Correspondence to: J.M. Shaw, Department of Biology, University of Utah, Salt Lake City, UT 84112. Tel:(801) 585-6205 Fax:(801) 585-9735
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Abstract |
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Yeast Dnm1p is a soluble, dynamin-related GTPase that assembles on the outer mitochondrial membrane at sites where organelle division occurs. Although these Dnm1p-containing complexes are thought to trigger constriction and fission, little is known about their composition and assembly, and molecules required for their membrane recruitment have not been isolated. Using a genetic approach, we identified two new genes in the fission pathway, FIS1 and FIS2. FIS1 encodes a novel, outer mitochondrial membrane protein with its amino terminus exposed to the cytoplasm. Fis1p is the first integral membrane protein shown to participate in a eukaryotic membrane fission event. In a related study (Tieu, Q., and J. Nunnari. 2000. J. Cell Biol. 151:353365), it was shown that the FIS2 gene product (called Mdv1p) colocalizes with Dnm1p on mitochondria. Genetic and morphological evidence indicate that Fis1p, but not Mdv1p, function is required for the proper assembly and distribution of Dnm1p-containing fission complexes on mitochondrial tubules. We propose that mitochondrial fission in yeast is a multi-step process, and that membrane-bound Fis1p is required for the proper assembly, membrane distribution, and function of Dnm1p-containing complexes during fission.
Key Words: Fis1p, Dnm1p GTPase, mitochondria, fission, fusion
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Introduction |
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Maintenance of the tubular mitochondrial reticulum in budding yeast requires opposing fission and fusion events that regulate organelle copy number and morphology (
In Saccharomyces cerevisiae, two evolutionarily conserved GTPases act on the outer mitochondrial membrane to regulate opposing fission and fusion reactions (Fig 1) (
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In a previous study (
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Materials and Methods |
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Strain and Plasmid Construction
Table 1 lists the strains used in this study. All strains are derivatives of the FY10 strain (
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For pRS415-MET25 + 9xMYC-FIS1 (Myc-Fis1p), a FIS1 PCR fragment flanked by HindIII/XhoI sites was cloned into pRS415-MET25 +9xMYC (J. Thatcher and J.M. Shaw, manuscript in preparation). For pRS415-MET25 + rsGFP-FIS1aa1-155 (GFP-Fis1p), an XbaI/HindIII digest was first used to remove 9xMYC from the pRS415-MET25 + 9xMYC-FIS1 plasmid described above. The 9xMyc was then replaced by a red-shifted green fluorescent protein (GFP)1 variant (rsGFP) PCR amplified from pQBI25-fc1 (Quantum Biotechnologies Inc.) and flanked by XbaI/HindIII sites. An ATG initiation codon was engineered into the 5' primer used to amplify rsGFP. For pRS416 + FIS1, a FIS1 PCR fragment flanked by XhoI sites was cloned into pRS416. For pGEX-4T-3 + FIS1aa-1-127, a PCR fragment encoding amino acids 1127 of FIS1 and flanked by BamHI/XhoI sites was cloned into pGEX-4T-3 (Amersham Pharmacia Biotech) to generate an in-frame GST-Fis1p fusion. For pRS414 + DNM1-rsGFP and pRS416 + DNM1-rsGFP, first a DNM1-containing PCR fragment flanked by SacII/SpeI sites was cloned into the SacII/NheI sites of pQBI25 (Quantum Biotechnologies Inc.), creating pQBI25 + DNM1-rsGFP. Second, an XhoI/BamHI fragment containing the DNM1-rsGFP sequence was cloned into pRS414 and pRS416. For pRS415-MET25 + rsGFP-FIS1aa1-127, a HindIII/XhoI digest was used to remove FIS1aa1-155 from pRS415-MET25 + rsGFP-FIS1aa1-155, which was then replaced by a HindIII/XhoI fragment containing FIS1aa1-127. For pRS416-GAL1 + PrF0ATP9-RFP (mito-RFP), a BamHI/XhoI fragment containing RFP (the red fluorescent protein from the pDsRed vector; CLONTECH Laboratories, Inc.) was cloned into pRS416-GAL1 + PrF0ATP9 (J. Thatcher and J.M. Shaw, manuscript in preparation). For pRS424-ADH1 + PrF0ATP9-RFP (mito-RFP), a SpeI/XhoI fragment containing PrF0ATP9-RFP from pRS416-GAL1 + PrF0ATP9-RFP was cloned into pRS424-ADH1.
GFP-Fis1p and Myc-Fis1p were expressed from CEN plasmids under control of the MET25 promoter. In the presence of methionine, this promoter is leaky and GFP-Fis1p and Myc-Fis1p are expressed at levels comparable with that of endogenous Fis1p in wild-type cells (verified by quantitative Western blotting). Under these conditions, GFP-Fis1p and Myc-Fis1p rescued the mitochondrial morphology defect in the fis1 mutant (83 and 55% wild type, respectively).
Identification of fis1 and fis2 and Cloning and Disruption of FIS1
14 spontaneous, second-site suppressors of the fzo1-1 temperature-sensitive (37°C) glycerol growth defect were isolated in JSY2788 (MATa ura3-52 his3200 leu2
1 trp1
63 fzo1::HIS3 + pRS414-fzo1-1). Standard genetic methods were used to show that suppression in each case was due to a single recessive mutation. Complementation and meiotic segregation analyses revealed that the 14 fzo1-1 suppressors defined three linkage groups. Seven suppressors comprising one linkage group failed to complement the dnm1
mutation in diploid cells and a DNM1-containing plasmid reversed the fzo1-1 suppression phenotype of the mutants in this group. Sequence analysis indicated that these suppressors contained mutant dnm1 alleles. The remaining seven suppressor strains fell into two linkage groups (later named fis1 and fis2).
FIS1 was cloned by complementation of a fis1 allele (restoration of the fzo1-1 no-growth-on-glycerol-at-37°C phenotype) using a yeast genomic library in YEP213 (
A fis1::HIS3 disruption that precisely replaced the FIS1 coding region was generated by gene replacement in a diploid strain as described (::HIS3. Sequence analysis indicated that the fis1 suppressors contained mutations in FIS1.
Quantification of Mitochondrial Morphology and Fusion Phenotypes
Mitochondrial morphology was scored in wild-type and mutant cells expressing a mitochondrial-targeted form of GFP (mito-GFP) either from the plasmid pVT100UGFP (provided by B. Westermann and W. Neupert, Universitaet Muenchen, Muenchen, Germany) or the plasmid pRS416 + preCox4-GFP (
Subcellular Localization of Fis1p and Dnm1p-GFP
To generate polyclonal serum specific for Fis1p, the soluble GST-Fis1paa1-127 fusion protein was expressed in Escherichia coli BL21-(DE3) cells from pGEX-4T-3 + FIS1aa-1-127 and batch purified on Glutathione Sepharose 4B beads (Amersham Pharmacia Biotech). After separation by SDS-PAGE, the fusion protein was excised and used to immunize rabbits (Covance Research Products, Inc.).
For the protease protection experiments, a fis1 strain (ADM549) expressing GFP-Fis1p was grown in S-galactose medium lacking leucine to select for the pRS415-MET25 + rsGFP-FIS1 plasmid. Differential sedimentation experiments and protease protection experiments were performed using either wild-type cells (ADM548) or the ADM549 strain, as described previously (
To localize Dnm1p in wild-type and mutant strains, Dnm1p-rsGFP expressed from either pRS414 + DNM1-rsGFP, pRS416 + DNM1-rsGFP, or pHS20 (
Immunoelectron microscopy was performed as described by
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Results |
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Mutations in FIS1 and FIS2 Suppress mtDNA Loss and Glycerol Growth Defects in fzo1-1
To identify yeast genes required for mitochondrial fission, we selected spontaneous, second-site suppressor mutations that restored glycerol growth at 37°C to fzo1-1 cells. The 14 suppressor strains isolated were recessive in heterozygous diploids and segregated 2:2 in backcrosses with the fzo1-1 mutant. Complementation analysis revealed that seven of these strains represented new alleles of the previously characterized DNM1 gene (
Like dnm1 mutations, fis1 and fis2 mutations rescued the fzo1-1 temperature-sensitive mtDNA loss and glycerol growth defects. Mitochondrial networks in wild-type cells retained their mtDNA nucleoids (DAPI staining, not shown) and these cells grew well on medium containing the nonfermentable carbon source glycerol at both 25° and 37°C (Fig 2 A, WT). In contrast, the fzo1-1 strain failed to grow on glycerol at 37°C where the mitochondrial reticulum fragments and mtDNA is lost (Fig 2 A, fzo1-1) ( mutation into fzo1-1 (Fig 2 A, dnm1
fzo1-1) or fzo1
cells (
, fis2-5, both mutations suppressed the temperature-sensitive glycerol growth defect (Fig 2 A, fis1
fzo1-1, fis2-5 fzo1-1) and mtDNA loss defect (not shown) of fzo1-1. (Note that fis2-5 is truncated after 191 of 714 amino acids and behaves like a null allele.)
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Mitochondrial Membranes Form Nets in fis1 and fis2 Mutant Strains
The morphology of mitochondrial membranes in fis1 and fis2 mutant strains was consistent with a role for these genes in fission. As shown previously (. These nets appeared identical when visualized with matrix-targeted, and inner and outer membrane-targeted forms of the red and green fluorescent proteins, indicating that fission of both the inner and outer membranes was abolished in these mutants (not shown). Mitochondrial nets were also observed in all combinations of dnm1, fis1, and fis2 double and triple mutant strains (Table 2 and not shown), suggesting that FIS1 and FIS2 act in the same pathway as DNM1 to regulate mitochondrial fission.
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fis1 and fis2 Mutations Block Mitochondrial Fission and Fragmentation in fzo1-1
We previously reported ( fzo1-1 in Q; Table 2). To determine whether fis1 and fis2 mutations also prevented mitochondrial fragmentation in fzo1-1, we examined mitochondrial morphology in fis1
fzo1-1 and fis2-5 fzo1-1 strains after a shift to the nonpermissive temperature. At 25°C, 80% of fis1
fzo1-1 and 44% of fis2-5 fzo1-1 cells contain mitochondrial nets similar to those observed in the single fis1
and fis2-5 mutants at 25°, 30°, and 37°C (Table 2). Upon shifting fis1
fzo1-1 (Fig 2 I; Table 2) and fis2-5 fzo1-1 (Fig 2 M; Table 2) cells to 37°C, mitochondrial fragmentation was blocked despite the absence of FZO1 function and mitochondrial membranes remained net-like. These results were similar to those observed for the fzo1-1 dnm1 double mutant (
fzo1-1 and fis2-5 fzo1-1 double mutants blocked mitochondrial fission and fragmentation in fzo1-1 cells at the nonpermissive temperature.
Because both fis1 and fis2-5 are null alleles, suppression of the fzo1-1 mitochondrial fragmentation, mtDNA loss and glycerol growth phenotypes cannot occur via direct interactions between Fzo1-1p and the FIS1 and FIS2 gene products. Indeed, the genetic interactions described above did not require expression of the Fzo1-1 protein, ruling out the possibility that fis1 and fis2 restore an Fzo1p-dependent fusion pathway. In fzo1
fis1
(Fig 3A, second row, and E) and fzo1
fis2-5 (not shown), double mutant strains, the fzo1
glycerol growth defect was suppressed and mitochondrial membranes remained tubular, although the tubules sometimes appeared ragged or frayed (Table 2). Interestingly, mitochondrial nets were largely absent when double mutants contained the fzo1
allele (Table 2). This observation indicates that formation of mitochondrial nets in fis1 and fis2 mutant strains requires FZO1-mediated fusion.
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dnm1, fis1 and fis2 Mutations Are General Suppressors of Mitochondrial Fragmentation Defects
To determine whether the dnm1, fis1, and fis2 mutations were specific suppressors of the fzo1 fusion defect or were general suppressors of mitochondrial fragmentation defects, we examined the effect of dnm1, fis1, and fis2 mutations in an mgm1 mutant (
mutant are shown in Fig 3 and Table 2. As observed for the fis1
fzo1
double mutant, fis1
is able to suppress the mgm1
mtDNA loss (not shown), glycerol growth defect (Fig 3 A, bottom two rows) and mitochondrial fragmentation defect (Fig 3 I; Table 2) in the fis1
mgm1
double mutant strain. Similar results were observed for the mgm1
fis2-5 double mutant strains (not shown) and mgm1
dnm1
(Gorsich, S., and J.M. Shaw, unpublished data), consistent with the idea that dnm1, fis1, and fis2 mutations prevent fragmentation of mitochondrial membranes by blocking the fission pathway.
Mutations in fis1 and fis2 Do Not Restore Mitochondrial Fusion in fzo1-1 Cells
The observation that fis1 and fis2 mutations block mitochondrial fragmentation in fzo1-1 cells is consistent with a role for Fis1p and Fis2p in mitochondrial fission. However, similar results could be obtained if mutations in fis1 and fis2 activated an FZO1-independent fusion pathway. To determine whether fis1 and fis2 mutations restored fusion in fzo1-1, we assayed mitochondrial fusion in zygotes formed from either fis1 fzo1-1 or fis2-5 fzo1-1 double mutants. Fusion was assayed essentially as described by
Both mito-GFP and -RFP colocalized in 100% of wild-type zygotes formed at 25° and 37°C, indicating that mitochondria from each haploid parent had fused and their contents had mixed (Table 3) ( fzo1-1 and fis2-5 fzo1-1 cells results from a block in mitochondrial fission rather than the activation of an FZO1-independent fusion pathway. Together, the results presented here indicate that FIS1 and FIS2 act in the same pathway as DNM1 to regulate mitochondrial fission.
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FIS1 Defines a Novel Gene Family
The FIS1 gene (YIL065C in the Saccharomyces Genome Database) was cloned by library complementation of the glycerol growth suppression phenotype in fis1 fzo1-1 strains. A fragment of the rescuing plasmid containing FIS1/YIL065C also rescued the mitochondrial morphology defects in a fis1 mutant strain. Integrative mapping studies and DNA sequencing were used to show that the original fis1 mutations were allelic to the YIL065C cloned open reading frame. FIS1 encodes a novel protein of 155 amino acids with an estimated molecular mass of 17,000 D (Fig 4). The only identifiable protein motif in the open reading frame is a single, predicted, COOH-terminal transmembrane domain (Fig 4, TM). A database search identified Fis1p orthologs in fission yeast, humans, plants, worms, and flies, suggesting that Fis1p function has been conserved during evolution (Fig 4). Whether these orthologs participate in membrane division events mediated by Dnm1p-like GTPases remains to be seen.
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FIS1 Encodes an Integral, Outer Mitochondrial Membrane Protein with Its NH2 Terminus Facing the Cytoplasm
The subcellular localization of Fis1p was investigated by fluorescence microscopy and cryoimmunoelectron microscopy using functional, amino-terminal GFP-tagged (GFP-Fis1p) and Myc-tagged (Myc-Fis1p) forms of the protein (see Materials and Methods; GFP-Fis1p and Myc-Fis1p were expressed at levels comparable with that of endogenous Fis1p in these experiments). When expressed in yeast, GFP-Fis1p and Myc-Fis1p rescued the mitochondrial net morphology defect in fis1 and the glycerol growth suppression phenotype in the fis1
fzo1-1 double mutant.
In dividing yeast cells, GFP-Fis1p colocalized completely with the MitoTracker red CMXRos-labeled mitochondrial reticulum (Fig 5, AC), indicating that Fis1p is a mitochondrial protein. The identical Fis1p localization pattern was observed in dnm1, fis2-5, and fis2-5 dnm1
mutants, suggesting that Fis1p's localization is not determined or affected by other known components of the fission machinery (not shown). In addition, the predicted COOH-terminal transmembrane domain (Fig 4) was required for Fis1p's mitochondrial localization and function. A GFP-Fis1p fusion protein lacking the transmembrane domain (GFP-Fis1paa1-127) failed to colocalize with MitoTracker red CMXRos-labeled mitochondria (Fig 5, DF) and did not rescue fis1 mitochondrial morphology defects or the glycerol growth suppression phenotype in a fis1
fzo1-1 strain (not shown). Carbonate and detergent extraction studies confirmed that Fis1p was an integral mitochondrial membrane protein (not shown). Interestingly, overexpression of cytoplasmic GFP-Fis1paa1-127 from a MET25-inducible promoter did not cause dominant mitochondrial morphology defects in wild-type cells, suggesting that this form of Fis1p is not competent to titrate essential components of the fission reaction (data not shown). Cryo-immunoelectron microscopy revealed that, unlike Dnm1p, which was found in discrete spots associated with mitochondrial constriction sites (
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Subcellular fractionation experiments using antiFis1p and antiGFP antibodies confirmed that Fis1p is a mitochondrial protein. AntiFis1p antibodies recognized a 17-kD protein in extracts prepared from FIS1 wild-type but not fis1 cells (Fig 5 H, compare 1 and 2, band c). Similarly, antiGFP antibodies detected a 47-kD fusion protein in extracts prepared from fis1
cells expressing GFP-Fis1p but not in extracts prepared from fis1
cells expressing untagged Fis1p (Fig 5 H, compare 3 and 4, band a). As shown in Fig 4 I, native Fis1p cofractionated predominantly with the mitochondrial pellet, along with the outer mitochondrial membrane protein porin, during differential centrifugation of a postnuclear cell extract. In extracts prepared from fis1
cells expressing GFP-Fis1p, GFP-Fis1p also cofractionated with porin, and not the cytoplasmic marker, 3-phosphoglycerate kinase (3-PGK; data not shown).
Protease protection experiments revealed that Fis1p is an outer mitochondrial membrane protein with its NH2 terminus exposed to the cytoplasm. AntiGFP and antiFis1p antibodies detected the full-length GFP-Fis1p fusion protein in untreated mitochondrial fractions (Fig 5 J, 0 µg/ml Trypsin). Treatment of intact mitochondria with 10 or 100 µg/ml trypsin resulted in the clipping and release of the NH2-terminal, 29-kD GFP tag from Fis1p (Fig 5 J, 10 and 100 µg/ml trypsin, IM; the linker region between GFP and Fis1p contains a predicted trypsin cleavage site). In the presence of 100 µg/ml trypsin, the intermembrane space protein cytochrome b2 was not digested, indicating that the outer mitochondrial membrane remained intact (Fig 5 J, 100 µg/ml trypsin, IM). When the outer membrane was disrupted by osmotic shock, however, cytochrome b2 was degraded by trypsin, although the matrix marker alpha-ketoglutarate dehydrogenase (KDH) was still protected (Fig 5 J, 100 µg/ml trypsin, OS). Protease clipping of KDH (*) only occurred when mitochondria were solubilized with Triton X-100 in the presence of trypsin (Fig 5 J, 100 µg/ml trypsin, TX). Interestingly, significant degradation of Fis1p was only observed after treatment of mitochondria with detergent, suggesting that Fis1p's small size and outer membrane association affects its protease susceptibility (Fig 5 J, note shifted and smeared Fis1p bands in TX lanes treated with trypsin). The fact that (a) the GFP tag is at the NH2 terminus of Fis1p, (b) Fis1p has only one predicted transmembrane domain, and (c) there are only nine amino acids COOH-terminal of the transmembrane domain suggests that the COOH-terminal nine amino acids of Fis1p face the mitochondrial intermembrane space.
Proper Assembly and Distribution of Dnm1p on the Outer Mitochondrial Membrane Requires Fis1p, but Not Fis2p Function
Two-hybrid, coimmunoprecipitation (Fukushima, N.H., B.R. Keegan, and J.M. Shaw. 1999. Mol. Biol. Cell. 10:315a. [Abstr.]), and genetic studies (Mozdy and Shaw, unpublished observations) indicate that Dnm1p interacts with itself and probably functions as a multimer. Our previous work demonstrates that these Dnm1p multimer-containing complexes assemble on the cytoplasmic face of the outer mitochondrial membrane at sites of future constriction and fission (
In 97% of wild-type cells and 92% of dnm1 cells examined, Dnm1p-GFP was organized in eight or more punctate structures that colocalized completely with wild-type mito-RFPlabeled networks (Fig 6, AD; Table 4). This pattern was consistent with the Dnm1p localization pattern determined previously by indirect immunofluorescence studies (
cells colocalized with mitochondrial membranes in this mutant. However, these structures were not able to catalyze fission, as evidenced by the mitochondrial nets formed in the fis1
strain (Table 2).
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Although Dnm1p-GFP complexes colocalized with mitochondria in the absence of Fis1p, Fis1p was clearly required to properly distribute these complexes into punctate structures along mitochondrial tubules. Analysis of one FIS1 allele, fis1-21, suggested that Fis1p may also be required for the function of these punctate Dnm1p-containing structures. The fis1-21 allele blocked mitochondrial fission and fragmentation in a fis1-21 fzo1-1 double mutant at 37°C (mitochondrial membranes remained tubular in 97% of the cells). However, the number and distribution of punctate Dnm1p-GFP structures on fis1-21 fzo1-1 mitochondria appeared more wild type (Table 4). These data suggested that the rate or extent of fission by properly distributed Dnm1p complexes was impaired in the fis1-21 fzo1-1 strain. Thus, while Fis1p plays a role in determining the punctate distribution of Dnm1p complexes on mitochondrial tubules, it is also required for Dnm1p complex function once that distribution is achieved.
Similar experiments demonstrated that wild-type Dnm1p-GFP distribution did not require FIS2 gene function. In the majority (86%) of fis2-5 cells, the number and distribution of Dnm1p-GFP structures was indistinguishable from wild type (Fig 6, IL; Table 4). Once again, these structures colocalized with fis2-5 mitochondrial tubules and nets, suggesting that the Fis2 protein was required for the function but not the localization of Dnm1p-containing complexes (see Discussion for more information about Fis2p). Finally, the number and distribution of Dnm1p-GFP structures in a fis1 fis2-5 double mutant was similar to that in a fis1
single mutant, indicating that fis1 is epistatic to fis2 with respect to Dnm1p-GFP localization (Fig 6, MP). Thus, Fis1p apparently functions upstream of Fis2p to localize Dnm1p.
Immunogold labeling of ultrathin cryosections confirmed that Dnm1p localization was altered in the fis1 mutant. In wild-type cells expressing the tagged Dnm1p-HAc protein, 89.3% of the 5-nm gold particles were associated with the tips and sides of mitochondrial tubules and with constriction sites in these tubules (Table 5). These results are similar to those reported previously for Dnm1p-HAc localization ( cells, only 8% of gold particles were found on the tips and sides of mitochondrial tubules, while the majority (92%) were found in the cytoplasm (Table 5). Quantitative Western blot analysis indicated that this difference was not due to reduced Dnm1p expression in fis1
relative to FIS1 wild type (not shown). Rather, in fis1
cells, the total amount of Dnm1p associated with mitochondria appeared to be reduced, and the distribution of this mitochondrial-associated Dnm1p appeared to be altered.
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Discussion |
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Our previous studies established that the S. cerevisiae dynamin-related GTPase, Dnm1p, associates with the outer mitochondrial membrane at sites of future constriction and fission ( and fis2-5 (null) alleles prevent fragmentation in an fzo1
strain and in mgm1
cells (Fig 3). Fis1p is a novel outer mitochondrial membrane protein essential for the proper distribution and function of the Dnm1p GTPase during the fission reaction. Fis1p is, to our knowledge, the first integral membrane protein known to function with a dynamin-related GTPase in a membrane fission event. In contrast, the FIS2 gene product is required for the function, but not the mitochondrial association and distribution, of Dnm1p.
The results described here raised the possibility that Fis1p functions early in the fission pathway to recruit Dnm1p or Dnm1p-containing complexes to the mitochondrial membrane. Although we do not exclude such a function for Fis1p, several lines of evidence argue against this model. First, we have been unable to detect interactions of Fis1p with Dnm1p using coimmunoprecipitation and yeast two-hybrid studies (not shown). It is possible that such interactions are simply too transient to detect by these methods. Alternatively, one or more additional molecules may serve as a bridge between Fis1p and its binding partner(s). It is also possible that the methods we used failed to mimic a guanine nucleotide-bound state of Dnm1p required for Fis1p binding interactions. Second, we found that one to three large structures containing Dnm1p-GFP continued to colocalize with mitochondria in the absence of Fis1p (Fig 6). This result suggests that Dnm1p contains Fis1p-independent signals for protein or lipid binding that mediate its mitochondrial attachment. A number of previously identified Dnm1p structural domains might function in this manner, including the middle domain, insert B, or the alpha-helical domain (
Even though punctate Dnm1p-GFP complexes are properly distributed in fis1-21 fzo1-1 cells (Table 4), mitochondrial fragmentation is still blocked in this double mutant at 37°C (97% tubular mitochondria), suggesting that the ability of these complexes to catalyze fission is impaired. Thus, Fis1p may be required (a) to distribute Dnm1p complexes on mitochondrial tubules and (b) to activate the fission activity of Dnm1p complexes. One intriguing possibility is that Fis1p functions late in the fission pathway (after mitochondrial recruitment of Dnm1p complexes) as an effector molecule to regulate some aspect of the Dnm1p GTPase cycle. Preliminary studies of wild-type Dnm1p-GFP localization in the presence of a dominant dnm1-T62A allele ( mutant, suggesting that the fis1
and dnm1-T62A mutations affect the same step in Dnm1p assembly, distribution, or activation.
A related study reported the cloning and characterization of the FIS2 gene, which they named MDV1 for mitochondrial division (
The data presented here and in the accompanying study by
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Whether or not this model is correct, it seems likely that Dnm1p's GTPase cycle will play an important role in regulating one or more steps in the division process. For example, GTP binding or hydrolysis by Dnm1p might regulate: (a) self assembly into a complex with itself or other proteins, (b) its association with the outer mitochondrial membrane, (c) the assembly of Dnm1p complexes into higher order structures, or (d) the recruitment of additional components to the site of fission. A model in which GTP hydrolysis by Dnm1p provides mechanical energy for membrane constriction and fission cannot yet be excluded. It is likely that lipid modification activities are also required to remodel the outer mitochondrial membrane during constriction and fission, similar to endophilin's proposed activity during endocytosis (
Although molecules required for outer mitochondrial membrane fission have been identified in a number of organisms (
Fis1p and Mdv1p are new additions to the list of players that regulate division of prokaryotic cells and eukaryotic organelles of prokaryotic origin, including mitochondria and chloroplasts. E. coli cell division is mediated by the FtsZ GTPase that forms a ring on the cytoplasmic face of the inner membrane and constricts during cytokinesis (
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Footnotes |
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1 Abbreviations used in this paper: GFP, green fluorescent protein; KDH, alpha-ketoglutarate dehydrogenase; RFP, red fluorescent protein.
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Acknowledgements |
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We are grateful to Q. Tieu and J. Nunnari (University of California, Davis, Davis, CA) for communicating their MDV1 results before publication. We also thank Steven Gorsich for providing the mgm1 and mgm1
dnm1
strains, C. Koehler (University of California, Los Angeles, Los Angeles, CA) for providing antiKDH and anti-cytochrome b2 antibodies, H. Sesaki and R. Jensen (Johns Hopkins University, Baltimore, MD) for the pHS20 plasmid, and members of the Shaw laboratory for stimulating discussions and careful review of the manuscript.
This work was supported by grants from the American Cancer Society (CB-97) and the National Institutes of Health (GM53466) awarded to J.M. Shaw. The Utah Health Sciences Sequencing Facility is supported by a National Cancer Institute grant (5-P30CA42014).
Submitted: 1 August 2000
Revised: 29 August 2000
Accepted: 8 September 2000
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References |
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