Article |
Address correspondence to Benedikt Westermann, Institut für Physiologische Chemie, Universität München, Butenandtstr. 5, D-81377 München, Germany. Tel.: 49-89-2180-77122. Fax: 49-89-2180-77093. E-mail: benedikt.westermann{at}bio.med.uni-muenchen.de
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Abstract |
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Key Words: membrane fission; mitochondria; mitochondrial dynamics; organelle morphology; Saccharomyces cerevisiae
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Introduction |
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Baker's yeast, Saccharomyces cerevisiae, turned out to be a powerful model organism to study the molecular machinery mediating mitochondrial distribution and morphology (Hermann and Shaw, 1998; Jensen et al., 2000; Boldogh et al., 2001; Shaw and Nunnari, 2002). Yeast mitochondria form a branched tubular network located immediately below the cell cortex (Hoffmann and Avers, 1973; Egner et al., 2002). The continuity of the mitochondrial reticulum is maintained by continuous membrane fusion and fission events (Nunnari et al., 1997; Bleazard et al., 1999; Sesaki and Jensen, 1999). Two proteins of the outer membrane, Fzo1 and Ugo1, were found to be involved in the fusion of mitochondria. Yeast mutants lacking either one of these proteins exhibit fragmented mitochondria because fusion is blocked in the presence of ongoing fission. As a secondary consequence of aberrant mitochondrial morphology, fzo1 and ugo1 mutants fail to inherit mitochondrial DNA (mtDNA),* resulting in a respiratory-deficient growth phenotype (Hermann et al., 1998; Rapaport et al., 1998; Sesaki and Jensen, 2001).
Division of the mitochondrial outer membrane in yeast depends on the dynamin-related GTPase Dnm1, which assembles on the surface of mitochondrial tubules at sites of organelle constriction and fission (Otsuga et al., 1998; Bleazard et al., 1999; Sesaki and Jensen, 1999). Dnm1 cooperates with the soluble protein Mdv1 (Fekkes et al., 2000; Tieu and Nunnari, 2000; Cerveny et al., 2001; Tieu et al., 2002) and the integral outer membrane protein Fis1 (Mozdy et al., 2000). Mutants lacking either one of these proteins exhibit long tubular mitochondria or closed planar nets of interconnected mitochondria, a phenotype indicative of a strong defect in mitochondrial division. Inheritance of mtDNA is not affected in these mutants. Mitochondrial fragmentation and the respiratory growth defect of mutants defective in fusion can be suppressed by deletion of components of the outer membrane division machinery (Bleazard et al., 1999; Sesaki and Jensen, 1999; Fekkes et al., 2000; Mozdy et al., 2000; Tieu and Nunnari, 2000; Cerveny et al., 2001; Sesaki and Jensen, 2001).
Mitochondria are complex organelles bounded by two membranes. Thus, mechanisms must exist that coordinate fusion and fission events of the outer and inner membranes. The fusion machinery of the outer membrane is in contact with as yet unknown factors of the inner membrane, and this connection appears to be critical to coordinate fusion of four mitochondrial membranes (Fritz et al., 2001). A second mitochondrial dynamin-related protein, Mgm1, is located in the intermembrane space. In contrast to Dnm1, it is not clear whether Mgm1 is involved in membrane fission events. However, it has been suggested that Mgm1 is involved in remodeling events of the mitochondrial inner membrane (Wong et al., 2000). Other proteins that might be involved in fusion and fission of the inner membrane have not been described.
We recently conducted a systematic genome-wide screen to identify novel yeast genes important for mitochondrial distribution and morphology, MDM. One of the mutants isolated in this screen, mdm33, exhibits giant ring-like mitochondrial structures, a phenotype that has never been described before for any other yeast mutant (Dimmer et al., 2002). Here, we report on the functional characterization of Mdm33 and discuss a possible role of this protein in fission of the mitochondrial inner membrane.
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Results |
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When the outer membrane was opened by osmotic swelling of mitochondria after import, a specific fragment was generated by protease treatment of mitoplasts (Fig. 2 B, lane 5). The apparent molecular mass of this fragment corresponds well to the predicted size of a segment ranging from the processing site to the end of the first predicted transmembrane domain (34 kD), suggesting that this part is protected by the inner membrane in mitoplasts. The fragment was not generated in the absence of a membrane potential (Fig. 2 B, lane 7), indicating that it was a derivative of imported Mdm33. As the imported protein was resistant to alkaline extraction (Fig. 2 C), it is an integral part of the membrane. We conclude that Mdm33 is located in the mitochondrial inner membrane with its NH2-terminal part in the matrix and its COOH-terminal part exposed to the intermembrane space.
To further investigate the intracellular localization, we expressed epitope-tagged versions of Mdm33 in a mdm33 background. A tagged version carrying 13 copies of the myc epitope fused to the COOH terminus of Mdm33 was exclusively detected in mitochondria upon subfractionation of cells (unpublished data). However, this construct was unable to rescue the mitochondrial morphology defect of the deletion mutant, suggesting that the COOH-terminal end of Mdm33 is important for function of the protein. Next, we constructed a chimeric protein carrying a GFP moiety inserted between the mitochondrial presequence and the NH2-terminal end of mature Mdm33. This construct, GFPMdm33, rescued the mitochondrial defect of the deletion mutant (unpublished data). Immunoelectron microscopy demonstrated an association of GFPMdm33 with the inner membrane, both at the cristae and the inner boundary membrane, the part of the inner membrane that faces the outer membrane (Fig. 2 D). The fact that a fraction of GFPMdm33 was found in cristae suggests that Mdm33 does not form stable contacts to the outer membrane.
mdm33 cells harbor aberrant mitochondria
We examined the phenotype of the mdm33 mutant by various methods. Cells harboring mitochondrial matrixtargeted GFP (mtGFP) were grown to mid-logarithmic growth phase in liquid cultures containing different carbon sources and examined by fluorescence microscopy. Under all conditions, the majority of
mdm33 cells harbored either large mitochondrial ring-like structures (Fig. 3 B) or two to four smaller mitochondrial rings that were often interconnected (Fig. 3 C). The remainder of the cells displayed either single elongated structures or aggregated or fragmented mitochondria. Branched tubular networks resembling wild-type cells (Fig. 3 A) could never be observed in
mdm33 cells. A quantification of mitochondrial morphology of
mdm33 cells and its isogenic wild type is presented in Table I.
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Aberrant mdm33 mitochondria form hollow spheres, interconnected sheets, and rings
To examine the three-dimensional organization of mdm33 mitochondria, cells expressing mtGFP were analyzed by confocal fluorescence microscopy. A representative
mdm33 cell containing a giant spherical organelle is depicted in Fig. 4. Large mitochondrial rings were observed in several consecutive focal planes (Fig. 4, AK). Ring-shaped structures similar to those seen in the x/y planes were observed also in sections corresponding to z/y (Fig. 4 L) and z/x planes (Fig. 4 M). Apparently, this mitochondrion formed a large hollow sphere of
4 µm diameter. A view onto a surface-rendered three-dimensional representation of this organelle is depicted in Fig. 4 O. Similar structures were observed in many other
mdm33 cells. Often these organelles appeared to be closed, sometimes they contained openings of varying sizes, or they formed sheets typically located at one side of the cell (see supplemental figures, Figs. S1S6, available at http://www.jcb.org/cgi/content/full/jcb.200211113/DC1).
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We examined the internal structure of mitochondria in cells overexpressing Mdm33 (Fig. 6, GJ). The most striking phenotype was the formation of inner membrane septa, membranous partitions separating the inner compartment into distinct chambers. Apparently, these partitions consisted of two parallel membranes in direct continuity with the inner membrane (Figs. 6, G and I, arrows). Furthermore, vesicular structures accumulated within the organelles, some of which appeared to be disconnected from the inner boundary membrane (Fig. 6, H and J). Some organelles were seen that were largely devoid of cristae (Fig. 6 H). All mitochondria were still bounded by a double membrane. We consider it likely that overexpression of Mdm33 initially results in the formation of inner membrane septa. With ongoing septation of the mitochondrial interior, smaller inner membrane fragments are generated that eventually become disconnected from the inner boundary membrane, leaving a largely cristae-free space. Our results demonstrate an important role of Mdm33 in maintenance of the structure of the mitochondrial inner membrane.
Epistatic relationships of mdm33,
fis1, and
fzo1 mutations
Establishment and maintenance of a tubular mitochondrial network depends on antagonistic fission and fusion events (Nunnari et al., 1997). Block of mitochondrial fission by deletion of any one of the DNM1, MDV1, or FIS1 genes leads to the formation of long tubular mitochondria or closed mitochondrial nets (Otsuga et al., 1998; Bleazard et al., 1999; Sesaki and Jensen, 1999; Fekkes et al., 2000; Mozdy et al., 2000; Tieu and Nunnari, 2000; Cerveny et al., 2001), whereas block of mitochondrial fusion by deletion of either one of the FZO1 or UGO1 genes leads to highly fragmented mitochondria (Hermann et al., 1998; Rapaport et al., 1998; Sesaki and Jensen, 2001). To investigate whether Mdm33 is involved in these processes, we constructed mdm33/
fis1 and
mdm33/
fzo1 double mutants by genetic crosses and examined their phenotypes.
The mdm33/
fis1 double mutant was viable on nonfermentable carbon sources (unpublished data). Mitochondrial morphology was very similar to the
mdm33 parent, and clearly different from
fis1. Most of the
mdm33/
fis1 cells displayed mitochondrial ring structures, whereas the elongated net-like mitochondria of
fis1 cells (Mozdy et al., 2000) could never be found in the double mutant (Table III). Thus, the
mdm33 mitochondrial morphology defect is epistatic to the
fis1 mitochondrial morphology defect. Similarly, overexpression of Mdm33 in
fis1 cells induced the same growth arrest and mitochondrial aggregation phenotypes that were observed in a FIS1 wild-type background (unpublished data). These results demonstrate that Mdm33 acts upstream of Fis1.
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Stable interactions of Mdm33 with outer membrane proteins known to be involved in mitochondrial morphogenesis appear unlikely, because Mdm33 is present both in the inner boundary membrane and in cristae (Fig. 2 D). If Mdm33 would be in contact to outer membrane proteins, it would be expected to be located exclusively in parts of the inner membrane that face the outer membrane. Moreover, genetic evidence suggests that Mdm33 function is not coupled to that of the outer membrane proteins Fis1 or Fzo1 (see above). Yet, we cannot exclude that Mdm33 interacts with other known or unknown proteins. However, the observed homotypic interactions of Mdm33 might explain why mdm33 was the only mutant displaying giant ring-shaped mitochondria in a comprehensive genome-wide screen for mitochondrial morphology mutants (Dimmer et al., 2002). Thus, no other known MDM component would be a likely interaction partner of Mdm33 based on its location, function, or phenotype. We suggest that Mdm33 performs homo-oligomeric interactions.
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Discussion |
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Second, deletion of the MDM33 gene leads to the formation of large organelles that typically contain long stretches of outer and inner membranes enclosing a very narrow matrix space. These extremely extended structures can form only in the absence of frequent organellar division events. As mitochondrial fusion is not compromised in the mdm33 strain, elongated organelles likely undergo self-fusion when two ends of the same organelle approach one another. Such a self-fusion event initially would form a ring structure, and this is indeed observed by electron microscopy. The enclosed spheres seen by confocal microscopy are probably generated when two-dimensional rings subsequently close also in the third dimension.
Third, overexpression of Mdm33 results in septation and vesiculation of the inner membrane and disappearance of inner membrane cristae. This phenotype can be best explained by greatly enhanced inner membrane fission activity in the presence of excess Mdm33. In particular, the appearance of septated mitochondria induced by Mdm33 overexpression bears striking similarity to dividing mitochondria in various tissues described in the literature (e.g., Tandler et al., 1969; Larsen, 1970; Tandler and Hoppel, 1972; Wakabayashi et al., 1974; Duncan and Greenaway, 1981; for review see Griparic and van der Bliek, 2001). Partitioning of mitochondria is likely due to enhanced fission activity of the inner membrane in the absence of division of the outer membrane.
Fourth, Mdm33 is an integral protein of the inner membrane and has extensive coiled-coil domains exposed to the matrix side. This topology and the observed homotypic proteinprotein interactions are in agreement with the following possible mode of action. Mdm33 protein complexes on apposing inner membranes would interact with each other from the matrix side, possibly by forming -helical bundles via Mdm33 coiled coils. Formation of
-helical bundles would then mediate constriction and/or fission of the inner membrane from the matrix side.
Maintenance of normal mitochondrial morphology by balanced membrane fission and fusion depends on several distinct, yet interdependent, processes. Based on the results reported herein and in previous studies by others, we propose a hypothetical cycle of sequential reactions of inner membrane fission, outer membrane fission, and mitochondrial fusion (Fig. 8). The first step of the cycle is inner membrane fission. Division of the mitochondrial outer and inner membranes can be uncoupled. Mitochondria of worms expressing dominant interfering mutants of DRP-1, the Caenorhabditis elegans homologue of yeast Dnm1, had swollen mitochondria with irregular shapes and sizes. Intriguingly, these mitochondria contained separated matrix compartments that were still connected by thin tubules of outer membrane. Thus, inner membrane fission persists, even when outer membrane fission is blocked by loss of DRP-1 function (Labrousse et al., 1999). An independent machinery appears to mediate fission of the mitochondrial inner membrane. Mdm33 is likely to play an important role to control this process in yeast.
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The cycle is completed by mitochondrial fusion. In yeast, mutations leading to deficiency in outer membrane fission are epistatic to mutations leading to deficiency in fusion, i.e., mitochondrial fragmentation and loss of mtDNA in fusion mutants is blocked by mutation of components of the outer membrane fission machinery (Bleazard et al., 1999; Sesaki and Jensen, 1999, 2001; Fekkes et al., 2000; Mozdy et al., 2000; Tieu and Nunnari, 2000; Cerveny et al., 2001). On the other hand, mitochondrial fusion is a prerequisite for the formation of extended mdm33 mitochondria, as we found that
fzo1 is epistatic to
mdm33. Why does deletion of the MDM33 gene not prevent fragmentation of mitochondria in
fzo1 cells, similar to deletion of genes encoding components of the outer membrane fission machinery? Interestingly, Sesaki and Jensen (1999) observed that mitochondrial fragments persisted in
40% of the cells when cells were first disrupted for FZO1 and subsequently for DNM1. This suggests that once the equilibrium of fission and fusion has been shifted in the direction of fission, it is difficult to rebuild larger mitochondrial structures without fusion activity. In case of the
mdm33/
fzo1 strain, the action of Dnm1 and its cofactors might well be sufficient to induce and maintain fragmentation of mitochondria, even in the absence of Mdm33.
Abnormal ring-shaped or cup-shaped mitochondria in various animal and human tissues have been described. These include, for example, parathyroid gland cells of senile dogs (Setoguti, 1977), renal oncocytes in rats (Krech et al., 1981), avian retinal pigment epithelium cells under continuous light (Lauber, 1982), Purkinje cells of the rat cerebrellar cortex after prolonged alcohol consumption (Tavares and Paula-Barbosa, 1983), and human salivary tumor cells (Kataoka et al., 1991). It will be interesting to see in the future whether there is a link between defects of mitochondrial division and various pathological conditions, and whether proteins similar to Mdm33 are involved.
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Materials and methods |
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Yeast strain constructions
Standard methods were used for growth and manipulation of yeast strains (Sherman et al., 1986; Gietz et al., 1992). Growth of yeast was always at 30°C. All strains used for microscopy and genetic studies were isogenic to BY4741, BY4742, and BY4743 (Brachmann et al., 1998). Haploid deletion strains mdm33,
fis1, and
fzo1 were obtained from EUROSCARF (Frankfurt, Germany). Double mutants
mdm33/
fis1 and
mdm33/
fzo1 were generated by mating of haploid strains, sporulation, and tetrad dissection. Genotypes of haploid progeny were determined by PCR. For overexpression of Mdm33, yeast strains were transformed with pYX223-MDM33 and grown on galactose-containing medium lacking histidine to select for the overexpressing plasmid. For expression of GFPMdm33 in yeast, pMM112 was transformed into
mdm33.
For visualization of mitochondria, yeast strains were transformed with plasmid pVT100U-mtGFP, pYX113-mtGFP, pYX122-mtGFP, pYX142-mtGFP (Westermann and Neupert, 2000), or pRS416-GAL1+PrF0ATP9RFP (Mozdy et al., 2000). For visualization of the ER, yeast strains were transformed with pWP1055 (Prinz et al., 2000), and transformants were grown in the absence of methionine to induce the MET25 promoter for expression of ER-targeted GFP. For visualization of the cytosol, yeast strains were transformed with pVT102U-cRFP.
Import of radiolabeled proteins into isolated mitochondria
Mitochondria of strain W303 were isolated by differential centrifugation as previously described (Diekert et al., 2001). Translation of proteins in vitro in the presence of [35S]methionine and import into isolated mitochondria were performed essentially as previously described (Ryan et al., 2001), with the exception that an energy-regenerating system was added, and 1 µM valinomycin was used to dissipate the mitochondrial membrane potential (Westermann et al., 1995). Hypotonic swelling, protease treatment of mitochondria (Diekert et al., 2001), and carbonate extraction of imported proteins (Fritz et al., 2001) were performed as previously described. Samples were analyzed by SDS-PAGE, blotting to nitrocellulose, and autoradiography.
Microscopy
Yeast cultures were grown in liquid YPD medium (yeast extract, peptone, dextrose) to mid-logarithmic growth phase, if not indicated otherwise. Epifluorescence microscopy was according to standard procedures (Westermann and Neupert, 2000). Quantification of mitochondrial morphology defects was performed without prior reference to strain identity. Confocal images were taken with a Leica TCS SP2 beam scanning confocal microscope equipped with a 1.4 NA oil immersion lens (Leica 100X; Planapo). For imaging, living cells were embedded in 1% low melting point agarose.
Electron microscopy and immunocytochemistry were performed as previously described (Kärgel et al., 1996). In brief, yeast cells were harvested by centrifugation and fixed for 1 h with 4% formaldehyde and 0.5% glutaraldehyde under culture conditions (pH and temperature were kept constant), cryoprotected by a mixture of 25% polyvinylpyrrolidone (PVP K15; Mr 10,000; Fluka) and 1,6 M sucrose for 3 h, and frozen in liquid nitrogen. Ultrathin cryosections were prepared with glass knifes and transferred to formvar-carboncoated copper grids using the cryoprotectant mixture. Labeling with anti-GFP IgG primary antibodies and secondary antibodygold complexes (10 nm; Dianova) was performed as described (Kärgel et al., 1996). Finally, the sections were stained and stabilized by a freshly prepared mixture of 3% tungstosilicic acid hydrate (Fluka) and 2.5% polyvinyl alcohol (Mr 10,000; Sigma-Aldrich).
Assay of mitochondrial fusion in vivo
Mitochondrial fusion was examined in vivo essentially as previously described (Nunnari et al., 1997; Mozdy et al., 2000) with some minor modifications. Cells of opposite mating types harboring plasmids encoding mtGFP or mtRFP under control of the GAL1 promoter were precultured in synthetic raffinose-containing medium under selection for the plasmids. Then, cells were grown to log phase in synthetic medium containing raffinose and galactose to induce expression of the fluorescent proteins. Then, yeast cells were incubated for 2 h in YPD medium (pH adjusted to 3.5) to shut off expression of the fluorescent proteins and to synchronize the cell cycle. Cultures were mixed, cells were transferred to nitrocellulose, placed for 3 h on YPD plates (pH 4.5) to allow mating, resuspended in water, and analyzed by fluorescence microscopy.
Miscellaneous
Gel filtration (Rapaport et al., 1998) and coimmunoprecipitation (Herrmann et al., 2001) were performed according to published procedures. Staining of the actin cytoskeleton with rhodamine-phalloidin (Molecular Probes) was performed as previously described (Amberg, 1998). Staining of the vacuole with 7-amino-4-chloromethylcoumarin, L-arginine amide (Molecular Probes) was performed according to the manufacturer's instructions.
Online supplemental material
The supplemental figures (Figs. S1S6) are available online at http://www.jcb.org/cgi/content/full/jcb.200211113/DC1. Mitochondrial morphology of wild-type and mdm33 cells expressing mtGFP was analyzed by confocal fluorescence microscopy as in Fig. 4.
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Footnotes |
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* Abbreviations used in this paper: cRFP, cytosolic red fluorescent protein; mtDNA, mitochondrial DNA; mtGFP, mitochondrial matrixtargeted GFP.
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Acknowledgments |
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This work was supported by the Deutsche Forschungsgemeinschaft through grants WE 2174/2-3 and SFB 413/B3.
Submitted: 25 November 2002
Revised: 7 January 2003
Accepted: 7 January 2003
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References |
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---|
Amberg, D.C. 1998. Three-dimensional imaging of the yeast actin cytoskeleton through the budding cell cycle. Mol. Biol. Cell. 9:32593262.
Andaluz, E., J.J. Coque, R. Cueva, and G. Larriba. 2001. Sequencing of a 4.3 kbp region of chromosome 2 of Candida albicans reveals the presence of homologues of SHE9 from Saccharomyces cerevisiae and of bacterial phosphatidylinositol-phospholipase C. Yeast. 18:711721.[CrossRef][Medline]
Bereiter-Hahn, J. 1990. Behavior of mitochondria in the living cell. Int. Rev. Cytol. 122:163.[Medline]
Bleazard, W., J.M. McCaffery, E.J. King, S. Bale, A. Mozdy, Q. Tieu, J. Nunnari, and J.M. Shaw. 1999. The dynamin-related GTPase Dnm1 regulates mitochondrial fission in yeast. Nat. Cell Biol. 1:298304.[CrossRef][Medline]
Boldogh, I.R., H.-C. Yang, and L.A. Pon. 2001. Mitochondrial inheritance in budding yeast. Traffic. 2:368374.[CrossRef][Medline]
Brachmann, C.B., A. Davies, G.J. Cost, E. Caputo, J. Li, P. Hieter, and J.D. Boeke. 1998. Designer deletion strains derived from Saccharomyces cerevisiae S288C: a useful set of strains and plasmids for PCR-mediated gene disruption and other applications. Yeast. 14:115132.[CrossRef][Medline]
Cerveny, K.L., J.M. McCaffery, and R.E. Jensen. 2001. Division of mitochondria requires a novel DNM1-interacting protein, Net2p. Mol. Biol. Cell. 12:309321.
Diekert, K., A.I. de Kroon, G. Kispal, and R. Lill. 2001. Isolation and subfractionation of mitochondria from the yeast Saccharomyces cerevisiae. Methods Cell Biol. 65:3751.[Medline]
Dimmer, K.S., S. Fritz, F. Fuchs, M. Messerschmitt, N. Weinbach, W. Neupert, and B. Westermann. 2002. Genetic basis of mitochondrial function and morphology in Saccharomyces cerevisiae. Mol. Biol. Cell. 13:847853.
Duncan, C.J., and H.C. Greenaway. 1981. The induction of septation and subdivision in muscle mitochondria. Comp. Biochem. Physiol. 69A:329331.
Egner, A., S. Jakobs, and S.W. Hell. 2002. Fast 100 nm resolution 3D-microscope reveals structural plasticity of mitochondria in live yeast. Proc. Natl. Acad. Sci. USA. 99:33703375.
Espinet, C., M.A. de la Torre, M. Aldea, and E. Herrero. 1995. An efficient method to isolate yeast genes causing overexpression-mediated growth arrest. Yeast. 11:2532.[Medline]
Fekkes, P., K.A. Shepard, and M.P. Yaffe. 2000. Gag3p, an outer membrane protein required for fission of mitochondrial tubules. J. Cell Biol. 151:333340.
Frank, S., B. Gaume, E.S. Bergmann-Leitner, W.W. Leitner, E.G. Robert, F. Catez, C.L. Smith, and R.J. Youle. 2001. The role of dynamin-related protein 1, a mediator of mitochondrial fission, in apoptosis. Dev. Cell. 1:515525.[Medline]
Fritz, S., D. Rapaport, E. Klanner, W. Neupert, and B. Westermann. 2001. Connection of the mitochondrial outer and inner membranes by Fzo1 is critical for organellar fusion. J. Cell Biol. 152:683692.
Gietz, D., A.S. Jean, R.A. Woods, and R.H. Schiestl. 1992. Improved method for high efficiency transformation of intact yeast cells. Nucleic Acids Res. 20:1425.[Medline]
Griparic, L., and A.M. van der Bliek. 2001. The many shapes of mitochondrial membranes. Traffic. 2:235244.[Medline]
Hales, K.G., and M.T. Fuller. 1997. Developmentally regulated mitochondrial fusion mediated by a conserved, novel, predicted GTPase. Cell. 90:121129.[Medline]
Hartl, F.-U., N. Pfanner, D.W. Nicholson, and W. Neupert. 1989. Mitochondrial protein import. Biochim. Biophys. Acta. 988:145.[Medline]
Hermann, G.J., and J.M. Shaw. 1998. Mitochondrial dynamics in yeast. Annu. Rev. Cell Dev. Biol. 14:265303.[CrossRef][Medline]
Hermann, G.J., J.W. Thatcher, J.P. Mills, K.G. Hales, M.T. Fuller, J. Nunnari, and J.M. Shaw. 1998. Mitochondrial fusion in yeast requires the transmembrane GTPase Fzo1p. J. Cell Biol. 143:359373.
Herrmann, J.M., B. Westermann, and W. Neupert. 2001. Analysis of protein-protein interactions in mitochondria by coimmunoprecipitation and chemical cross-linking. Methods Cell Biol. 65:217230.[Medline]
Hoffmann, H.-P., and C.J. Avers. 1973. Mitochondrion of yeast: ultrastructural evidence for one giant, branched organelle per cell. Science. 181:749750.[Medline]
Hofmann, K., and W. Stoffel. 1993. TMbase - a database of membrane spanning protein segments. Biol. Chem. Hoppe-Seyler. 347:166.
Jensen, R.E., A.E. Aiken Hobbs, K.L. Cerveny, and H. Sesaki. 2000. Yeast mitochondrial dynamics: fusion, division, segregation, and shape. Microsc. Res. Tech. 51:573583.[CrossRef][Medline]
Kärgel, E., R. Menzel, H. Honeck, F. Vogel, A. Böhmer, and W.-H. Schunck. 1996. Candida maltosa NADPH-cytochrome P450 reductase: cloning of a full-length cDNA, heterologous expression in Saccharomyces cerevisiae and function of the N-terminal region for membrane anchoring and proliferation of the endoplasmic reticulum. Yeast. 12:333348.[CrossRef][Medline]
Kataoka, R., Y. Hyo, T. Hoshiya, H. Miyahara, and T. Matsunaga. 1991. Ultrastructural study of mitochondria in oncocytes. Ultrastruct. Pathol. 15:231239.[Medline]
Krech, R., H. Zerban, and P. Bannasch. 1981. Mitochondrial anomalies in renal oncocytes induced in rat by N-nitrosomorpholine. Eur. J. Cell Biol. 25:331339.[Medline]
Labrousse, A.M., M.D. Zappaterra, D.A. Rube, and A.M. van der Bliek. 1999. C. elegans dynamin-related protein DRP-1 controls severing of the mitochondrial outer membrane. Mol. Cell. 4:815826.[Medline]
Larsen, W.J. 1970. Genesis of mitochondria in insect fat body. J. Cell Biol. 47:373383.
Lauber, J.K. 1982. Retinal pigment epithelium: ring mitochondria and lesions induced by continuous light. Curr. Eye Res. 2:855862.[Medline]
Lupas, A., M. Van Dyke, and J. Stock. 1991. Predicting coiled coils from protein sequences. Science. 252:11621164.[Medline]
Mozdy, A., J.M. McCaffery, and J.M. Shaw. 2000. Dnm1p GTPase-mediated mitochondrial fusion is a multistep process requiring the novel integral membrane component Fis1p. J. Cell Biol. 151:367379.
Nunnari, J., W.F. Marshall, A. Straight, A. Murray, J.W. Sedat, and P. Walter. 1997. Mitochondrial transmission during mating in Saccharomyces cerevisiae is determined by mitochondrial fusion and fission and the intramitochondrial segregation of mitochondrial DNA. Mol. Biol. Cell. 8:12331242.[Abstract]
Ono, T., K. Isobe, K. Nakada, and J.I. Hayashi. 2001. Human cells are protected from mitochondrial dysfunction by complementation of DNA products in fused mitochondria. Nat. Genet. 28:272275.[CrossRef][Medline]
Otsuga, D., B.R. Keegan, E. Brisch, J.W. Thatcher, G.J. Hermann, W. Bleazard, and J.M. Shaw. 1998. The dynamin-related GTPase, Dnm1p, controls mitochondrial morphology in yeast. J. Cell Biol. 143:333349.
Prinz, W.A., L. Grzyb, M. Veenhuis, J.A. Kahana, P.A. Silver, and T.A. Rapoport. 2000. Mutants affecting the structure of the cortical endoplasmic reticulum in Saccharomyces cerevisiae. J. Cell Biol. 150:461474.
Rapaport, D., M. Brunner, W. Neupert, and B. Westermann. 1998. Fzo1p is a mitochondrial outer membrane protein essential for the biogenesis of functional mitochondria in Saccharomyces cerevisiae. J. Biol. Chem. 273:2015020155.
Ryan, M.T., W. Voos, and N. Pfanner. 2001. Assaying protein import into mitochondria. Methods Cell Biol. 65:189215.[Medline]
Sambrook, J., E.F. Fritsch, and T. Maniatis. 1989. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. 1659 pp.
Scheffler, I.E. 2001. A century of mitochondrial research: achievements and perspectives. Mitochondrion. 1:331.[CrossRef]
Sesaki, H., and R.E. Jensen. 1999. Division versus fusion: Dnm1p and Fzo1p antagonistically regulate mitochondrial shape. J. Cell Biol. 147:699706.
Sesaki, H., and R.E. Jensen. 2001. UGO1 encodes an outer membrane protein required for mitochondrial fusion. J. Cell Biol. 152:11231134.
Setoguti, T. 1977. Electron microscopic studies of the parathyroid gland of senile dogs. Am. J. Anat. 148:6583.[Medline]
Shaw, J.M., and J. Nunnari. 2002. Mitochondrial dynamics and division in budding yeast. Trends Cell Biol. 12:178184.[CrossRef][Medline]
Sherman, F., G.R. Fink, and J. Hicks. 1986. Methods in Yeast Genetics: A Laboratory Course. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. 198 pp.
Sikorski, R.S., and P. Hieter. 1989. A system of shuttle vectors and host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. Genetics. 122:1927.
Tandler, B., and C.L. Hoppel. 1972. Possible division of cardiac mitochondria. Anat. Rec. 173:309323.[Medline]
Tandler, B., R.A. Erlandson, A.L. Smith, and E.L. Wynder. 1969. Riboflavin and mouse hepatic cell structure and function. II. Division of mitochondria during recovery from simple deficiency. J. Cell Biol. 41:477493.
Tavares, M.A., and M.M. Paula-Barbosa. 1983. Mitochondrial changes in rat Purkinje cells after prolonged alcohol consumption. A morphologic assessment. J. Submicrosc. Cytol. 15:713720.[Medline]
Tieu, Q., and J. Nunnari. 2000. Mdv1p is a WD repeat protein that interacts with the dynamin-related GTPase, Dnm1p, to trigger mitochondrial division. J. Cell Biol. 151:353365.
Tieu, Q., V. Okreglak, K. Naylor, and J. Nunnari. 2002. The WD repeat protein, Mdv1p, functions as a molecular adaptor by interacting with Dnm1p and Fis1p during mitochondrial fission. J. Cell Biol. 158:445452.
Vernet, T., D. Dignard, and D.Y. Thomas. 1987. A family of yeast expression vectors containing the phage f1 intergenic region. Gene. 52:225233.[CrossRef][Medline]
Wakabayashi, T., M. Asano, and C. Kurono. 1974. Some aspects of mitochondria having a "septum." J. Electron Microsc. (Tokyo). 23:247254.[Medline]
Warren, G., and W. Wickner. 1996. Organelle inheritance. Cell. 84:395400.[Medline]
Westermann, B. 2002. Merging mitochondria matters. Cellular role and molecular machinery of mitochondrial fusion. EMBO Rep. 3:527531.
Westermann, B., and W. Neupert. 2000. Mitochondria-targeted green fluorescent proteins: convenient tools for the study of organelle biogenesis in Saccharomyces cerevisiae. Yeast. 16:14211427.[CrossRef][Medline]
Westermann, B., C. Prip-Buus, W. Neupert, and E. Schwarz. 1995. The role of the GrpE homologue, Mge1p, in mediating protein import and folding in mitochondria. EMBO J. 14:34523460.[Abstract]
Wong, E.D., J.A. Wagner, S.W. Gorsich, J.M. McCaffery, J.M. Shaw, and J. Nunnari. 2000. The dynamin-related GTPase, Mgm1p, is an intermembrane space protein required for maintenance of fusion-competent mitochondria. J. Cell Biol. 151:341352.
Yaffe, M.P. 1999. The machinery of mitochondrial inheritance and behavior. Science. 283:14931497.