Correspondence to: Jodi Nunnari, Section of Molecular and Cellular Biology, University of California Davis, Davis, California 95616. Tel:530-754-9774 Fax:530-752-7522
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Abstract |
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Mutations in the dynamin-related GTPase, Mgm1p, have been shown to cause mitochondrial aggregation and mitochondrial DNA loss in Saccharomyces cerevisiae cells, but Mgm1p's exact role in mitochondrial maintenance is unclear. To study the primary function of MGM1, we characterized new temperature sensitive MGM1 alleles. Examination of mitochondrial morphology in mgm1 cells indicates that fragmentation of mitochondrial reticuli is the primary phenotype associated with loss of MGM1 function, with secondary aggregation of mitochondrial fragments. This mgm1 phenotype is identical to that observed in cells with a conditional mutation in FZO1, which encodes a transmembrane GTPase required for mitochondrial fusion, raising the possibility that Mgm1p is also required for fusion. Consistent with this idea, mitochondrial fusion is blocked in mgm1 cells during mating, and deletion of DNM1, which encodes a dynamin-related GTPase required for mitochondrial fission, blocks mitochondrial fragmentation in mgm1 cells. However, in contrast to fzo1 cells, deletion of DNM1 in mgm1 cells restores mitochondrial fusion during mating. This last observation indicates that despite the phenotypic similarities observed between mgm1 and fzo1 cells, MGM1 does not play a direct role in mitochondrial fusion. Although Mgm1p was recently reported to localize to the mitochondrial outer membrane, our studies indicate that Mgm1p is localized to the mitochondrial intermembrane space. Based on our localization data and Mgm1p's structural homology to dynamin, we postulate that it functions in inner membrane remodeling events. In this context, the observed mgm1 phenotypes suggest that inner and outer membrane fission is coupled and that loss of MGM1 function may stimulate Dnm1p-dependent outer membrane fission, resulting in the formation of mitochondrial fragments that are structurally incompetent for fusion.
Key Words: fission, membrane remodeling, inner membrane, organelle, morphology
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Introduction |
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During cell division, copies of both the mitochondrial organelle and its genome must be inherited for progeny to survive and be respiratory competent. The accurate partitioning of these components into daughter cells is dependent on the morphology of the mitochondrial organelle. In S. cerevisiae, mitochondria form a reticulum distributed at the cell cortex (
The continuity and reticular nature of yeast mitochondria is also established and maintained by balanced fission and fusion events (
Mitochondrial fission is mediated by the dynamin-related GTPase, Dnm1p (
In the case of double-membraned organelles such as mitochondria and chloroplasts, mechanisms likely exist to coordinate fission and fusion events of the outer and inner membranes, and to maintain the separation of these membranes. Data suggest that coordination of chloroplast outer and inner membrane fission is accomplished by the differential localization and action of two distinct homologues of the bacterial cell division GTPase, FtsZ (FtsZ1 and FtsZ2;
Despite the structural similarities between Dnm1p and Mgm1p, the phenotypes associated with dnm1 cells and mgm1 cells are distinct. Unlike dnm1 cells, mutations in MGM1 cause mitochondrial aggregation and mtDNA loss in cells (
In contrast to published observations, our localization data indicate that Mgm1p is in the intermembrane space compartment of mitochondria, associated with the inner membrane. We also show that loss of MGM1 function results in the rapid fragmentation of mitochondrial reticuli with secondary aggregation of mitochondrial fragments. In addition, we have found that mitochondrial fusion in mgm1 cells is blocked during mating. However, MGM1 does not appear to function directly in fusion, because its requirement can be bypassed by the deletion of DNM1. Instead, our data are consistent with the idea that loss of MGM1 function triggers Dnm1p-dependent outer membrane fission, resulting in the formation of fusion incompetent mitochondrial fragments. Taken together, our data suggest a model where Mgm1p functions in the intermembrane space in coordination with Dnm1p-dependent outer membrane fission to regulate inner membrane division and/or structure.
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Materials and Methods |
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Yeast Genetic Techniques
Media preparations and genetic techniques were performed as described (
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Isolation and Characterization of mgm1 Alleles
Mutant alleles of MGM1 were isolated in a previously described screen for conditional, recessive nuclear mutations that cause loss of mtDNA at the nonpermissive temperature of 37°C (
To characterize mgm1 alleles, cultures were grown at the permissive temperature (25°C) to log phase in YPG, cells were plated onto YPD plates, and colony color was assessed and quantified. Because all strains analyzed contained the ade2 mutation, on YPD, white colonies were classified as respiratory deficient and red colonies were classified as respiratory competent. To directly visualize mtDNA, cells were fixed in 70% ethanol containing 10 ng/ml DAPI and washed with PBS. Mitochondrial morphology was visualized and quantified either by incubating cells in YPG with MitoTracker CMXR (Molecular Probes) or by mitochondrial matrix targeted GFP (mito-GFP, kindly provided by B.Westerman and W. Neupert, Ludwig Maximilians Universitaet Muenchen, Muenchen, Germany).
Sequence Analysis of mgm1-5
The mutation at the MGM1 locus in mgm1-5 cells was identified by DNA sequence analysis. Yeast genomic DNA prepared from mgm1-5 and W303 strains as described (
In Vivo Mitochondrial Fusion Assay
To assess mitochondrial fusion, haploid strains of opposite mating type were labeled with either mito-GFP or MitoTracker CMXR, mated at 25 or 37°C, and analyzed as described by
Construction of HA-tagged Mgm1p
JSY2519 was generated by integrating a 3X-HA-URA3-3X-HA (hemagglutinin) cassette between codons 173 and 174 of the MGM1 open reading frame in JSY836 (
Biochemical Analyses
Mitochondria were prepared from JSY2519 and protease protection experiments were conducted on enriched mitochondrial fractions as described (
Indirect Immunofluorescence
JSY2519 cells were grown to log phase in YPG and stained with MitoTracker CMXR for 30 min at 25°C as described (
Microscopy
All samples were imaged using a Leica confocal microscope with a 100x 1.4 NA objective (Leica, Inc.). Cryoimmunoelectron microscopy was performed as described (
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Results |
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The Primary Defect Associated with Loss of MGM1 Function Is Fragmentation of Mitochondrial Reticuli
Previous work showed that MGM1 is required for wild-type mitochondrial morphology and respiratory competence. Specifically, mtDNA loss and mitochondrial aggregation and inheritance defects were observed in temperature sensitive mgm1 and mgm1 cells (
To determine the primary defect associated with loss of MGM1 function, we focused on one conditional allele of MGM1, mgm1-5. This allele was chosen because analysis of our collection of mgm1 mutants indicated that mgm1-5 cells exhibited the greatest and most rapid phenotypic change upon shifting to nonpermissive temperature. Specifically, under permissive conditions, no difference in the generation of respiratory incompetent colonies was observed between mgm1-5 and wild-type cells, indicating that Mgm1p is functional under these conditions (99% respiratory competent, n = 100). In contrast, under restrictive conditions, 100% of mgm1-5 cells were respiratory incompetent and devoid of mtDNA as compared with 3% of wild-type cells, indicating a complete loss of MGM1 function (n = 100). In heterozygous mgm1-5 MGM1 strains, mgm1-5 phenotypes were recessive, indicating that the mutation causes a loss of MGM1 function at nonpermissive temperature. The MGM1 locus in mgm1-5 cells contains a single point mutation that changes G408 to D408. This residue is contained within the GTPase domain of the protein, consistent with previously published observations indicating its importance for MGM1 function (
To gain more insight into MGM1 function, we examined mitochondrial morphology in mgm1-5 cells using mito-GFP. Under permissive conditions, the majority of mgm1-5 cells contained reticular mitochondria, similar to that observed in wild-type cells (Fig 1, compare B to D). Within 20 min after shifting to the nonpermissive temperature, mitochondrial reticuli fragmented into many smaller mitochondria that remained distributed at the cell cortex in mgm1-5 cells (Fig 1, compare D to F). Extended exposure (60 min) of mgm1-5 cells to nonpermissive temperature resulted in aggregation of mitochondrial fragments (Fig 1 H). This terminal morphological phenotype resembles the mitochondrial morphology defect observed in mgm1 cells (Fig 1, compare H to J). Thus, the primary phenotype associated with loss of MGM1 function is mitochondrial fragmentation and a secondary phenotype is aggregation of these mitochondrial fragments (see Fig 1 K for quantification).
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A previous study reported a mitochondrial inheritance defect in mgm1 cells and suggested that Mgm1p might be involved in the movement of mitochondria into daughter cells (
Mitochondrial Fusion Is Blocked in mgm1-5 Cells during Mating
Both mitochondrial fragmentation and mtDNA loss phenotypes observed in mgm1-5 cells are similar to the phenotypes associated with loss of FZO1 function (
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Deletion of DNM1 in mgm1-5 Cells Blocks Mitochondrial Fragmentation and Restores Mitochondrial Fusion during Mating
To further test the hypothesis that fragmentation of mitochondrial reticuli in mgm1-5 cells is the result of a block in fusion, we determined whether mitochondrial tubules were restored in mgm1-5 cells under conditions where mitochondrial fission is abolished. To block mitochondrial fission in mgm1-5 cells, we deleted DNM1, which encodes a dynamin-related GTPase required for mitochondrial division. Mitochondrial morphology was examined in mgm1-5 dnm1 cells at permissive and nonpermissive conditions using mito-GFP. Under permissive conditions, where Mgm1p is functional, mitochondrial net-like structures are observed in mgm1-5
dnm1 cells (Table 3). These net-like mitochondrial structures are characteristic of
dnm1 cells and likely arise because mitochondrial tubules fuse and new tubule ends cannot be generated by fission (Fig 3E and Fig F; Table 3). At the nonpermissive temperature in mgm1-5
dnm1 cells, where mitochondrial fragmentation is observed in mgm1-5 cells, mitochondrial tubules and net structures are maintained (Fig 3, compare C and D to G and H; Table 3). Thus, abolishing Dnm1p-dependent mitochondrial fission blocks mitochondrial fragmentation resulting from loss of MGM1 function. In addition, the mgm1-5 glycerol growth and mtDNA loss phenotypes observed at nonpermissive temperature are suppressed in mgm1-5
dnm1 cells (not shown). Deletion of DNM1 in fzo1 cells also has been shown to block mitochondrial fragmentation and mtDNA loss.
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Although these data suggest that MGM1 might play a role in the fusion process, the structural similarity of Mgm1p to dynamin-like proteins suggests that it is involved in membrane remodeling and/or fission events. Thus, we considered the alternative explanation, that mitochondrial fragmentation in mgm1-5 cells results from an increase in Dnm1p-dependent mitochondrial fission. In this scenario, mitochondrial fragments might fail to fuse because of their geometry/structure and not as a primary consequence of loss of MGM1 function. To test this possibility, we assayed mitochondrial fusion during mating in mgm1-5 dnm1 cells, where deletion of DNM1 blocks mitochondrial fragmentation and restores mitochondrial tubular structures (Fig 3G and Fig H; Table 3). As shown previously, deletion of DNM1 has no effect on mitochondrial fusion during mating, consistent with its role in fission (
dnm1 haploids (Table 2). In contrast to mgm1-5 cells at nonpermissive temperature, where both mitochondrial fragmentation and a block in mitochondrial fusion are observed, deletion of DNM1 in mgm1-5 cells restored mitochondrial fusion during mating (Fig 4, EH; Table 2). Thus, the presence of mitochondrial tubular and net structures in mgm1-5
dnm1 cells correlates with the restoration of mitochondrial fusion (Table 3). In contrast, FZO1 function is still required for mitochondrial fusion, even when tubules are restored in fzo1-1
dnm1 zygotes, consistent with Fzo1p's proposed direct role in the fusion process (
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To test whether restoration of mitochondrial fusion in mgm1-5 dnm1 zygotes was FZO1-dependent and not due to activation of an alternate fusion pathway, we examined mitochondrial fusion in the mgm1-5 fzo1-1
dnm1 triple mutant. Morphologically, mitochondria in these triple mutant cells were similar to those in both
dnm1 and mgm1-5
dnm1 cells (Fig 4, MP). As expected, at permissive temperature, mitochondria fused in zygotes formed from mgm1-5 fzo1-1
dnm1 haploids (Table 2). However, at nonpermissive temperature, mitochondria failed to fuse in mgm1-5 fzo1-1
dnm1 zygotes, confirming previous evidence indicating a direct role of Fzo1p in mitochondrial fusion (
Mgm1p Is Localized to the Mitochondrial Intermembrane Space
Given that MGM1 does not play a direct role in mitochondrial fusion, we suggest three possible explanations for the mitochondrial fragmentation observed in mgm1-5 cells. In wild-type cells, Mgm1p may function indirectly in mitochondrial fusion by remodeling and/or maintaining mitochondrial membranes in fusion competent tip and tubular structures. Alternatively, given that both Mgm1p and Dnm1p are dynamin-related GTPases predicted to assemble into oligomeric structures, Mgm1p may interact directly with Dnm1p to negatively regulate mitochondrial fission. This latter possibility is potentially supported by a recent report that Mgm1p is localized to the mitochondrial outer membrane (
To detect and examine its subcellular localization, we epitope-tagged Mgm1p. Neither NH2- or COOH-terminal tagged Mgm1p complemented the mitochondrial morphology and growth defects of mgm1 cells (not shown). A tagged version of Mgm1p that retained function was created by inserting three tandem copies of the HA epitope (3XHA) within the coding sequence between amino acids 216217, directly proceeding the predicted GTPase domain (Mgm1:3XHAp). In a strain where Mgm1:3XHAp replaced the wild-type MGM1 locus (JSY2519), mitochondrial morphology was indistinguishable from wild-type cells (n = 443).
Western blot analysis of extracts made from JSY2519 with anti-HA antibodies detected a predominant 92-kD species (Fig 5 A, lane 1). In contrast, no species were detected by Western blotting in the absence of tagged Mgm1p, indicating that anti-HA antibodies specifically recognize Mgm1:3XHAp (Fig 5 A, lane 2).
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To examine Mgm1p's subcellular localization, JSY2519 extracts were fractionated by differential centrifugation and analyzed by SDS-PAGE and Western blotting. Consistent with the previously reported mitochondrial localization of Mgm1p, Mgm1:3XHAp was highly enriched in mitochondrial pellet (Fig 5 B, lane 2). In addition to the predominant 92-kD form of Mgm1:3XHAp, we also detected a less abundant, slower migrating 116-kD form of Mgm1:3XHAp in enriched mitochondrial fractions (Fig 5 B, lane 2). These data are in agreement with a previous study of Mgm1p localization, where two forms of Mgm1p were detected with antibodies raised against a COOH-terminal Mgm1p-derived peptide (
Indirect immunofluorescence of JSY2519 cells using anti-HA antibodies revealed that Mgm1p:3XHAp was localized to reticular structures at the cell cortex (Fig 6 B). Mgm1:3XHAp staining colocalized with structures labeled with the covalent vital mitochondrial fluorescent probe, MitoTracker (Fig 6B and Fig C). Thus, localization of Mgm1p by both biochemical and indirect immunofluorescence indicate that it is a mitochondrial protein, consistent with previous observations (
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We examined the submitochondrial localization and topology of Mgm1:3XHAp by treating isolated mitochondria with exogenous proteases. In intact mitochondria, both the 92- and 116-kD forms of Mgm1:3XHAp were inaccessible to even high concentrations of proteinase K (PK; Fig 5 B, lanes 24) and trypsin (not shown). Under these conditions, however, the outer mitochondrial marker, Fzo1p, was digested completely, indicating that the outer membrane was fully accessible to protease (Fig 5 B, lanes 24). In addition, both intermembrane space (AAC and Tim23p) and matrix (KDH) marker proteins were protected from proteolysis, confirming that the mitochondrial outer and inner membranes were intact (Fig 5 B, lanes 24). When both the inner and outer mitochondrial membranes were disrupted by treatment with detergent or by sonication, complete proteolysis of Mgm1:3XHAp and marker proteins in all mitochondrial compartments was observed (Fig 5 B, lane 5, and C, lanes 1 and 2). These results indicate that Mgm1p is not a component of the mitochondrial outer membrane and is localized inside mitochondria. Thus, Mgm1p does not directly interact with Dnm1p to regulate mitochondrial outer membrane fission.
To determine whether Mgm1p resides in the intermembrane space or matrix compartment, we converted intact mitochondria to mitoplasts by selectively rupturing the mitochondrial outer membrane by hypoosmotic shock. Upon osmotic shock, a significant fraction of the soluble intermembrane space protein, cytochrome b2, was released into the supernate fraction after centrifugation of treated mitochondria (Fig 5 D, lanes 1 and 2). In contrast, the inner membrane markers, AAC and Tim23p, and the soluble matrix protein, KDH, was recovered in the pellet fraction, indicating that the inner membrane of the mitoplasts remained intact (Fig 5 D, lanes 1 and 2). Both forms of Mgm1:3XHAp also were recovered in the pellet fraction, indicating that they are either membrane-associated or soluble in the matrix.
To determine if Mgm1:3XHAp is localized to the matrix compartment or membrane-associated and exposed to the intermembrane space, we treated mitoplasts with PK. In mitoplasts, the matrix marker, KDH, was protected from PK digestion, indicating that the inner membrane was impermeant to protease and intact (Fig 5 D, lanes 24). Although both AAC and Tim23p are inner membrane proteins, they contain epitopes exposed to the intermembrane space. Thus, as predicted, both were accessible to PK in mitoplasts (Fig 5 D, lanes 24). Under these conditions, both forms of Mgm1:3XHAp behaved exactly like these intermembrane space markers and were also sensitive to PK digestion in mitoplasts (Fig 5 D, lanes 24). In addition, identical results were observed for both forms of Mgm1:3XHAp and marker proteins when mitoplasts were treated with trypsin (not shown), indicating that the substrate specificity did not influence the susceptibility to proteolysis. These results indicate that both forms of Mgm1p are not localized to the matrix compartment, but instead, are membrane-associated and exposed to the intermembrane space.
To determine whether Mgm1p is associated with the outer or inner mitochondrial membrane, we examined Mgm1:3XHAp's localization by immunoelectron microscopy using anti-HA antibodies. Immunogold-labeling was performed as described on cryosections of JSY2519 cells (
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To determine whether Mgm1p is an integral inner membrane protein, we examined Mgm1p's sensitivity to sodium carbonate extraction. Mitochondria containing Mgm1:3XHAp were extracted with 0.1 M Na2CO3 (pH 10.5) or mock-treated and fractionated into supernate and pellet fractions by centrifugation (Fig 5 E, lanes 14). As expected, the inner membrane protein Tim23p was resistant to sodium carbonate extraction and was quantitatively recovered in the pellet fraction (Fig 5 E, compare lanes 3 and 4). In contrast, both forms of Mgm1:3XHAp and the soluble intermembrane space protein cytochrome b2 were released into the supernatant upon sodium carbonate treatment, but not in control samples treated with buffer (Fig 5 E, lanes 14). Based on this analysis, Mgm1:3XHAp is peripherally associated with the inner membrane, and not an integral membrane protein.
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Discussion |
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S. cerevisiae Mgm1p was previously reported to be an integral mitochondrial outer membrane protein (
The picture that emerges from these data is that the dynamin-related GTPases, Dnm1p and Mgm1p, are regulating the morphology of the outer and inner mitochondrial membranes, respectively (
Precedence for a role of Mgm1p in inner membrane remodeling also can be found in observed similarities between mitochondria and chloroplast fission. Chloroplast fission requires the coordinate action of two homologues of the bacterial cell division GTPase FtsZ, called FtsZ1 and FtsZ2 (
Although FtsZ1 and Mgm1p may play related roles in remodeling inner organellar membranes, their topologies with respect to the inner membrane are different. FtsZ1 localizes to the stromal (matrix-equivalent) face of the chloroplast inner membrane, while Mgm1p localizes to the intermembrane space side of the mitochondrial inner membrane (
This Mgm1p-mediated pinching activity could affect inner membrane structure in several ways. First, Mgm1p might function in the formation or stabilization of inner membrane cristae by self-assembling into ring-like structures around the inner membrane (Fig 8A and Fig B). A second possibility is that Mgm1p facilitates inner membrane fission, similar to dynamin and Dnm1p (Fig 8 B). Finally, a third possibility is that Mgm1p is required sequentially for cristae formation and inner membrane fission as depicted in Fig 8 B.
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If inner membrane division is mediated by machinery separate from the Dnm1p-dependent outer membrane machinery, as is suggested from observations in C. elegans, then mechanisms that coordinate inner and outer membrane fission must exist. Taking this into account, the mitochondrial fragmentation we observe in mgm1 cells may indicate that the loss of MGM1 function can somehow be sensed by the outer membrane, causing the rate of Dnm1p-dependent outer membrane fission to increase. Consistent with this model, mitochondrial fragmentation is blocked and fusion is restored during mating in mgm1 cells when fission is abolished by deletion of DNM1. Alternatively, it is possible that loss of MGM1 function could either directly or indirectly affect the structure of the mitochondrial membrane tubules, rendering them fusion incompetent, and ultimately resulting in fission-mediated fragmentation observed in mgm1 cells. In this case, an Mgm1p-independent pathway for tubule formation could be operating in cells lacking Dnm1p, explaining the observed restoration of mitochondrial tubules and fusion in the dnm1 mgm1 double mutant.
Independent of the exact mechanism of mitochondrial fragmentation in mgm1 cells, our data are consistent with a role of Mgm1p in inner membrane remodeling events. We recently used genetic approaches to identify several new components of the Dnm1p outer mitochondrial membrane fission machinery (
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Footnotes |
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Edith D. Wong and Jennifer A. Wagner contributed equally to this work.
1 Abbreviations used in this paper: mito-GFP, mitochondrial matrix-targeted green fluorescent protein; mtDNA, mitochondrial DNA; PK, proteinase K.
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Acknowledgements |
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We thank Voytek Okreglak and T.J. Holewinske for their technical and intellectual contributions, and the Nunnari lab for discussions and critical reading of the manuscript. We also thank C. Koehler and R. Jensen for antibodies.
This work was supported by the National Science Foundation (MCB-9724143) to J. Nunnari and the National Institutes of Health (NIH; GM-53466) to J. Shaw. E. Wong was supported by an NIH Training Grant (GM-07377).
Submitted: 10 July 2000
Revised: 17 August 2000
Accepted: 21 August 2000
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References |
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