated for several days at 37°. Isolated revertants able to respire at high temperatures were colony purified, rescreened on YPEG at 37°, analyzed for suppression of other yme1-1::URA3 phenotypes, and screened for recessive collateral phenotypes by scoring for growth on different carbon sources at different temperatures. Each suppressed strain was mated to every other suppressed strain. Diploids were analyzed to identify complementation groups. Tetrad analysis of matings between the suppressed strains and a wild-type strain provided confirmation that any collateral phenotype segregated with the suppressing mutation. Dominant suppressors were isolated in the same manner as the recessive suppressors except that the screen was performed using the diploid strain PTY52 x PTY60. Isolation of suppressors of the yme1 petite-negative phenotype was performed as described (WEBER et al. 1995 ).

Nucleic acid techniques and DNA sequencing:
All manipulations of DNA were performed using standard techniques (MANIATIS et al. 1982 ). Restriction enzymes and DNA modification enzymes were purchased from New England Biolabs (Beverly, MA). Double-stranded DNA templates were prepared for sequencing by boiling lysis (MANIATIS et al. 1982 ) and were sequenced by the nucleotide chain termination method (SANGER et al. 1977 ) using the Sequenase Version 2.0 DNA sequencing kit (United States Biochemical, Cleveland). DNA-blot hybridization analysis was performed using standard techniques (MANIATIS et al. 1982 ). Blots were washed in 2x SSC with 0.5% SDS for 2 hr at 50°. Random primed probes corresponding to the entire open reading frames (ORFs) of YME1 and ATP1 from S. cerevisiae were prepared using the NEBlot kit from New England Biolabs.

Isolation of ATP1-75:
Briefly, genomic DNA was prepared as described (ROSE and BROACH 1991 ) from the yeast strain PTY78 bearing the ATP1-75 mutation. Genomic DNA was partially digested with Sau3A; 6–10-kb DNA fragments were isolated and cloned into the yeast CEN-vector pRS316 (SIKORSKI and HIETER 1989 ). Library DNA was prepared by large-scale alkaline lysis (MANIATIS et al. 1982 ) and was used to transform PTY62 using the alkali cation treatment method (ITO et al. 1983 ). Approximately 2 x 10⁵ transformants were inoculated into 10 ml of selective SD and incubated with agitation at 30° for 2 hr. Aliquots (1 ml) of the SD culture were inoculated into each of 10 tubes containing 10 ml selective SD media plus ethidium bromide (25 µg/ml) and grown to saturation at 30° (8 days). Ten microliters of each culture was inoculated into 10 ml of fresh SD plus ethidium bromide media and grown to saturation at 30° (12 days). Dilutions of each culture were plated on YPD media. Colonies were replica plated to media containing 5-fluoroorotic acid (5-FOA; BOEKE et al. 1984 ) to identify cells unable to grow without plasmid DNA. Thirty-two colonies unable to grow on 5-FOA were recovered from the YPD plates and the phenotype was rechecked. Plasmid DNA was prepared from four isolates. Three clones contained identical inserts of ~2.4 kb. One of these, pDK1, was sequenced and found to contain ATP1. This clone was sent to Macromolecular Resources (Fort Collins, CO) for sequencing of the entire ATP1-75 allele.

In vitro mutagenesis of plasmid DNA:
In vitro mutagenesis of plasmid DNA was performed using the Altered Sites in vitro mutagenesis system from Promega. Oligonucleotides were obtained from Integrated DNA Technology. The ATP1-75 allele was cloned into pALTER-Ex1 as follows. pDK1 was digested with KpnI and the ends were made blunt with mung bean nuclease. After digestion with EcoRI, the 2.2-kb ATP1-75 fragment was isolated and inserted into pALTER-Ex1 that had been digested with EcoRI and StuI. The resulting construct, pDK3, was the starting substrate for site-specific mutagenesis. The mutant ATP1 allele was restored to wild type using the oligonucleotide 5'-GGCTTTGAACTTGGAGCC-3', generating the plasmid pDK5. The sequencing oligonucleotide 5'-GCAGTCGGTGATGGTATTGC-3' was used to verify that the wild-type ATP1 sequence was restored in pDK5. This plasmid was then used as a substrate to recreate the ATP1-75 allele using the oligonucleotide 5'-GGCTTTGATCTTGGAGCC-3'. The sequence of the resulting plasmid, pDK6, was again verified using the sequencing oligonucleotide described above. The 2.2-kb EcoRI-HpaI inserts in pDK5 and pDK6 were transferred to the yeast CEN vector pRS316, creating the plasmids pDK9 and pDK10, respectively. These constructs, along with pDK1, were transformed into PTY62 to verify that the ATP1-75 mutation was able to suppress the petite-negative yme1 phenotype.

Creation of an ATP1 null allele:
The ATP1 null mutant was created using the plasmid pG50/ST6, a gift from Dr. Sharon Ackerman. The gene disruption was made by removing an internal 200-bp BglII fragment of the ATP1 gene and replacing it with a 3-kb BglII fragment containing the LEU2 gene. The chromosomal disruption was made by digesting pG50/ST6 partially with KpnI and then digesting completely with HindIII. The 4-kb KpnI-HindIII fragment containing the atp1::LEU2 construct was gel purified and used to transform PTY44. The resulting strain, DKY30, was then tested for the ability to respire and whether it was petite-positive or petite-negative. Creation of the null mutant was verified by performing PCR with the oligonucleotides 5'-TAAAGGTCCTATTGACGCTGC-3' and 5'-TATTGTAGAGGAGCGGCTTCA-3'.

S. pombe expression clones:
The S. pombe expression constructs were made using the plasmid pART1, a gift from Dr. Paul Nurse. To isolate the YME1 coding sequence, the plasmid pYME1-NdeI was digested with NdeI and the ends were made blunt by filling in the site with Klenow. After digestion with SacI, the 2-kb YME1 fragment was isolated and ligated into pART1 that had been digested with SacI and SmaI, generating pART1-YME1. The plasmid pyme1m was constructed by digesting pART1-YME1 with MluI. This site was then filled in using Klenow and the plasmid was religated. Transformation of S. pombe was performed by treatment of cells with alkali cations (ITO et al. 1983 ).

Preparation of cellular extracts and detection of Yme1p and Atp1p:
Protein extracts were prepared as described (PILLUS and SOLOMON 1986 ). Mitochondrial fractions were isolated by differential centrifugation (DAUM et al. 1982 ). Protein fractions were separated by SDS-PAGE, transferred to a nitrocellulose membrane, and probed with antisera directed against Yme1p or Atp1p as described previously (HANEKAMP and THORSNESS 1996 ).

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Isolation and characterization of bypass suppressors of yme1:
To identify gene products that interact with Yme1p, suppressors of a yme1-1::URA3 null allele were isolated. The yme1 null allele was used for this analysis because suppressors of yme1-1, a missense mutation, were all intragenic. Mutants were isolated either as dominant or recessive suppressors of the yme1 pet-ts phenotype or as suppressors of the yme1 petite-negative phenotype. These mutants were then screened for their ability to suppress other yme1 phenotypes, as well as for inherent collateral phenotypes. Nine strains were identified with mutations that suppress various yme1 phenotypes (Fig 1). Complementation analysis indicates that these suppressors represent mutations at distinct genetic loci. These mutations can be placed in classes based on the yme1 phenotypes suppressed and collateral phenotypes linked to the suppressing mutation. Class 1 contains the recessive suppressor ynt1, which suppresses all of the yme1 phenotypes and has a recessive collateral phenotype of cold-sensitive growth on the nonfermentable carbon sources ethanol and glycerol (CAMPBELL et al. 1994 ). Class 2 includes the recessive mutants ynt4 and ynt5. These mutations suppress the yme1 pet-ts, the high rate of mtDNA escape, and cold-sensitive growth on rich-glucose phenotypes. These suppressing mutations display no collateral phenotypes. The third class of suppressors includes the recessive mutants ynt7 and ynt8, which suppress the pet-ts and cold-sensitive growth on rich-glucose media phenotypes of yme1 strains. Additionally, these mutations have a recessive collateral phenotype of temperature-sensitive growth on rich-glucose media. The fourth class of recessive yme1-1::URA3 suppressors includes the mutant ynt6, which suppresses only the pet-ts phenotype. This mutation has a recessive collateral phenotype of pet-ts growth in a YME1 background. Also shown in Fig 1 is the mutation YNT9-1, which is presumably a dominant suppressor of the yme1 pet-ts phenotype since it was isolated in a yme1-1::URA3 homozygous diploid. However, further characterization of this mutation has proven difficult because this strain grows poorly on glucose media and does not sporulate. The final class of suppressors includes the dominant mutations ATP3-1 and ATP1-75, previously referred to as YNT3-1 (WEBER et al. 1995 ). These mutants suppress only the petite-negative phenotype of yme1. Cells grown in the presence of ethidium bromide rapidly lose all DNA from their mitochondria (SLONIMSKI et al. 1968 ; FOX et al. 1991 ) and are termed rho⁰. Unlike wild-type yeast, which readily grows in the presence of ethidium bromide, yme1 rho⁰ yeast are very slow growing. To better understand the basis for this petite-negative phenotype of yme1 strains, we pursued analysis of the ATP1-75 mutation.

View larger version (57K): In this window In a new window Download PPT slide   Figure 1. Suppression of yme1 phenotypes. Top, the indicated yeast strains were scored for mtDNA escape by patching on a YPEG plate, incubating at 30° for 2 days, and then replica plating to synthetic glucose lacking tryptophan. The plate was then scored after 4 days of incubation at 30°. Strains that have an increased rate of mtDNA escape have an increase in the relative number of Trp+ papillae (THORSNESS et al. 1993 ). Middle, the indicated strains were patched to YPD, incubated for 1 day and then replica plated to YPD and YPEG and incubated at the temperatures shown for 1–7 days, depending on temperature and media. Bottom, the indicated strains were streaked on a synthetic glucose plate containing 25 µg/ml ethidium bromide and incubated for 5 days at 30°. Strains: yme-1 (PTY52), yme1 ynt1 (NTY1), yme1 ynt4 (DKY21), yme1 ynt5 (DKY22), yme1 ynt6 (DKY25), yme1 ynt7 (DKY26), yme1 ynt8 (DKY27), yme1 ATP1-75 (PTY93), yme1 YNT9-1 (DKY1), WT (PTY44).

Isolation of ATP1-75, a dominant suppressor of the yme1 rho⁰ slow-growth phenotype:
Previous work in our laboratory demonstrated that mutations in the ATP-synthase -subunit suppress the petite-negative phenotype of yme1 yeast (WEBER et al. 1995 ). Additionally, work in the petite-negative yeast K. lactis has shown that mutations in the ATP-synthase -, ß-, and -subunits convert this yeast to petite-positive (CHEN and CLARK-WALKER 1995 , CHEN and CLARK-WALKER 1996 ). To assess the similarities of petite negative suppressors in yme1 S. cerevisiae and K. lactis, we cloned the ATP1-75 mutation. A genomic DNA library was constructed from a yme1-1::URA3 ATP1-75 strain. This genomic library was transformed into a yme1-1 strain, and transformed cells were screened for the ability to grow upon mtDNA loss. Several clones were isolated from this screen. Fig 2 shows the growth of a rho⁰ yme1-1 mutant after introduction of one of these plasmids, pDK1. DNA sequence analysis demonstrated that the plasmid insert contained the complete open reading frame of ATP1, the structural gene for the -subunit of mitochondrial ATP synthase. Sequence analysis of the entire ORF identified a single nucleotide change that resulted in the conversion of the strictly conserved asparagine residue at position 102 to isoleucine. To demonstrate that the amino acid change at position 102 in ATP1-75 was responsible for suppression of the petite-negative phenotype, site-directed mutagenesis was performed. First, the residue was changed to the wild-type asparagine, followed by reversion of the asparagine residue back to the mutant isoleucine. As shown in Fig 2, a plasmid bearing the reverted wild-type ATP1 allele is unable to rescue the petite-negative phenotype, while both the original ATP1-75 allele and the reverted ATP1-75 allele restore growth on ethidium bromide.

View larger version (49K): In this window In a new window Download PPT slide   Figure 2. Complementation of the yme1 petite-negative phenotype by cloned ATP1-75. Wild-type yeast (PTY44) or a strain bearing the yme1-1 mutation (PTY62) was transformed with the indicated plasmids and then streaked on a synthetic glucose plate (A) and incubated for 3 days at 30°, or streaked on a synthetic glucose plate containing 25 µg/ml ethidium bromide (B) and incubated for 5 days at 30°. The indicated plasmids are the vector (pRS316), the cloned ATP1-75 allele (pATP1-75), the cloned ATP1 allele reverted to wild type via site directed mutagenesis (pATP1r), and the ATP1 wild-type clone reverted back to the ATP1-75 allele (pATP1-75r).

A requirement of functional mitochondrial ATP-synthase F₁ component in rho⁰ yeast:
It has been reported that null mutations in the ATP-synthase -subunit (WEBER et al. 1995 ) and the -subunit (GIRAUD and VELOURS 1997 ) cause a petite-negative phenotype in S. cerevisiae. These observations have been taken as evidence for a requirement of assembled and functional F₁ subunits in mitochondria that lack DNA. To determine whether an ATP1 null mutation would confer a similar phenotype, an ATP1-1::LEU2 strain, DKY30, was constructed and streaked on media containing ethidium bromide. Additionally, another strain that had an ATP12 null mutation was also cultured on media containing ethidium bromide. Atp12p is involved in the assembly of ATP-synthase F₁ subunits (BOWMAN et al. 1991 ). As shown in Fig 3, both null mutant strains exhibit a petite-negative phenotype, demonstrating the requirement for an assembled and functional F₁ portion of the ATP-synthase complex, even in the absence of a functional F₀ component.

View larger version (57K): In this window In a new window Download PPT slide   Figure 3. Growth of rho0 yeast bearing yme1, atp1, or atp12 null alleles. Yeast cells were streaked on a synthetic glucose plate (A) and incubated for 3 days at 30° and on a synthetic glucose plate containing 25 µg/ml ethidium bromide (B) and incubated for 5 days at 30°. The indicated yeast strains are wild type (PTY44), yme1 (PTY52), atp1 (DKY30), and atp12 (W303ATP12).

An absence of closely related YME1 genes in S. pombe and K. lactis:
The results presented above suggest that the activity of Yme1p in S. cerevisiae is essential to maintain the petite-positive phenotype of this yeast. Because of the striking similarities between a yme1 mutant and the petite-negative yeasts S. pombe and K. lactis, we wondered if this growth characteristic was due to the absence of a Yme1p-like activity. To address this issue DNA-blot hybridization analysis was performed to determine whether the genomes S. pombe and K. lactis encode a YME1 homolog. Total DNA prepared from the strains PTY44, PTY52, PNY10, and Y11401 was digested with EcoRI or SspI, blotted, and probed with a random-primed probe prepared from a PCR product corresponding to the YME1 ORF under low stringency conditions. Fig 4 shows that a strong signal was detected for the S. cerevisiae DNA (Fig 4, lanes 5, 6, 9, and 10), corresponding to YME1. An additional weak signal was detected in the SspI-digested S. cerevisiae DNA at ~6.5 kb (Fig 4, lanes 9 and 10). These fragments correspond to the AFG3 gene (data not shown). Digestion of the AFG3 locus with EcoRI produces a fragment of ~15 kb; thus the AFG3 signal in lanes 5 and 6 was masked by the intense YME1 signal at the same position. The AFG3 gene is the nearest homolog to YME1 in the S. cerevisiae genome, sharing an internal coding region that is 61% identical. Weak signals were also detected in the S. pombe DNA (Fig 4, lanes 7 and 11), but not in K. lactis (lanes 8 and 12). The relative strength of the hybridization signal generated with the S. pombe DNA indicates that the YME1 probe recognizes a DNA sequence in S. pombe that is no more homologous to the YME1 sequence than is the AFG3 gene. As a control, the highly conserved ATP1 genes in S. pombe and K. lactis were easily detected when S. cerevisiae ATP1 sequences were used to probe genomic DNA (Fig 4, lanes 13–18).

View larger version (22K): In this window In a new window Download PPT slide   Figure 4. Detection of DNA sequences homologous to YME1. DNA-blot hybridization was performed on genomic DNA prepared from S. cerevisiae, S. pombe, and K. lactis. Designations for the DNA sources are as follows: Sc (lanes 1, 5, 9, 13, 16), wild-type S. cerevisiae (PTY44); Sc' (lanes 2, 6, 10), yme1-1::URA3 S. cerevisiae (PTY52); Sp (lanes 3, 7, 11, 15, 18), S. pombe (PNY10); Kl (lanes 4, 8, 12, 14, 17), K. lactis (Y11401). Restriction enzyme digestions were as indicated. Lanes 1–12 were probed with S. cerevisiae YME1 sequences and lanes 13–18 with S. cerevisiae ATP1 sequences. Hybridization signals were detected with a Bio-Rad (Richmond, CA) phosphorimager.

To identify a closely related YME1 gene product in S. pombe, whole cell extracts were prepared from a S. pombe culture, proteins were separated on polyacrylamide gels and blotted to a nitrocellulose filter, and the filter was probed with polyclonal antisera directed against Yme1p. No cross-reaction with the Yme1p antisera was observed (Fig 6, lane 4). In contrast, antisera directed against the conserved S. cerevisiae Atp1p gene product detected S. pombe Atp1p (Fig 6, lanes 4 and 5).

View larger version (44K): In this window In a new window Download PPT slide   Figure 5. Heterologous expression of YME1 in S. pombe. S. cerevisiae and S. pombe were transformed with the indicated plasmids and streaked on a synthetic glucose plate (A) and incubated for 5 days at 30° or streaked on a synthetic glucose plate containing 25 µg/ml ethidium bromide (B) and incubated for 7 days at 30°. The indicated strains are as follows: S. c. wild type, S. cerevisiae wild-type strain PTY44; S. c. yme1, S. cerevisiae yme1 strain PTY52; S. p. wild-type, S. pombe strain PNY10. The indicated plasmids are as follows: pART1, S. pombe expression vector; pYME1, the vector plasmid pART1 bearing wild-type YME1; pyme1m, the vector plasmid pART1 bearing an inactivated yme1 allele.

View larger version (55K): In this window In a new window Download PPT slide   Figure 6. Detection of Yme1p in S. cerevisiae and S. pombe. Mitochondrial fractions prepared from the indicated strains bearing the indicated plasmids were subjected to SDS-PAGE, transferred to nitrocellulose filters, and probed with antisera directed against Yme1p and Atp1p. Lane 1, PTY44/pART1; lane 2, PTY52/pART1; lane 3, PTY52/pYME1; lane 4, PNY10/pART1; lane 5, PNY10/pYME1. Whole cell extracts and postmitochondrial supernatants prepared from PTY52/pART1, PNY10/pART1, or PNY10/pYME1 do not produce a cross-reacting signal with Yme1p antisera.

Heterologous expression of YME1 in S. pombe:
To determine if Yme1p activity was able to convert a petite-negative yeast to petite-positive, YME1 was expressed in S. pombe. The YME1 gene was cloned into the S. pombe expression vector pART1. As an additional control, the pART1-YME1 construct was digested with MluI, filled in, and religated. This plasmid, pyme1m, has a frame-shift in the YME1 ORF that creates a nonfunctional gene product. These two plasmids, along with the vector, were transformed into a S. cerevisiae yme1 mutant and the S. pombe strain PNY10. As shown in Fig 5, expression of YME1 in both the S. cerevisiae yme1 and S. pombe strains allows growth in the absence of mtDNA, while the pART1 vector and the inactivated yme1 allele do not. Thus, Yme1p provides an activity that is essential for viability of two yeast when they lack mtDNA. Western blot analysis using whole cell extracts from these strains detected Yme1p in mitochondria of S. pombe transformed with pART1-YME1 (Fig 6).

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

To identify possible Yme1p substrates and to elucidate the role of Yme1p in yeast mitochondria, we undertook a suppressor analysis of a yme1 null mutant. Several genes were identified that, when mutated, lead to suppression of various yme1 phenotypes (Fig 1). Because mutation of different genes suppress different spectrums of yme1 phenotypes, we anticipate multiple roles for Yme1p in mitochondrial biogenesis and function.

Previous work in our laboratory identified mutations in the ATP3 gene that suppress the petite-negative phenotype of yme1 strains (WEBER et al. 1995 ). Here we identify a mutation in the ATP1 gene that suppresses the same phenotype. The F₁ complex of the mitochondrial ATP synthase is a multi-subunit structure with a subunit stoichiometry of ₃ß₃. Deletion of ATP1 leads to the inability to use nonfermentable carbon sources (TAKEDA et al. 1986 ) and causes a petite-negative phenotype (Fig 3). Mutational inactivation of the - and -F₁ subunits (WEBER et al. 1995 ; GIRAUD and VELOURS 1997 ) and of Atp12p, a protein involved in F₁ subunit assembly (BOWMAN et al. 1991 ), also cause this phenotype (Fig 3). These results indicate that Yme1p, as well as the F₁ portion of the ATP synthase, are important for the growth of S. cerevisiae in the absence of mtDNA. One extensively characterized mutation that causes S. cerevisiae to become petite-negative is op1, a mutation in the major ADP/ATP translocator encoded by AAC2 (KOVACOVA et al. 1968 ; LAWSON and DOUGLAS 1988 ; KOLAROV et al. 1990 ). Both op1 and a null mutation of ACC2 render cells unable to grow on nonfermentable carbon sources (KOVACOVA et al. 1968 ; LAWSON et al. 1990 ). The petite-negative nature of op1 strains has been taken as evidence for the absolute requirement for ATP in the matrix of mitochondria.

The yeasts K. lactis and S. pombe are petite-negative. Work in K. lactis has shown that specific mutations in the ATP-synthase -, ß-, and -subunits convert this yeast to a petite-positive organism (CHEN and CLARK-WALKER 1995 , CHEN and CLARK-WALKER 1996 ). Based on the similarities between these petite-negative strains and an S. cerevisiae yme1 mutant, we surmised that these yeasts may be petite-negative because they lack a Yme1p-like activity. DNA-blot analysis indicated that neither K. lactis nor S. pombe encode a close YME1 homolog (Fig 4), nor is there an antigenically related Yme1p homolog in S. pombe (Fig 6). Additionally, heterologous expression of YME1 in S. pombe converted this petite-negative yeast to petite-positive. Thus, Yme1p has an activity that is missing in S. pombe, and presumably also in K. lactis, that results in these yeast being petite-negative.

We utilized the bovine F₁-ATPase crystal structure (ABRAHAMS et al. 1994 ) to identify the relative location of mutations in the - and -subunits of mitochondrial ATP synthase that convert petite-negative yeast to petite-positive. The amino acid changed in ATP1-75, Asn102, is located near the interface of the - and -subunits in the region referred to as the "dimple" of the F₁ complex, distal to the stalk connecting F₁ to F₀ (ABRAHAMS et al. 1994 ). The two amino acid positions mutated in the K. lactis -subunit, Ala333 and Phe443, are also located along this interface, in direct contact with the -subunit (CHEN and CLARK-WALKER 1996 ). Thus, it is likely that the interaction of the - and -subunits is altered in these mutant F₁ complexes.

The information presented here suggests that Yme1p must be involved in the regulation of mitochondrial ATP synthase, although the nature of this regulation is unknown. The consequences of the absence of Yme1p in yeast that contain a complete mitochondrial genome is not deleterious at 30°, as these cells can use nonfermentable carbon sources. Upon loss of mtDNA, however, the absence of Yme1p leads to the petite-negative phenotype. It has previously been shown that Yme1p is a metallo-protease that is involved in the turnover of cytochrome oxidase subunit II (NAKAI et al. 1995 ; PEARCE and SHERMAN 1995 ; WEBER et al. 1996 ). One possibility is that Yme1p may also have a direct role in the turnover of F₁ subunits. In mitochondria lacking mtDNA, which encodes the F₀ subunits Atp6p, Atp8p, and Atp9p, excess F₁ subunits might be degraded in a Yme1p-dependent manner. Without Yme1p, F₁ subunits may accumulate, leading to aberrant complex formation. This scenario seems unlikely since the accumulation of Atp3p has been shown to be independent of Yme1p (WEBER et al. 1995 ). Alternatively, Yme1p may affect the F₁ complex indirectly by interacting with F₀ subunits, at least six of which are encoded in the nucleus. In the absence of the mitochondrially encoded F₀ subunits, the nuclearly encoded subunits may be subject to degradation by Yme1p, allowing the F₁ portion of the ATP synthase to function properly. In the absence of Yme1p, these F₀ subunits may accumulate and interfere with an F₁ activity that is essential for cells lacking mtDNA. The mutations that have been identified in ATP1 and ATP3 would thus allow the F₁ complex to assume a conformation that is not subject to interference by inhibitory factors that exist in the absence of Yme1p activity.

	ACKNOWLEDGMENTS

We thank Dr. Sharon Ackerman, Dr. Paul Nurse, and Dr. Claudia Abeijon for generously providing plasmids and yeast strains. We also thank the members of the Thorsness laboratory for critical review of this manuscript. This work was supported by Public Health Service grant GM-47390.

Manuscript received September 11, 1998; Accepted for publication September 27, 1999.

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