Correspondence to: Robert E. Jensen, Department of Cell Biology and Anatomy, The Johns Hopkins University School of Medicine, 725 N. Wolfe Street, Baltimore, MD 21205. Tel:(410) 955-7291 Fax:(410) 955-4129 E-mail:rjensen{at}jhmi.edu.
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Abstract |
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In yeast, mitochondrial division and fusion are highly regulated during growth, mating and sporulation, yet the mechanisms controlling these activities are unknown. Using a novel screen, we isolated mutants in which mitochondria lose their normal structure, and instead form a large network of interconnected tubules. These mutants, which appear defective in mitochondrial division, all carried mutations in DNM1, a dynamin-related protein that localizes to mitochondria. We also isolated mutants containing numerous mitochondrial fragments. These mutants were defective in FZO1, a gene previously shown to be required for mitochondrial fusion. Surprisingly, we found that in dnm1 fzo1 double mutants, normal mitochondrial shape is restored. Induction of Dnm1p expression in dnm1 fzo1 cells caused rapid fragmentation of mitochondria. We propose that dnm1 mutants are defective in the mitochondrial division, an activity antagonistic to fusion. Our results thus suggest that mitochondrial shape is normally controlled by a balance between division and fusion which requires Dnm1p and Fzo1p, respectively.
Key Words: mitochondrial division, mitochondrial fusion, dynamin, GTPase, yeast
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Introduction |
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MITOCHONDRIA undergo regulated fusion and division in many cell types (
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Materials and Methods |
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Strain Construction
Strain YHS2, which expresses red-shifted GFP (F64L, T65C, and I167T) fused to the Cox4p presequence (residues 121) under the ADH1 promoter (ADH1-COX4-GFP), was constructed as follows. First, pHS1 was constructed by replacing wild-type GFP in pOK29, a HIS3-CEN plasmid which carries ADH1-COX4-GFP (Kerscher, O., unpublished information), with a NcoI-BamHI fragment carrying red-shifted GFP from pQBI25 (Quantum Biotechnologies). The EcoRV-BamHI fragment from pHS1 was inserted into pDH9, which carries 5' and 3' untranslated regions of MFA2 (a gift from S. Michaelis), forming pHS2. To integrate ADH1-COX4-GFP at chromosomal MFA2, a XhoI-SmaI fragment carrying 5'-MFA2-ADH1-COX4-GFP-3'-MFA2 from pHS2 was transformed into strain SM1235, which carries mfa2::URA3 ( mfa2::ADH1-COX4-GFP, was selected on 5-fluoro-orotic acid medium (
Mutant Isolation
YHS2 was mutagenized with 3% ethane methylsulfonate to ~30% survival (
Crosses to wild-type strain 1002 showed that all class I, II, and III mutations were recessive and caused by a defect in a single gene. Complementation tests revealed that all eight recessive class IV mutants were defective in the same gene. Crosses between class IV mutants and TRP1 strain 194 (a gift from E. Schweizer) or dnm1 strain JSY1361 (
Gene Disruption
Complete disruptions of the DNM1 and FZO1 were constructed by PCR-mediated gene replacement as described (, we used HIS3 plasmid pRS303 (
we used kanMX4 plasmid pRS400 (
fzo1
strain YHS27 and MAT
dnm1
fzo1
strain YHS23 were constructed by crossing MATa dnm1
strain YHS19 to MAT
fzo1
strain YHS22. Mitochondria in the disruption strains were visualized using pHS12, a CEN-LEU2 plasmid containing ADH1-COX4-GFP. pHS12 was created by inserting the XhoI-NotI fragment from pHS1 into pRS315 (
Plasmid Construction
The DNM1 gene with a NotI site immediately preceding its termination codon was PCR amplified from yeast genomic DNA and subcloned into pAA3, a CEN-LEU2 plasmid which contains the HA epitope with a NotI site at its NH2 terminus (Aiken, A., unpublished data), forming pDNM1-HA (pHS14). DNM1-GFP plasmid pHS20 was constructed as described above except that pAA1, a CEN-LEU2 plasmid which contains GFP with a NotI site at its NH2 terminus (Aiken, A., unpublished data), was used instead of pAA3. To form pHS15, DNM1-HA coding sequences were PCR amplified from pHS14 with 50 bp of flanking sequences homologous to the GAL1-URA3 promoter in pRS314GU (
Quantitation of Dnm1p-GFP Localization
dnm1 fzo1
cells carrying pGAL1-DNM1-GFP were incubated in galactose media for 12 h, stained with MitoTracker Red CMXRos (Molecular Probes). 12 cells were examined by fluorescence microscopy and the mitochondrially associated Dnm1p-GFP dots (82 total) were assigned to one of two locations: (a) the end of a tubule (50 dots), or (b) the side of a tubule (32 dots). The end of a tubule was defined as when the center of a Dnm1p-GFP dot was located within 0.15 µm from the end. The average length of the mitochondrial tubules was estimated to be 2.7 ± 1.9 µm and the diameter ~0.3 µm (n = 44). We calculate that the side of the tubule represents 89% of the mitochondrial surface area and the remainder (11%) represents the end.
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Results and Discussion |
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We screened for yeast mutants defective in mitochondrial shape using a novel strategy in which mitochondria are visualized by the green fluorescent protein (GFP) and mutants were isolated by micromanipulation. GFP was fused to the presequence (residues 121) of mitochondrial cytochrome oxidase subunit IV (COX4;
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Of ~72,000 cells screened, we isolated 20 mutants, which were classified into four categories (Figure 1 B). Class I mutants (two isolates) contained one or two large, spherical mitochondria instead of the normal tubules seen in wild-type cells. Genetic crosses showed that both carried mdm10 mutants (
Genetic crosses showed that our 14 class IV mutants comprised 8 recessive, 5 dominant and 1 semi-dominant mutations. Mapping studies showed that all 14 mutations were centromere linked (1.1 cM) and located on chromosome XII. We noted that DNM1 ( strain and a plasmid containing DNM1 (kindly provided by J. Shaw), we found that all 14 class IV mutants carried dnm1 alleles. These results were unexpected since mitochondrial shape in our mutants was strikingly different from previously seen in dnm1 mutants, where mitochondria collapse to one side of the cell and form a single tubule (
A complete disruption of DNM1 coding sequences was constructed, and examined for mitochondrial shape (Figure 1 C). ~90% of dnm1 cells showed a single highly branched mitochondrial network. ~10% of dnm1
cells displayed a single mitochondrial tubule localized to one side of the cell, similar to that seen earlier (
In yeast, mitochondria are very dynamic, fusing or dividing on average every two minutes ( and fzo1
by genetic crosses. We found that normal mitochondrial shape was restored (Figure 2). ~85% of double mutants contained multiply-branched, tubular mitochondria very similar to those seen in wild-type cells (Table 1). This was in marked contrast to dnm1
mutants, which usually had a single organelle, and fzo1
mutants, with numerous mitochondrial fragments (Figure 2;
fzo1
cells were not always completely normal; the tubules tended to be longer and more curved than in wild-type cells, and occasionally formed bundles. Nonetheless, our observations suggest that excess mitochondrial division in fzo1
cells is suppressed by inactivating DNM1, and that excess mitochondrial fusion in dnm1
cells is rescued by fzo1
. We propose that division, which requires Dnm1p, and fusion, controlled by Fzo1p, have antagonistic effects on mitochondrial shape and number. Our results also suggest that mitochondrial tubule formation occurs by a mechanism independent of fusion and division.
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Interestingly, mitochondrial shape and number in dnm1 fzo1
cells was dependent upon the order of gene disruption. When cells were first disrupted for FZO1 and subsequently for DNM1 (Table 1, fzo1
dnm1
), ~40% of cells carried mitochondrial fragments similar to those seen in fzo1
single mutants. In contrast, when cells were first disrupted for DNM1 and then for FZO1 (Table 1, dnm1
fzo1
), ~30% of cells displayed a mitochondrial network like that seen in dnm1
cells. Our results indicate that the mitochondrial networks found in dnm1
mutants persist in the absence of fusion activity, and fragments formed in the fzo1
mutant persist in the absence of fission activity. We also found tubular mitochondria in many of the double mutant cells formed by consecutive gene disruption (~50% for fzo1
dnm1
; ~60% for dnm1
fzo1
). These results further indicate that mitochondrial tubules form in the absence of division and fusion. It is not clear why dnm1
fzo1
double mutants generated by crossing a dnm1
cell to a fzo1
cell contained mostly (>80%) tubular mitochondria and essentially no mitochondrial networks or fragments (Table 1). During germination and growth of a dnm1
fzo1
spore, it is possible that cells are simultaneously depleted of Dnm1p and Fzo1p, leading to the formation of tubules, but not networks or fragments.
To further test the role of Dnm1p in division, we induced Dnm1p expression in dnm1 fzo1
cells and observed its effect on mitochondria. Dnm1p was fused to the HA epitope (Dnm1p-HA) (
phenotype on galactose medium (not shown). When dnm1
fzo1
cells containing pGAL1-DNM1-HA were grown in the absence of galactose, no Dnm1p-HA was detected (Figure 3 B) and ~70% of cells displayed the tubular mitochondria typical of dnm1
fzo1
mutants (Figure 3 A). Upon transfer to galactose medium, Dnm1p-HA levels gradually increased, while the level of hexokinase, a control protein, remained constant (Figure 3 B). Concomitant with the accumulation of Dnm1p-HA, mitochondrial shape changed dramatically (Figure 3 A). The number of cells with tubular mitochondria decreased, and those with fragmented mitochondria increased. By 5 h, ~65% of the cells contained completely fragmented mitochondria. At intermediate times (2 h) after inducing Dnm1p-HA, cells contained partially fragmented tubules, and many mitochondrial tubules were adjacent to small fragments. Our results clearly show that the division of mitochondria in dnm1
fzo1
cells coincides with the expression of Dnm1p.
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Further supporting a role for Dnm1p in mitochondrial fission, we found that the Dnm1 protein was preferentially localized to sites of mitochondrial division. We constructed a fusion between Dnm1p and the green fluorescent protein (GFP). Consistent with previous results ( fzo1
mutant and examined cells at early times after induction. We found a tight correlation between the appearance of Dnm1p-GFP and fragmentation of mitochondria. Two representative cells are shown in Figure 4B and Figure C; both cells contained two Dnm1p-GFP dots, one of which was located at the end of a tubule, the other appeared to be reside near a constricted region of the mitochondrion. After analysis of additional cells, we found that Dnm1p-GFP was localized to ends of mitochondrial fragments much more frequently (>60%) than predicted if Dnm1p-GFP was randomly distributed on mitochondria (~11%). These results suggest that Dnm1p acts at the site of mitochondrial fission. We note that Dnm1p-GFP is not exclusively found at the ends of mitochondria. We surmise that Dnm1p on the sides of the tubules may mark future sites of division, or represent Dnm1p-containing complexes that have diffused away from the end of the tubule. More definitive experiments (e.g., time-lapse videomicroscopy) to determine the role of Dnm1p in fission are in progress.
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The relatively normal mitochondria seen in dnm1 fzo1
mutants could be explained by a restoration of fusion activity; for example, if Dnm1p were an inhibitor of mitochondrial fusion. To test this possibility, we monitored mitochondrial fusion during mating (
cells, which did not carry pCLbGFP, and allowed to mate on glucose medium. Mitochondria were visualized in zygotes using MitoTracker. If mitochondrial fusion occurred, GFP and MitoTracker fluorescence would completely overlap, because the matrix-localized CS1-GFP from the MATa mitochondria diffused into the mitochondrial matrix of the MAT
cell. If no fusion occurred, GFP-labeled mitochondria would be seen in only one half of the zygote.
Zygotes formed by two wild-type cells, or two dnm1 mutants, exhibited efficient mitochondrial fusion, with GFP fluorescence and MitoTracker overlapping in all mitochondria (Figure 4 A). In contrast, fusion was defective in fzo1
mutants, consistent with previous observations (
cells and individual fragments were difficult to distinguish. Nonetheless, in matings between two fzo1
cells, MitoTracker showed clusters of fragmented mitochondria in the zygote and diploid bud, but we detected GFP fluorescence in only half of the mitochondrial clusters. Like fzo1
mutants, dnm1
fzo1
double mutants failed to fuse their mitochondria. Although mitochondria in dnm1
fzo1
/dnm1
fzo1
diploid cells had normal shape, only half of the organelles contained GFP. Our results indicate that dnm1
fzo1
cells are defective in mitochondrial fusion.
To eliminate the possibility that low, basal levels of fusion occur in dnm1 fzo1
cells, we used a more sensitive fusion assay using the matrix markers, CS1-GFP and mitochondrial DNA (mtDNA;
cells, which contained mtDNA, but not the plasmid (Figure 5 B). The DAPI and GFP fluorescence overlapped in all the mitochondrial tubules in wild-type zygotes (52 zygotes examined). When 500 dnm1
fzo1
zygotes were examined, we found no overlap between DAPI and GFP. The fusion activity in dnm1
fzo1
mutants is therefore at least 500-fold less than that in wild-type cells. Even after the zygotes were allowed to grow and divide, we found no fusion in the mutant cells (Figure 5 B). When 100 dnm1
fzo1
/dnm1
fzo1
diploid cells were examined 24 h after mating, none contained an overlap between GFP and DAPI, whereas a complete GFP and DAPI overlap was seen in 43 wild-type diploids. dnm1
fzo1
cells clearly lack significant mitochondrial fusion activity. Our results above also suggest that dnm1
fzo1
cells lack fission activity. We therefore propose that in cells lacking Fzo1p and Dnm1p, mitochondrial tubule formation occurs by a mechanism independent of fusion and division, such as growth from the ends of preexisting organelles.
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Dynamin has been proposed to work as a mechanochemical enzyme that pinches off plasma membrane invaginations, forming intracellular vesicles (
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Footnotes |
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1 Abbreviations used in this paper: GFP, green fluorescent protein; mtDNA, mitochondrial DNA.
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Acknowledgements |
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We especially thank J. Shaw for generous gifts of strains and plasmids. We also thank J. Holder, O. Kerscher, S. Michaelis, J. Boeke, A. Aiken, A. Davis, R. Butow, K. Okamoto, D. Murphy, R. Bustos, C. Machamer, and T. Kai for reagents, equipment and technical advice. We thank C. Machamer, K. Wilson, and the members of the Jensen lab for valuable comments on the manuscript.
This work was supported by a PHS grant (R01-GM54021) to R.E. Jensen and a JSPS fellowship to H. Sesaki.
Submitted: 27 July 1999
Revised: 1 October 1999
Accepted: 4 October 1999
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References |
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