Department of Biology, University of California, San Diego, La Jolla, California 92093-0347
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Abstract |
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The mdm17 mutation causes temperature-dependent defects in mitochondrial inheritance, mitochondrial morphology, and the maintenance of mitochondrial DNA in the yeast Saccharomyces cerevisiae. Defects in mitochondrial transmission to daughter buds and changes in mitochondrial morphology were apparent within 30 min after shifting cells to 37°C, while loss of the mitochondrial genome occurred after 4-24 h at the elevated temperature. The mdm17 lesion mapped to MGM1, a gene encoding a dynamin-like GTPase previously implicated in mitochondrial genome maintenance, and the cloned MGM1 gene complements all of the mdm17 mutant phenotypes. Cells with an mgm1-null mutation displayed aberrant mitochondrial inheritance and morphology. A version of mgm1 mutated in a conserved residue in the putative GTP-binding site was unable to complement any of the mutant defects. It also caused aberrant mitochondrial distribution and morphology when expressed at high levels in cells that also contained a wild-type copy of the gene. Mgm1p was localized to the mitochondrial outer membrane and fractionated as a component of a high molecular weight complex. These results indicate that Mgm1p is a mitochondrial inheritance and morphology component that functions on the mitochondrial surface.
Key words: mitochondrial inheritance; mitochondrial morphology; yeast; dynamin; outer membrane ![]() |
Introduction |
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MITOCHONDRIA perform essential cellular functions yet cannot be synthesized de novo (Attardi
and Schatz, 1988). Instead, these organelles are
derived from preexisting mitochondria and specific cellular mechanisms act to ensure the faithful transmission of mitochondria to progeny. The molecular details of the inheritance process are largely unknown, but a growing list
of key protein components is emerging from analysis of
conditional mutants of the budding yeast Saccharomyces
cerevisiae that are defective for mitochondrial distribution
and morphology mutant (mdm)1 (Berger and Yaffe, 1996
;
Hermann and Shaw, 1998
).
Thus far, most characterized MDM gene products have
fallen into two basic categories. The first consists of cytoplasmic or cytoskeletal components such as: Mdm1p, an
intermediate filament-like protein required for both mitochondrial and nuclear inheritance (McConnell and Yaffe,
1992, 1993
); and Mdm20p, a protein of unknown function
that is important for the structure of the actin cytoskeleton (Hermann et al., 1997
). The second category consists of
proteins of the mitochondrial outer membrane: Mdm10p,
Mdm12p, and Mmm1p. These proteins play roles both in
maintaining normal mitochondrial morphology and mediating mitochondrial inheritance (Burgess et al., 1994
; Sogo
and Yaffe, 1994
; Berger et al., 1997
). Genetic analysis suggests that a number of additional inheritance components remain to be identified. This report describes the isolation
and characterization of a new mutant, mdm17, which displays conditional defects in mitochondrial inheritance, mitochondrial morphology, and maintenance of the mitochondrial genome.
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Materials and Methods |
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Strains and Genetic Techniques
Yeast strains used in this study are listed in Table I. Culture and genetic
analysis of yeast were performed by standard procedures (Rose et al.,
1990). Semisynthetic lactate medium was prepared as described (Daum
et al., 1982
). Plasmid DNA was prepared from Escherichia coli strain
DH5
.
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Isolation of mdm17
The mdm17 mutant was isolated in a screen for novel alleles of another mitochondrial inheritance gene, mdm13. Strain MYY900, containing the mdm13 lesion, was crossed to the SL collection of temperature-sensitive strains which were derived from strain MYY290, and the resulting diploids were tested for growth at 37°C on yeast extract/peptone/glucose (YPD) medium. Diploids that failed to grow were sporulated and the meiotic progeny were analyzed by backcrossing and allelism tests. The recovered mdm17 spores were backcrossed three times to the wild-type parental strain, and the temperature-sensitive growth and mitochondrial distribution defects were shown to cosegregate in a 2:2 pattern. The backcrossed strain no longer displayed nonallelic noncomplementation with mdm13 and that property apparently depended on the presence of a third, unlinked mutation in the original mdm17 mutant strain. The MDM13 gene has yet to be isolated.
Gene Cloning and Mapping
mdm17 cells were transformed with a yeast genomic DNA library in the centromere-based LEU2 vector p366 (M. Hoekstra, ICOS Inc.). 10,000 Leu+ transformants were screened for complementation of the temperature-sensitive growth defect by replica plating to YPD medium and to yeast extract/peptone/glycerol (YPG) medium at 37°C. Four different complementing clones were isolated, and restriction analysis demonstrated that they contained overlapping yeast DNA inserts. The identity of the inserts was determined by nucleotide sequence analysis of the ends of the inserts and comparison to sequences in the Saccharomyces Genome Database.
A full-length version of the MGM1 gene was synthesized using PCR.
The high-fidelity polymerase, Pfu (Stratagene), was used with primers
5'-CTCTCTAGAGTTCTTCTGCTCGCTAATGGTAAATG-3' and 5'-CTCCTCGAGGCAAGAAGATGAGTTGGATGAAGG-3' to amplify
the MGM1 open reading frame together with ~500 bp of flanking DNA
sequences. The PCR product was phosphorylated with T4 DNA kinase
and ligated into vector pRS313 (Sikorski and Hieter, 1989) that had been
digested with SmaI and EcoRV and treated with calf intestinal phosphatase. The resulting plasmid was designated pRS313-MGM1.
For integrative mapping, a 2.6-kb HindIII fragment adjacent to the
MGM1 locus was subcloned into the YIp5 vector (Struhl et al., 1979). The
plasmid was linearized by digestion with HpaI and the DNA was transformed into strain MYY290. Ura+ transformants were crossed to strain
MYY971 containing the mdm17 mutation. The meiotic progeny of the resulting diploid consisted of 19 parental ditype and 1 tetratype, mapping
the mdm17 lesion to within 2.5 cM of the MGM1 locus.
Gene Replacement
A gene replacement cassette was created by PCR as described by Baudin
et al. (1993). The oligonucleotides 5'-ATGAGTAATTCTACTTCATTAAGGGCCATCCCAAGAGTGGATTGTACTGAGAGTGCACC-3' and 5'-TCATAAATTTTTGGAGACGCCCTTGTAGCTTTTCTTGAAAGTGCGGTATTTCACACCGC-3' were used as PCR primers, and
the LEU2 gene in plasmid pRS305 (Sikorski and Hieter, 1989
) served as
the template. The resulting PCR product was used to transform the diploid strain MYY298. Leu+ transformants were screened by PCR to identify strains containing a replacement of one of two copies of MGM1 with
LEU2. The heterozygous diploid strain was sporulated to obtain haploid
segregants with the mgm1::LEU2 (mgm1-null) mutation.
Phenotypic Analysis
For phenotypic analysis, cultures were first grown overnight in YPD-liquid medium at 23°C and diluted to 0.5 A600/ml before incubation at 37°C for varying times. To evaluate respiration competence, cells were plated on YPD-agar medium, cultured at 23°C, and replica-plated to YPG medium. Colonies that failed to grow on YPG at 23°C were scored as having lost respiratory function. At least 700 colonies were scored for each time point.
To characterize mitochondrial distribution and morphology in living
cells, mitochondria were stained with the membrane potential-sensitive dye 2-(4-dimethylaminostryl)-1-methylpyridium iodide (DASPMI), and
cells were examined by fluorescence microscopy (Yaffe, 1995). Indirect
immunofluorescence microscopy was performed on fixed yeast cells as described (McConnell et al., 1990
).
Antibody Preparation
Antibodies were raised against a peptide, CGGYKGVSKNL, of which the last eight residues correspond to the extreme COOH terminus of Mgm1p. Peptide synthesis, coupling of peptide to keyhole-limpet hemocyanin, and immunization of rabbits was carried out by Research Genetics, Inc. The antibodies were purified on an affinity column prepared by coupling the peptide antigen to Affigel 10 (Bio-Rad Laboratories).
Subcellular Fractionation and Analysis
Cells were grown in semisynthetic lactate medium (Daum et al., 1982),
converted to spheroplasts, homogenized, and fractionated by differential
centrifugation as previously described (Schauer et al., 1985
; Yaffe, 1991
).
Mitochondrial subfractions were isolated as described (Daum et al., 1982
).
Mitochondria were subjected to trypsin treatment or extraction with sodium carbonate or sodium chloride as previously described (Sogo and
Yaffe, 1994
).
Pulse-Chase Analysis
Wild-type cells were grown in minimal medium lacking methionine and
cysteine to an A600 = 0.5-0.8. Cells were harvested, resuspended in a solution of 40 mM KPi, pH 6.0, 1% glucose, and labeled for 5 min at 30°C by
addition of 0.1 mCi [35S]-Translabel (ICN) per ml. The chase was initiated
by adding unlabeled methionine and cysteine to 20 µM and, in some cases, cycloheximide to 1 µg/ml. Cells were collected and processed for immunoprecipitation as previously described (Yaffe, 1991).
Gel Filtration Analysis
Gel filtration analysis of Mgm1p was similar to that described by Rapaport et al. (1998). Briefly, 5 mg of mitochondria was pelleted and resuspended in 1 ml buffer A (150 mM potassium acetate, 1% Triton X-100,
4 mM magnesium acetate, 0.5 mM EDTA, 0.5 mM PMSF, and 30 mM
Tris-HCl, pH 7.4). The resuspended mitochondria were sonicated for 15 min in an ice-water bath, and the mixture was centrifuged for 15 min at
90,000 g. The supernatant was loaded onto a 100-ml Sephacryl-300 column
(Pharmacia Biotech Inc.) that had been equilibrated with buffer A and
fractionated with an FPLC system (Pharmacia Biotech Inc.) at a flow rate
of 0.5 ml/min. 1-ml fractions were collected, proteins were precipitated
with TCA, and analyzed by SDS-PAGE and immunoblotting. Protein
bands on the immunoblots were scanned and quantified using NIH Image software.
Characterization of the mdm17 Mutation
The mdm17 gene was amplified by PCR from genomic DNA isolated
from mutant cells using Taq polymerase. DNA products were cloned into
the TA vector (Invitrogen Corp.) and sequenced by the dideoxy method
(Sanger et al., 1977). Additionally, portions of mdm17 were amplified
from genomic DNA using Pfu polymerase (Stratagene) and sequenced directly using the CircumVent Kit (New England Biolabs, Inc.).
In Vitro Mutagenesis of MGM1
Site-directed mutagenesis to create point mutations in MGM1 was carried
out using the "PCR SOEing" technique (Horton, 1995). The PCR products containing the mutations were used to replace corresponding wild-type sequences in pRS313-MGM1 via standard cloning techniques. DNA
sequence analysis confirmed the presence of the novel mutations and the
absence of any PCR-generated errors. Plasmids containing the mutant
genes were transformed into the heterozygous (MGM1/mgm1::LEU2)
diploid strain MYY973. Transformants were sporulated, tetrads dissected,
and mgm1-null spores harboring each of the mutant constructs were identified and analyzed.
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Results |
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mdm17 Cells Display Conditional Defects in Mitochondrial Inheritance, Mitochondrial Morphology, and Maintenance of the Mitochondrial Genome
The mdm17 mutant was isolated in a screen for novel alleles of mdm13, an uncharacterized gene that is required for normal mitochondrial inheritance and morphology. This screen involved crossing a haploid strain harboring the temperature-sensitive mdm13 lesion to a collection of temperature-sensitive strains and analyzing growth of the resulting diploids at the nonpermissive temperature (37°C). One diploid strain displayed temperature-sensitive growth, and analysis of its meiotic progeny indicated that one of two mutations conferring temperature-sensitive growth mapped to a genetic locus unlinked to mdm13. Therefore, rather than being an allele of mdm13, the new mutation defined a distinct gene. Analysis of cells harboring only the new mutation (after its genetic isolation from mdm13) by staining with the mitochondria-specific, vital dye DASPMI and fluorescence microscopy revealed defects in mitochondrial distribution and morphology after incubation at the nonpermissive temperature (Fig. 1). Complementation and allelism tests indicated that the new mutation was unlinked to previously characterized mdm mutations. Genetic analysis further demonstrated that the defects in growth at 37°C, mitochondrial distribution, and mitochondrial morphology were caused by a single nuclear mutation. This novel mutation was designated mdm17.
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To quantify the effect of mdm17 on mitochondrial distribution and morphology, cells were stained with DASPMI and examined by fluorescence microscopy. In wild-type cells incubated at either 23°C or 37°C, mitochondria appeared as extended, tubular structures, distributed throughout the cytoplasm of both mother and bud portions of the cell (Fig. 1, A and B). Mitochondria in cells harboring the mdm17 mutation appeared very similar to wild-type at 23°C (Fig. 1 A) but displayed dramatic changes in mitochondrial distribution and morphology after incubation at 37°C (Fig. 1 B). In particular, mitochondrial aggregation and empty daughter buds were apparent in many cells. These alterations in mitochondrial distribution and morphology were confirmed by indirect immunofluorescence microscopy (data not shown).
To examine the development of mutant phenotypes at 37°C, populations of mutant cells were analyzed by indirect immunofluorescence microscopy at various times after shifting to the nonpermissive temperature (Fig. 2, A and B). After 1 h at 37°C, most cells (87%) contained aggregated mitochondria, and a significant fraction of cells (18%) possessed buds devoid of mitochondria. Some of the cells with empty buds still possessed mitochondria with normal tubular morphology (data not shown). By 2 h, 90% of cells displayed aggregated mitochondria and a majority (56%) possessed empty buds. These results demonstrate that the mdm17 mutation rapidly causes defects in mitochondrial distribution and morphology after shifting cells to the nonpermissive temperature.
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The fraction of cells in the mutant population that showed mitochondrial staining with the potential-dependent dye DASPMI decreased during incubation at the nonpermissive temperature, suggesting that the cells were becoming respiration deficient. To analyze this phenotype, mdm17 cells were incubated at 37°C for various times, plated on glucose (YPD) medium, and cultured at 23°C. Colonies were tested for respiratory competence by replica plating onto glycerol (YPG) medium. At early times, almost all cells were respiration competent (Fig. 2 C). Significant numbers of respiration-deficient cells began appearing in the population only after 4 h at 37°C. After 24 h, >95% of the cells were respiration deficient (Fig. 2 C). When cells from the 16-h time point were stained with the DNA-binding dye 4,6-diamino-2-phenylindole and examined by fluorescence microscopy, mitochondrial DNA staining was no longer evident in a majority of cells (data not shown), indicating that the respiration deficiency reflected a loss of mitochondrial DNA.
mdm17 Is Allelic to MGM1, a Gene Implicated in Maintenance of the Mitochondrial Genome
To identify the molecular basis for the mdm17 phenotypes, the wild-type MDM17 gene was cloned by complementation of the temperature-sensitive growth defect of
mdm17 mutant cells. From 10,000 transformants, four
strains harboring complementing plasmids were isolated.
Restriction mapping of the yeast genomic DNA inserts in
these plasmids indicated that all contained overlapping
clones. DNA sequence analysis and comparison with sequences in the Saccharomyces Genome Database indicated that the genomic inserts in the plasmids corresponded to a region of chromosome XV. Deletion analysis
of the plasmids localized complementing activity to a single open reading frame corresponding to the previously
identified MGM1 gene (Jones and Fangman, 1992). Transformation of mdm17 cells with a centromere-based plasmid containing only a single copy of MGM1 complemented all of the mutant phenotypes. Finally, the mdm17
mutation was localized to the MGM1 locus by integrative
transformation and mapping (described in Materials and
Methods). These results demonstrate that mdm17 is a mutant allele of MGM1.
Previous studies reported that mgm1 mutant cells
readily lost mitochondrial DNA and contained aggregated
mitochondria but showed normal mitochondrial distribution to buds (Guan et al., 1993). This latter property differs
from the phenotype of the mdm17 mutant, where a large
fraction of cells exhibited buds devoid of mitochondria.
Accordingly, the phenotype of a null mutant in mgm1 generated in the same strain background as the mdm17 mutant was examined. Indirect immunofluorescence microscopy revealed many mgm1-null cells with buds devoid of
mitochondria, even in cells cultured at 23°C (Fig. 3). As
previously described (Guan et al., 1993
), mitochondria in
the mgm1-null strain were extensively aggregated.
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Mgm1p Is Localized to the Mitochondrial Outer Membrane
Previously reported phenotypes of the mgm1 mutant, together with the resemblance of the protein's predicted
NH2 terminus to a mitochondrial targeting sequence, suggested that Mgm1p is a mitochondrial protein (Guan et al.,
1993). However, direct evidence of the protein's subcellular location was lacking. To determine the functional location of Mgm1p, antibodies were generated that were specific for a peptide corresponding to the extreme COOH terminus. These antibodies were used to detect the protein
in subcellular fractions. Immunoblotting of total cellular
extracts indicated that the affinity-purified antibodies recognized two polypeptides of ~100 and 90 kD (Fig. 4 A).
Neither of these species was apparent in mgm1-null cells
(Fig. 4 A), indicating that both polypeptides are products
of the MGM1 gene. Furthermore, both species displayed
substantially increased levels in cells harboring a multicopy (2 µ) plasmid encoding MGM1 (Fig. 4 A). These increased levels had no apparent effect on mitochondrial
distribution and morphology (data not shown).
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The exact relationship between the two Mgm1p species is unclear, but the 90-kD form appears to be derived from the 100-kD form by proteolysis. The relative amounts of the two species varied randomly in different experiments, and pulse-chase analysis in intact cells failed to reveal a biosynthetic precursor-product relationship (data not shown). However, prolonged incubation of subcellular fractions in vitro (even at 0°C in the presence of a cocktail of protease inhibitors) led to conversion of the larger species to the smaller form (data not shown). These results suggest that the 90-kD form is a product of the partial proteolytic degradation of the 100-kD species.
Earlier characterization of MGM1 failed to identify
which of five NH2-terminal methionine residues in the
predicted open reading frame actually constituted the
translational start of the protein (Jones and Fangman,
1992; Guan et al., 1993
). To resolve this uncertainty, in
vitro mutagenesis was used to generate versions of mgm1
in which individual methionine codons were converted to alanine codons. These mutant versions were tested for
their complementation of the phenotypes of mgm1-null
cells. No complementation was observed (and the protein
was not detected) when methionine-2 was changed to alanine (data not shown). Mutation of the remaining four methionines did not affect complementation of the mutant
defects, and both 100- and 90-kD forms of Mgm1p were
detected in cells harboring these genes. These results indicate that the authentic translational start residue is methionine-2 (referred to henceforth as residue 1 of the coding region).
The subcellular distribution of Mgm1p was characterized by immunoblotting of proteins extracted from subcellular fractions isolated from wild-type cells. Mgm1p was localized predominantly to a subcellular fraction enriched in mitochondrial proteins (Fig. 4 B). Analysis of purified submitochondrial fractions revealed that Mgm1p was associated with the mitochondrial outer membrane (Fig. 4 C). Furthermore, Mgm1p was susceptible to protease treatment of intact mitochondria under conditions where internal proteins, such as the matrix-localized Mas2p, were protected (Fig. 4 D).
To examine the nature of Mgm1p association with the
outer membrane, isolated mitochondria were extracted
with sodium chloride and with sodium carbonate. The latter treatment strips peripheral proteins but leaves integral
proteins embedded in the membrane (Fujiki et al., 1982).
Mgm1p was resistant to extraction with 1 M NaCl (Fig. 5),
indicating that the protein was tightly associated with the
membrane. After carbonate treatment, the 90-kD form of Mgm1p was recovered in the soluble fraction, while the
100-kD form remained associated with the membrane
(Fig. 5). This result indicates that the full-length Mgm1p is
an integral membrane protein. The behavior of the 90-kD
fragment suggests that Mgm1p is embedded in the membrane via the putative membrane-spanning domain in the NH2 terminus, since both Mgm1p species are recognized
by the antibody specific for the extreme COOH terminus.
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The state of Mgm1p in the mitochondrial outer membrane was further characterized by gel filtration analysis of mitochondrial proteins solubilized in 1% Triton X-100. About 60% of Mgm1p eluted in a peak corresponding to a molecular mass of ~400 kD, while the remainder of the protein eluted in a peak appropriate to the monomeric size (90-100 kD) (Fig. 6). The 90-kD (fragment) form of the protein eluted in a distinct peak with an apparent molecular mass of ~120 kD (data not shown). The elution behavior of Mgm1p was distinct from that of two other integral proteins of the mitochondrial outer membrane, Tom70p and OM45 (Fig. 6). These results suggest that Mgm1p is part of a high molecular mass complex either in a homo-oligomeric state or complexed with other proteins on the mitochondrial surface.
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Mgm1p Function Depends on the Conserved GTP-binding Domain
To explore further the function of Mgm1p, the identity of
the mdm17 mutation was determined. The mutant gene
contained a single change: a transition of G to A at nucleotide 880, resulting in a change of Glu294 to Lys. This lesion mapped near the conserved, tripartite GTP-binding motif, suggesting a role for this site in Mgm1p function. To
test the importance of the GTP-binding site, a mutant
form of MGM1 incorporating a change in a conserved residue, S224N, was created (the numbering of this residue is
based on the designation of the second methionine in
MGM1 as the NH2-terminal residue as described above).
The equivalent mutation in the H-ras-GTPase (S17N)
eliminates the protein's binding of GTP (Feig and Cooper, 1988), and a corresponding mutation in rat dynamin
(S45N) blocks the protein's function in endocytosis (Herskovits et al., 1993
). Immunoblot analysis of cellular extracts detected Mgm1p in mgm1-null cells harboring the
mutant gene (Fig. 7 A). However, the mgm1-S224N gene
failed to complement any of the mutant phenotypes of a
cell containing an mgm1-null lesion (Fig. 7 B and data not
shown). In addition, the mgm1-S224N gene did not complement the growth or mitochondrial distribution and
morphology defects of the mdm17 mutant cells (Fig. 7 C).
These results indicate that an intact GTP-binding domain
is essential for Mgm1p function.
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Overexpression of dynamin family members mutated in
critical residues of the GTP-binding site was previously
found to cause dominant-negative effects in wild-type cells
(Warnock and Schmid, 1996). To test whether this is also
true for MGM1, high-copy plasmids encoding either wild-type MGM1 or mgm1-S224N were transformed into wild-type cells. Only a small number of transformants harboring the mutant gene was recovered, and Western blot analysis
indicated that the mutant protein was not overexpressed
in these cells (perhaps reflecting a regulatory or suppression phenomenon). However, both wild-type and mutant
proteins were overexpressed in mgm1-null cells harboring
the respective plasmids (data not shown). These transformed cells were mated to wild-type cells, and the effect of overexpression in the resulting zygotes was analyzed by
fluorescence microscopy. High levels of wild-type MGM1
had no apparent effect on mitochondrial distribution or
morphology (Fig. 8 A), but zygotes with elevated levels of
the mutant protein displayed defects very similar to those
caused by the original mdm17 mutation (Fig. 8 A). In particular, 90% of the latter zygotes displayed aggregated mitochondria (Fig. 8 B), and 52% of the buds produced by
these zygotes contained no mitochondria (Fig. 8 B). These
results demonstrate that the mgm1-S224N mutant gene
product confers dominant-negative effects on mitochondrial distribution and morphology.
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Discussion |
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We have identified a role for Mgm1p in mitochondrial inheritance through the analysis of cells harboring mdm17, a
mutation causing temperature-sensitive growth and alterations in mitochondrial distribution and morphology. Genetic analysis revealed that mdm17 is a mutant allele of
MGM1, a nuclear gene originally identified by its role in
maintenance of the mitochondrial genome (Jones and
Fangman, 1992). Two key observations indicate that the
primary functions of Mgm1p are to facilitate the maintenance of mitochondrial morphology and to mediate mitochondrial inheritance and that mitochondrial genome
maintenance is a secondary role of the protein. First, analysis of mdm17 cells revealed that alterations in mitochondrial morphology and distribution appeared very quickly
after a shift to elevated temperature, whereas loss of respiratory competence and the mitochondrial genome occurred only much later. Second, the location of Mgm1p on
the mitochondrial surface is consistent with a primary function of the protein in morphology and distribution, but not
with a direct role in maintenance of the matrix-localized mitochondrial DNA. In addition, several other mutations
that cause aberrant mitochondrial morphology also cause
increased loss of mitochondrial DNA (Burgess et al., 1994
;
Sogo and Yaffe, 1994
; Berger et al., 1997
). This loss appears
to be a secondary effect of the morphological changes.
Mgm1p is one of a small group of outer membrane proteins that have been shown to be required for mitochondrial inheritance. Mdm10p, Mdm12p, and Mmm1p are
also integral proteins of the outer membrane whose loss
leads to dramatic alterations in mitochondrial morphology
and defects in transmission of mitochondria into developing daughter buds (Burgess et al., 1994; Sogo and Yaffe,
1994
; Berger et al., 1997
). Although the specific molecular
activities of these other components are unknown, their
location in the mitochondrial outer membrane and their
exposed cytoplasmic domains suggest two potential models of function. One possibility is that these proteins serve
as binding sites or anchor points for interaction with cytoskeletal structures or molecular motors. Such interactions might pull mitochondria into tubular structures and
mediate mitochondrial movement into buds. A second
possibility is that the outer membrane components regulate structural properties of the outer membrane, such as membrane fluidity, which are themselves essential for normal mitochondrial morphology and inheritance. Additionally, mitochondrial inheritance may depend on a tubular
morphology that is maintained by these outer membrane
proteins. Mgm1p may function in concert with the other
outer membrane proteins, or it may fulfill a unique role. This latter possibility is suggested by the observation that
phenotypes caused by mdm10, mdm12, and mmm1 are
suppressed by the SOT1 mutation (Berger et al., 1997
) but
defects caused by mutations in mgm1 are not (data not shown).
Mgm1p is a member of the dynamin family of large
GTP-binding proteins. The best characterized member of
this family is dynamin itself, which plays an essential role
in clathrin-mediated endocytosis in animal cells (Herskovits et al., 1993; van der Bleik et al., 1993). Although S. cerevisiae does not have a true dynamin orthologue, this yeast
possesses two other dynamin-like proteins, Vps1p (Vater et al., 1992
) and Dnm1p (Gammie et al., 1995
). Vps1p is
localized to the Golgi apparatus and participates in the vesicular transport of proteins to the vacuole (Vater et al.,
1992
). Dnm1p may function in the endosomal pathway
(Gammie et al., 1995
), but was recently found to play an
important role in mitochondrial distribution and morphology (Otsuga et al., 1998
). In particular, cells deleted for
Dnm1p displayed mitochondrial tubules collapsed along
one side of the cell and extending from the mother portion
of the cell into the daughter bud. A related phenotype, the
collapse of mitochondrial tubules into perinuclear aggregates, was observed in an independent study in which a
mutant form of the human dynamin-like protein, Drp1,
was expressed in cultured mammalian cells (Smirnova et al.,
1998
). These observations suggest that Dnm1p and its
mammalian homologue Drp1 may mediate the lateral distribution or branching of mitochondrial tubules.
Many molecular details of dynamin's function in endocytosis remain to be clarified, but key features of the
protein's activity include GTP hydrolysis and assembly of
dynamin into oligomeric structures (Herskovits et al.,
1993; Hinshaw and Schmid, 1995
). Dynamin has been proposed to act as a mechanical constrictor for the neck of an
invaginating coated pit (Warnock and Schmid, 1996
), or as
a regulator or recruiter of the constriction machinery
(Roos and Kelly, 1997
). Although it is not apparent that a
constricting activity is necessary for the maintenance of
mitochondrial morphology and distribution, our results
suggest that Mgm1p shares two key functional features
with dynamin. First, mutational analysis revealed that the
GTP-binding site is essential for Mgm1p function, suggesting the importance of GTP hydrolysis. Second, the dominant-negative effect of the S224N mutation and the wild-type protein's gel filtration profile suggest that Mgm1p
functions as part of an oligomeric complex. Mgm1p might
play a structural role in locally influencing the properties
of the outer membrane. For example, Mgm1p could assemble into a structure that promotes a tubular mitochondrial morphology. Alternatively, Mgm1p might fulfill a
regulatory function in controlling the activity of other
membrane components. More details of the protein's
function should emerge from identification of interacting proteins.
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Footnotes |
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Received for publication 30 April 1998 and in revised form 23 December 1998.
Address correspondence to Dr. Michael Yaffe, Department of Biology,
0347, University of California, San Diego, La Jolla, CA 92093-0347. Tel.:
619-534-4769. Fax: 619-534-4403. E-mail: myaffe{at}ucsd.edu
This paper is dedicated to the memory of Segall Livneh (1973-1991), who
generated the collection of temperature-sensitive strains from which the
mdm17 mutant was isolated. We are grateful to Randy Hampton, Karen
Berger, and Peter Fekkes for valuable comments on the manuscript and
members of the Yaffe lab for their insightful advice and helpful suggestions. We thank Rick Roberts for purification of membrane fractions, Jim
Kadonaga for use of the FPLC apparatus, and Alan Kutach for his invaluable help in FPLC analysis.
This work was supported by grant GM44614 from the National Institutes of Health.
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Abbreviations used in this paper |
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DASPMI, 2-(4-dimethylaminostryl)- 1-methylpyridinium iodide; mdm, mitochondrial distribution and morphology mutant; YPD, yeast extract/peptone/glucose; YPG, yeast extract/ peptone/glycerol.
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