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Previous Article | Next Article 
Mol Cell Biol, July 1998, p. 4043-4052, Vol. 18, No. 7
Department of Biology, University of
California, San Diego, La Jolla, California
Received 4 February 1998/Returned for modification 17 March
1998/Accepted 21 April 1998
0270-7306/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Prohibitin Family Members Interact Genetically with
Mitochondrial Inheritance Components in Saccharomyces
cerevisiae
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
ACKNOWLEDGMENTS
REFERENCES
SUMMARY
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Phb2p, a homolog of the tumor suppressor protein prohibitin, was identified in a genetic screen for suppressors of the loss of Mdm12p, a mitochondrial outer membrane protein required for normal mitochondrial morphology and inheritance in Saccharomyces cerevisiae. Phb2p and its homolog, prohibitin (Phb1p), were localized to the mitochondrial inner membrane and characterized as integral membrane proteins which depend on each other for their stability. In otherwise wild-type genetic backgrounds, null mutations in PHB1 and PHB2 did not confer any obvious phenotypes. However, loss of function of either PHB1 or PHB2 in cells with mitochondrial DNA deleted led to altered mitochondrial morphology, and phb1 or phb2 mutations were synthetically lethal when combined with a mutation in any of three mitochondrial inheritance components of the mitochondrial outer membrane, Mdm12p, Mdm10p, and Mmm1p. These results provide the first evidence of a role for prohibitin in mitochondrial inheritance and in the regulation of mitochondrial morphology.
INTRODUCTION
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Mitochondrial inheritance is an essential and active process by which daughter cells receive mitochondria prior to the completion of cytokinesis. In budding yeast, factors specifically required for mitochondrial inheritance have been identified and characterized through the analysis of conditional mutants (7, 25). Three distinct proteins of the mitochondrial outer membrane, Mdm10p, Mmm1p, and Mdm12p, have been found to constitute one class of mitochondrial inheritance factors. Each protein is required for normal mitochondrial morphology and inheritance, and mdm10, mmm1, and mdm12 loss-of-function mutants exhibit similar phenotypes of temperature-sensitive growth and enlarged, round mitochondria (6, 9, 39). At least one of these proteins, Mdm12p, has been evolutionarily conserved and possesses a homolog in the fission yeast Schizosaccharomyces pombe (6). While the location of these proteins in the mitochondrial outer membrane suggests that they may interact with cytoskeletal elements to mediate normal mitochondrial distribution, their molecular activity remains to be defined.
To explore Mdm12p function, high-copy-number plasmid-borne suppressors able to bypass the cellular requirement for Mdm12p were identified. This paper describes the characterization of a plasmid-borne suppressor encoding a prohibitin-related protein localized to the mitochondrial inner membrane and exhibiting genetic interactions with mitochondrial outer membrane inheritance components. Prohibitins are a family of conserved proteins whose first member was identified as a negative regulator of cell division in cultured animal cells (29). Prohibitin homologs have been identified in diverse organisms and cell types and have been localized to mitochondria in animal and plant cells (20, 38). The function of prohibitin at the molecular level is unknown.
MATERIALS AND METHODS
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Strains and media.
The Saccharomyces cerevisiae
strains used in this work are listed in Table
1. All strains were derived from MYY290
or MYY291 (37). Media for yeast were prepared as described
previously (33). Yeast were transformed with lithium acetate
(21). Respiration-deficient isolates were obtained by
plating cells on YPDG medium (1% yeast extract, 2% Bacto Peptone,
0.1% glucose, 3% glycerol) and screening for small colonies.
Candidate respiration-deficient strains were shown to be unable to grow
on YPG medium (1% yeast extract, 2% Bacto Peptone, 3% glycerol). The
mitochondrial DNA of these respiration-deficient cells is likely to be
partially or fully deleted, as DAPI (4,6-diamidino-2-phenylindole) staining of cells failed to reveal any mitochondrial fluorescence. We
refer to such respiration-deficient cells as
[rho
] cells. For inhibition of mitochondrial
protein synthesis, cells were grown for several generations in 1%
yeast extract-2% Bacto Peptone-2% galactose at 30°C, with either
no drug, erythromycin (2 mg/ml), or chloramphenicol (4 mg/ml).
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Genetic and molecular biological techniques.
The plasmids
used are described below. Escherichia coli DH5
was used
to propagate plasmid DNA. General molecular biological methods were as
described previously (34). PCR amplification was performed
with Taq DNA polymerase (Fisher Scientific, Pittsburgh, Pa.)
in Taq reaction buffer supplemented with 2.5 mM
MgCl2 and 0.2 mM concentrations of deoxynucleoside
triphosphates (Boehringer Mannheim Corp., Indianapolis, Ind.) by using
an ERICOMP (San Diego, Calif.) thermal cycler. Specific
oligonucleotides were synthesized by Operon Technologies (Alameda,
Calif.). Site-directed mutagenesis was carried out by
oligonucleotide-mediated mutagenesis with the Transformer kit (Clontech
Laboratories, Palo Alto, Calif.). Isolation of total yeast RNA and
Northern analysis were performed essentially as described previously
(37). DNA probes for the PHB1, PHB2, and MDM1 genes were prepared from PCR products corresponding
to the respective coding sequences and labeled with
[32P]dCTP by random priming with a DNA labeling kit
(Boehringer Mannheim). NIH Image (version 1.61) software was used for
quantitative comparison of RNA levels following Northern analysis and
of protein levels following immunoblotting. For yeast genomic library
construction, yeast genomic DNA from strain MYY629 was partially
digested with BamHI and BglII and ligated into
the 2µm plasmid YEp13 (8), which had been digested with
BamHI and treated with calf alkaline phosphatase.
Isolation of high-copy-number plasmid suppressors of mdm12. Cells of mdm12-null strain MYY623 were transformed with a yeast genomic DNA library constructed in the LEU2 2µm vector YEp13 and plated onto selective media lacking leucine. Plates were incubated at 23°C (permissive temperature) until colonies had formed and were replica plated to yeast extract-peptone-dextrose (YPD) at 37°C (nonpermissive temperature). Colonies able to grow at 37°C after replica plating were further analyzed to identify those for which growth at high temperature was plasmid dependent. Of ~3,400 Leu+ transformants screened, one clone that provided partial suppression of the mdm12 mutant phenotype was identified.
Construction of phb1-null and phb2-null strains. Replacement of wild-type chromosomal DNA corresponding to PHB1 and PHB2 coding sequences with prototrophic markers was performed by one-step gene replacement with PCR-generated cassettes essentially as described previously (4). Gene replacement oligonucleotide sequences are available on request. All gene replacements were generated in diploid cells and subsequently verified by PCR analysis of haploid progeny following sporulation. Independently, PHB1 was replaced with LEU2 and with HIS3 to generate phb1::LEU2 and phb1::HIS3, respectively, and PHB2 was replaced with HIS3 to generate phb2::HIS3.
Determination of replicative life span. Replicative life span was determined essentially as described previously (27). Briefly, virgin mother cells were isolated as new daughters and were separated by micromanipulation from each daughter. The original mother was retained until it lysed or stopped dividing. The number of generations for a given mother was equal to the number of buds put forth by that cell. Replicative life spans were determined for cells grown on YPD at 30°C, with plates refrigerated overnight for the course of the experiment. Life spans were determined for at least 54 mother cells for each strain analyzed, and these data were used to determine mean and maximum replicative life spans. Statistical analysis was performed by using Student's two-tailed t test.
Gene constructions. For site-directed oligonucleotide mutagenesis of the YEp13-based PHB2-tetA suppressing clone (plasmid pKB38), the selection primer in each case was oligonucleotide XhoI-KILL (5'GAAGTTCTCCTGGAGGATTTAG-3') (mutated nucleotides are underlined throughout), which destroys the unique XhoI site in this plasmid. An in-frame stop codon was created between the 3'-truncated PHB2 and the 5'-truncated tetA in plasmid pKB38 with the mutagenic primer PT-STOP (5'-CGATGCGTCCGGTCTAGAGGATCTTTGC-3'), yielding plasmid pKB39 (PHB2-STOP). To destroy TetA transporter activity, Asp-469 of Phb2p-TetA (corresponding to Asp-287 of native TetA from pBR322) was mutated to Ala with the mutagenic oligonucleotide TetA-DA (5'-CCCAGCGCGGCGGCCGCCAT-3'). Mutations were confirmed by nucleotide sequence analysis.
For construction of a hemagglutinin (HA)-tagged version of PHB2-tetA, a unique BamHI site at the 3' end of tetA in plasmid pKB38 was created by oligonucleotide-directed mutagenesis using the mutagenic primer TetA-BamHI (5'-CGAGGTGGCCCGGATCCATGCACCGCG-3') to yield plasmid pKB40. Sequences encoding three tandem repeats of the influenza virus HA epitope were liberated from plasmid pGTEP1 (36) by digestion with NotI, fragment ends were filled with Klenow fragment, and the fragment was ligated into plasmid pKB40, which had been digested with BamHI and treated with Klenow fragment and calf alkaline phosphatase, yielding plasmid pKB41. Full-length wild-type versions of PHB1 and PHB2 were cloned following PCR amplification from genomic DNA of strain MYY290. For PHB1, primers PHB1-U2 (5'-GGGGATTCTTCAGGGAAAGGGAGTTTGACGAT-3') and PHB1-L2 (5'-GGGGATTCAGCAGAAGGAGGGCAAGAAGACAA-3') were used to amplify the coding region along with approximately 150 bp of 5' noncoding sequence and 400 bp of 3' noncoding sequence, flanked by BamHI sites. For PHB2, the coding region and approximately 570 bp of 5' noncoding sequence and 250 bp of 3' noncoding sequence were amplified with flanking BamHI sites by using primers PHB2-U2 (5'-GGGGATCCAACAAGGAAGGTTTGGAGTGTAGC-3') and PHB2-L2 (5'-GGGGATCCGAATATACCACGCAAGCCGAATGT-3'). A centromere-based plasmid containing the HA-tagged PHB2-tetA suppressor was constructed by digestion of pKB41 with NheI and PvuII, which recognize unique sites 5' and 3' of PHB2-tetA-HA in YEp13 sequences, and ligation with plasmid pRS315 (36a), which was digested with XbaI and SmaI. Expression of the centromere vector-based Phb2p-TetA-HA at reduced levels relative to the 2µm-based suppressing clone was verified by Western analysis using anti-HA antibodies. A plasmid for high-level expression of both PHB1 and PHB2 was generated by subcloning the BamHI fragment containing PHB2 into YEp13::PHB1. High-level expression of Phb1p and Phb2p from cells harboring the 2µm-PHB1-PHB2 plasmid was verified by Western analysis.Preparation of antibodies. To generate antibodies specific for Phb1p and Phb2p, vectors were constructed to express fusion proteins LacZ-Phb1p and LacZ-Phb2p, respectively. For plasmid pKB48, the 3' portion of PHB1 (from the unique ClaI site to the unique PstI site 3' of the gene) was introduced into plasmid pTRB1 (10), which was digested with BamHI, treated with Klenow fragment, and then digested with PstI. Plasmid pKB49 consisted of the 3' portion of PHB2 (from the unique ApaLI site) introduced into the BamHI site of plasmid pTRB1 following modification with Klenow fragment. The fusion proteins were expressed in bacterial cells, purified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and electroelution, and injected into rabbits (18). The LacZ-Phb1p fusion protein contained the C-terminal 279 amino acids of Phb1p, and the LacZ-Phb2p protein contained the C-terminal 265 amino acids of Phb2p. At the antibody concentrations used for immunoblot detection of Phb1p and Phb2p, antiserum cross-reactivity was not observed.
Western analysis, indirect immunofluorescence, and fluorescence
microscopy.
Whole-cell protein extracts were prepared by glass
bead lysis in radioimmunoprecipitation assay buffer (150 mM NaCl, 1%
Nonidet P-40, 0.5% deoxycholate, 0.1% SDS, 50 mM Tris-HCl [pH 8.0])
(18) with the addition of 1 mM phenylmethylsulfonyl
fluoride. SDS-PAGE and immunoblotting were performed as described
previously (39, 42). Protein concentrations were determined
by bicinchoninic acid assays (Pierce, Rockford, Ill.). Methods for
indirect immunofluorescence, staining with
2-(4-dimethylaminostryl)-1-methylpyridinium iodide (DASPMI) and DAPI,
and fluorescence microscopy were as described previously
(25). Additional antisera were specific for Tom70p (31), OM45 (44), F1
(22), Mdm10p (39), or Mas2p (22), and
have been described previously. The monoclonal antibody 12CA5 (Berkeley
Antibody Co., Richmond, Calif.) was used for immunodetection of the HA
epitope (15).
Subcellular fractionation. Yeast cells were grown in semisynthetic lactate medium (12) at 30°C, converted to spheroplasts, homogenized, and subjected to differential centrifugation to isolate subcellular fractions as previously described (35, 43). Purified inner and outer mitochondrial membranes were obtained following osmotic shock, sonication, and sucrose density gradient centrifugation (12). Mitoplasts, possessing largely intact mitochondrial inner membranes and osmotically disrupted outer membranes, were prepared as described previously (12).
RESULTS
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Identification of a high-copy-number suppressor of mdm12. Cells lacking Mdm12p are viable at 23°C but fail to grow at 37°C (6). To investigate further the cellular role of Mdm12p, a screen for high-copy-number plasmid-borne suppressors of the temperature-sensitive growth defect of mdm12-null cells was initiated (see Materials and Methods). A single clone which conferred improved growth of mdm12-null cells at elevated temperature was identified (Fig. 1A). Mutant cells harboring the suppressing clone also showed a partial restoration of normal mitochondrial morphology (Fig. 1B). In unsuppressed mdm12-null cells, the wild-type mitochondrial reticulum appeared to have collapsed into one or two giant spherical organelles which were largely defective for entry into buds even at the permissive temperature, 23°C (6) (Fig. 1B). In mdm12-null cells harboring the suppressor plasmid, mitochondria appeared smaller and more fragmented than in unsuppressed mutant cells and also sometimes exhibited elongated morphologies (Fig. 1B). In addition, while mitochondria were typically restricted to the mother portions of unsuppressed mdm12-null cells (6), mitochondria were often distributed in both mother and bud portions of suppressed cells. Cells harboring the mdm10-null mutation, which exhibit phenotypes extremely similar to those of mdm12-null cells, were also transformed with the suppressing plasmid. The mdm10-null transformants also exhibited partial suppression of aberrant mitochondrial morphology and distribution (Fig. 1B) as well as of temperature-sensitive growth (data not shown). Cells with an mmm1-null mutation, which are phenotypically similar to mdm10-null and mdm12-null cells (9), also showed improved growth and mitochondrial morphology when harboring the suppressing plasmid (data not shown). Mitochondrial morphology in otherwise wild-type (MDM10 MDM12) cells did not appear altered by the suppressing plasmid (Fig. 1B). The temperature sensitivity of other mutants functionally unrelated to the mdm10 and mdm12 mutants, namely the mas1 (45), mas3 (37), and mas5 (2) mutants, and of the pep12/vps6- (5) and vps18-null (32) mutants was not suppressed by transformation of these strains with the plasmid, indicating that suppressing activity was restricted to a limited class of temperature-sensitive mutants.
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Suppression is conferred by a prohibitin homolog. An analysis of sequences in the suppressing plasmid revealed that suppression was conferred by a single S. cerevisiae open reading frame. The cloned S. cerevisiae sequences corresponded to a previously uncharacterized open reading frame, recently designated PHB2 (Saccharomyces Genome Database, Stanford, Calif.) (11). PHB2 encodes a predicted protein product of 315 amino acids and 34.1 kDa which is homologous to a second S. cerevisiae protein, prohibitin, the product of PHB1. Prohibitin (Phb1p) and the prohibitin-related protein encoded by PHB2 (Phb2p) in yeast display 52% amino acid identity and 63% similarity, with conserved residues distributed throughout the lengths of the proteins (data not shown). The proteins show the greatest divergence at their N and C termini. Phb1p and Phb2p are homologous to prohibitins and prohibitin-related proteins previously identified in a wide range of organisms (11, 14, 28, 29, 38, 41). In mammalian cells, prohibitin has been implicated as a regulator of cell growth, and this protein may function in diverse cellular processes including development and tumor suppression (24, 29).
DNA sequence analysis of the suppressing plasmid revealed that genomic library construction had fortuitously generated an in-frame gene fusion between PHB2 and the bacterial tetracycline resistance gene, tetA, carried by the pBR322-derived vector YEp13 (Fig. 2). The 5' PHB2 portion of the PHB2-tetA gene fusion encoded all but the C-terminal 34 amino acids of the 315-amino-acid Phb2p; the 3' tetA sequences encoded the last 297 amino acids of the 396-amino-acid product of the bacterial tetracycline resistance gene (30). The predicted PHB2-tetA product is a 578-amino-acid protein of 62.3 kDa. To determine whether tetA sequences were required for PHB2-tetA-mediated suppression of mdm12, an in-frame stop codon between PHB2 and tetA sequences in the suppressing clone was generated by site-directed mutagenesis. The resulting clone no longer suppressed mdm12 defects (PHB2-STOP; Fig. 1A). Furthermore, the high-level expression of a wild-type copy of PHB2 also did not confer suppression of mutant growth (Fig. 1A) or mitochondrial morphology, nor did expression of these constructs appear to affect mitochondrial morphology in wild-type (MDM12) cells (data not shown). Finally, suppression could not be mediated by lower-level expression of PHB2-tetA from a centromere-based plasmid, as shown by the failure of such a construct to improve growth or mitochondrial morphology of mdm12-null transformants (data not shown).
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Characterization of phb1 and phb2
mutants.
To investigate prohibitin function, null alleles of both
PHB1 and PHB2 were generated by integrative
transformation and gene replacement. Either singly or together,
phb1-null and phb2-null mutations did not confer
any apparent phenotype on otherwise wild-type cells. Growth of
phb1-null and/or phb2-null mutant cells on
various carbon sources (ethanol, galactose, glycerol, glucose, and
raffinose) and at different temperatures (14, 23, 30, and 37°C)
appeared indistinguishable from that of the parental (PHB1
PHB2) strain. Mitochondrial morphology and distribution,
visualized by DASPMI staining and fluorescence microscopy, appeared
unaltered by the loss of Phb1p and/or Phb2p (data not shown).
Homozygous phb1/phb1 phb2/phb2 diploids remained competent
for sporulation and did not exhibit reduced spore viability. Finally,
phb1-null and phb2-null mutations did not
increase the frequency of loss of respiratory capacity associated with
deletion of the mitochondrial genome (generation of
[rho] cells) compared to that for the
parental strain (data not shown).
]) were examined. These cells did not
exhibit any apparent difference from otherwise isogenic
PHB1 PHB2 [rho] cells in growth
on fermentable carbon sources at 14, 23, 30, or 37°C. However,
indirect immunofluorescence microscopy revealed that the phb1
phb2 [rho] cells displayed substantial
alterations of mitochondrial morphology and distribution (Fig.
3). Wild-type (PHB1 PHB2)
[rho] cells contained mitochondria with
typical tubular morphology and peripheral distribution, and most cells
(77%; n = 207) contained at least one snake-like
mitochondrion that spanned at least one-fourth of the length of the
cell. In contrast, most mitochondria in phb1-null phb2-null [rho] cells lacked the
normal reticular morphology and organized mitochondrial distribution,
and only a minority of cells (20%; n = 303) exhibited even one tubular mitochondrion that was at least one-fourth the length
of the cell. In single-mutant phb1-null or
phb2-null [rho] cells,
mitochondria also appeared fragmented and disorganized compared to
those in PHB1 PHB2 [rho] cells,
similar to what was found for the double-null mutant (data not shown).
These results indicate that Phb1p and Phb2p are important for normal
mitochondrial morphology and distribution specifically in cells which
lack a functional mitochondrial genome.
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cells was related to the absence of the mitochondrial genome or to
defective mitochondrial function, phb1 phb2 mutant cells which were respiration deficient due to a mutation in the
cox4 gene were examined by indirect immunofluorescence
microscopy. These cells exhibited mitochondrial morphology
indistinguishable from that of PHB1 PHB2 cox4 cells (data
not shown). Mitochondrial morphology was also examined in PHB1
PHB2 and phb1 phb2 [rho+]
cells following inhibition of mitochondrial protein synthesis with
either chloramphenicol or erythromycin. These treatments did not affect
mitochondrial morphology in either PHB1 PHB2 or phb1
phb2 cells (data not shown). These results suggest that
mitochondrial DNA (or associated proteins) may play a structural role
which is revealed in the absence of Phb1p and Phb2p.
At the time of this work, the function of PHB1 and
PHB2 in budding yeast had not been previously described. In
unpublished data, loss of function of the PHB1-encoded
prohibitin in yeast was reported to increase replicative life span,
defined as the number of times a single yeast cell produces daughter
buds before ceasing to divide (3). The effect of
phb1-null and phb2-null mutations on replicative
life span for both cells containing a wild-type mitochondrial genome
([rho+]) and cells with the mitochondrial DNA
deleted ([rho]) was examined. In these
cells, loss of Phb1p and/or Phb2p function did not increase the
replicative life span. In fact, loss of Phb1p and/or Phb2p function
reduced replicative life span, as measured by either mean or maximum
life span (Table 2). For
[rho+] cells, either the phb1-null
or the phb2-null mutation significantly reduced mean
replicative life span (P < 0.01). The double
phb1-null phb2-null mutant also showed a
similarly reduced life span relative to wild-type cells
(P < 0.001). The single phb1-null mutation had a more severe effect on replicative life span than the
phb2-null or the double phb1-null
phb2-null mutation (P < 0.001). In
[rho] cells, both the phb2-null
and the phb1-null phb2-null mutants showed
reduced life spans compared to wild-type
[rho] cells (P < 0.01), but
the [rho] phb1-null mutant did
not. These data are substantially in agreement with another recent
analysis of Phb1p and Phb2p function in budding yeast (11),
although we did not observe an enhanced effect on replicative life span
in the double mutant.
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Phb1p and Phb2p are proteins of the mitochondrial inner membrane. Phb1p and Phb2p homologs have been localized to mitochondria in higher animal and plant cells (20, 38) but have not been previously localized in yeast. Further, the proteins' submitochondrial distribution has not been firmly established in any cell type. To address the site of activity of Phb1p and Phb2p, polyclonal antisera recognizing each of the proteins were generated and used for immunoblot analysis of different subcellular fractions. Both Phb1p and Phb2p were concentrated in fractions enriched for mitochondrial proteins (Fig. 4A). In subfractionation of purified mitochondrial membranes, both Phb1p and Phb2p behaved as components of the mitochondrial inner membrane (Fig. 4B). Additionally, neither Phb1p nor Phb2p was extracted from the membranes with alkali carbonate treatment (see below), indicating that both polypeptides behaved as integral membrane proteins. Identical subcellular and submitochondrial distributions of Phb1p and Phb2p in Phb2p-TetA-suppressed mdm12 cells (Fig. 4) and wild-type cells were observed (data not shown).
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Phb1p and Phb2p are interdependent. The characterization of Phb1p and Phb2p localization revealed that the presence of these two proteins in the cell was interdependent. In phb1-null cells, Phb2p was no longer detectable by Western analysis, and Phb1p was similarly reduced to undetectable levels in phb2-null cells (Fig. 5A). Phb1p levels in phb2-null cells were restored by expression of PHB2 from a centromere-based plasmid, and Phb2p was likewise restored in phb1-null cells by PHB1 expression (Fig. 5A). To determine whether the interdependence of Phb1p and Phb2p occurred at the level of gene expression, levels of PHB1 and PHB2 mRNA were examined by Northern analysis (Fig. 5B). In phb2-null cells, PHB1 RNA levels were slightly reduced, and PHB2 RNA levels were slightly decreased in phb1-null cells (Fig. 5B). These modest reductions in RNA levels seemed unlikely to account for the corresponding absence of detectable polypeptide subunits, and the interdependence of Phb1p and Phb2p therefore appeared to be largely posttranscriptional.
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Genetic interaction of prohibitin mutations with mdm mutations. Because PHB2 was identified in a genetic screen for high-copy-number suppressors of mdm12 loss of function, we examined whether the phb2 or phb1 mutation might confer a synthetic phenotype in combination with the mdm12 mutation. A genetic analysis of progeny of a cross of single-mutant phb2-null and mdm12-null strains indicated that the double-mutant phb2 mdm12 strain was inviable (Table 3). A cross of phb1-null and mdm12-null mutant strains yielded similar results, indicating that the phb1 mdm12 double mutant was either inviable or marginally viable, forming only extremely small colonies. Mutations in either PHB1 or PHB2 also appeared deleterious in combination with the loss of function of either Mdm10p or Mmm1p, two other mitochondrial outer membrane proteins which, like Mdm12p, are required for normal mitochondrial inheritance and morphology (9, 39) (Table 3 and data not shown). Finally, phb1-null phb2-null mdm10-null and phb1-null phb2-null mdm12-null triple mutants were generated and also were inviable or extremely sick (data not shown). Viable phb2 mdm12 cells could be obtained if the heterozygous diploid parent strain harbored a plasmid encoding wild-type PHB2; such cells were unable to lose the plasmid even on rich medium. The phb1- and phb2-null mutations did not produce synthetic phenotypes when combined with other mdm mutations in genes which encode factors not localized to mitochondria, specifically, different mutant alleles of mdm1 (16) or an mdm2 mutation (40). Additionally, a phb1-null or phb2-null mutation did not produce synthetic inviability when combined with the loss of OM45, a mitochondrial outer membrane protein which does not play a role in mitochondrial inheritance (44), or when combined with a mutation in TOM70, encoding an outer membrane component of the mitochondrial protein import apparatus (19) (data not shown).
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Synthetic lethality of the mdm12 phb2 double mutant is suppressed by the SOT1 mutation. Previously, a novel mutation, the SOT1 mutation, was shown to suppress the growth and mitochondrial morphology defects of mdm12, mmm1, and mdm10 mutant cells (6). To examine the effect of the SOT1 mutation on the synthetic lethality of mdm12 and phb2 mutations, the haploid progeny of a cross between mdm12 SOT1 and phb2 strains were examined by tetrad analysis. A majority (13 of 21 cells) of the triple-mutant mdm12 phb2 SOT1 cells were viable, indicating that the SOT1 mutation could at least partially suppress the synthetic lethality. In these cells, mitochondrial morphology and inheritance appeared indistinguishable from those in otherwise isogenic cells with wild-type PHB2 (Fig. 7). The plasmid-encoded PHB2-tetA suppressor was also tested for its ability to abrogate the synthetic lethality of phb2 mdm12 cells. Double-mutant phb2 mdm12 cells could be obtained at a low frequency (2 of 7 cells) following sporulation if they harbored the PHB2-tetA suppressor, but these haploid progeny appeared extremely sick indicating, at most, very limited suppression. A similar experiment indicated that the PHB2-tetA suppressor likewise failed to rescue the phb1 mdm12 double mutant (data not shown). Finally, high-copy-number expression of PHB1 could not substitute for that of PHB2 in the phb2 mdm12 double mutant, nor could high-copy-number PHB2 rescue the phb1 mdm12 double mutant.
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DISCUSSION
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While investigating the role of Mdm12p in mitochondrial inheritance, we identified a genetic interaction between a class of inheritance components of the mitochondrial outer membrane and proteins belonging to the prohibitin family. This interaction was uncovered through the characterization of a fusion protein that partially suppresses an mdm12-null mutation when expressed from a high-copy-number plasmid. The fusion protein consists of the Phb2p protein fused to the bacterial tetracycline resistance protein, TetA, this fusion having arisen fortuitously through the construction of a yeast genomic library in a yeast-E. coli shuttle vector. The Phb2p-TetA fusion protein altered the mitochondrial morphology of mdm12 and mdm10 mutant cells but did not appear to affect mitochondria in otherwise wild-type cells. Although the mechanism of suppression is unknown, the Phb2p-TetA fusion protein could not bypass the cellular requirement for Phb1p and Phb2p in mdm10 and mdm12 mutant cells. Also, the fusion protein localized to the same submitochondrial site as the authentic Phb1p and Phb2p. One model of suppression consistent with these observations is that the suppressing protein activates or augments some normal activity of Phb1p and Phb2p. The effect of the fusion protein might be to cause changes in the physical properties of the mitochondrial membranes or to alter the interaction of the membranes with specific peripheral proteins, leading to more tubular mitochondria and a more dispersed mitochondrial distribution.
Prohibitin was originally identified in mammalian cells as the product of a tumor suppressor gene (24, 29). In the yeast S. cerevisiae, prohibitin was proposed to play a role in cellular senescence (3). Contrary to earlier reports that a mutation in Phb1p increased replicative life span (3), Coates et al. (11) have recently presented data indicating that loss of either Phb1p or Phb2p decreased replicative life span. Our results demonstrating a modest reduction in replicative life span in both phb1 and phb2 mutants are consistent with the latter study.
Prohibitin has now been identified in a wide range of species and localized to mitochondria in animal and plant cells (11, 20, 38). Our results have extended this localization to demonstrate that both Phb1p and Phb2p are integral proteins of the mitochondrial inner membrane in S. cerevisiae. Although we were not able to demonstrate the direct binding of Phb1p and Phb2p (as was shown by coimmunoprecipitation for prohibitin and prohibitin-related protein in animal cells [11]), our finding that the stability or maintenance of Phb1p depends on Phb2p (and vice versa) supports the idea that the two polypeptides function as a complex. In addition, each of the proteins appears to play distinct molecular roles, since overexpression of either species led to significant steady-state levels of the respective protein but could not complement the loss of the homolog.
The specific cellular role or activity of prohibitin or
prohibitin-related protein is unknown, but our identification of
genetic interactions between PHB genes and a subset of
mitochondrial inheritance components, along with the altered
mitochondrial morphology of [rho]
phb1 and phb2 mutants, suggests that Phb1p and
Phb2p function in the regulation of mitochondrial morphology and
distribution. Loss of Mdm12p, Mdm10p, or Mmm1p results in giant
mitochondria and severe defects in mitochondrial distribution, yet
daughter buds occasionally receive small mitochondria, and cultures
grow, although slowly, at 23°C (6, 9, 39). The
"backup" mechanism that facilitates this inefficient mitochondrial
inheritance may depend on the function of Phb1p and Phb2p, accounting
for the inviability of the double-mutant strains. The molecular
activity of these proteins may be further clarified by the
identification of additional interacting components.
ACKNOWLEDGMENTS
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We thank Jennifer Whistler (University of California, Berkeley) for advice on PCR-mediated gene disruption and Randy Hampton (University of California, San Diego [UCSD]) for the gift of plasmid pGTEP1. We thank Immo Scheffler (UCSD) for the sdh2-null mutant, Peter Thorsness (University of Wyoming, Laramie) for the yme2 mutant, Scott Emr (UCSD) for pep12 and vps18 mutants, and members of the Emr laboratory for their helpful advice. We are grateful to Randy Hampton, Anne-Laure Danquigny-Genestier, Harold Fisk, and Kelly Shepard for valuable comments on the manuscript.
This work was supported by grant GM44614 from the National Institutes of Health. K.H.B. was supported by NIH grant GM16173 and a fellowship from the American Heart Association.
FOOTNOTES
* Corresponding author. Mailing address: University of California, San Diego, Department of Biology, 0347, La Jolla, CA 92093-0347. Phone: (619) 534-4769. Fax: (619) 534-4403. E-mail: myaffe{at}ucsd.edu.
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