The dynamin-related GTPase Dnm1 controls mitochondrial morphology in yeast. Here we show that dnm1 mutations convert the mitochondrial compartment into a planar 'net' of interconnected tubules. We propose that this net morphology results from a defect in mitochondrial fission. Immunogold labelling localizes Dnm1 to the cytoplasmic face of constricted mitochondrial tubules that appear to be dividing and to the ends of mitochondrial tubules that appear to have recently completed division. The activity of Dnm1 is epistatic to that of Fzo1, a GTPase in the outer mitochondrial membrane that regulates mitochondrial fusion. dnm1 mutations prevent mitochondrial fragmentation in fzo1 mutant strains. These findings indicate that Dnm1 regulates mitochondrial fission, assembling on the cytoplasmic face of mitochondrial tubules at sites at which division will occur.The mitochondrion is a complex organelle with a double membrane, its own genome and an independent protein-synthetic machinery. Although the role of mitochondria in metabolism and ATP production is more widely recognized, alterations in mitochondrial shape and abundance are also important for cellular function and differentiation. For example, mitochondrial morphology and copy number change in response to nutrient availability in yeast cells 1 , cell damage and apoptosis in mammalian cells 2 and developmental cues in Xenopus and Drosophila 3,4 . Cytological studies indicate that the 'steady-state' mitochondrial morphology and copy number can vary dramatically in different cell types, ranging from multiple, spherical organelles to the branched, tubular networks found in budding yeast and some mammalian cells [5][6][7] . These differences in mitochondrial morphology and copy number are largely determined by the balance between ongoing mitochondrial fission and fusion events.Little is known about molecules that regulate mitochondrial fission and fusion. The fuzzy onions (fzo) family of transmembrane GTPases was recently shown to control mitochondrial fusion in different organisms and cell types. In Drosophila, fzo is required for a developmentally regulated mitochondrial fusion event during spermatogenesis 8 . In budding yeast, mutations in fzo1 cause mitochondrial networks to fragment 9,10 and prevent mitochondrial fusion during yeast mating 9 . Molecules required for mitochondrial fission © 1999 Macmillan Magazines Ltd § Correspondence and requests for materials should be addressed to J.M.S. shaw@bioscience.utah.edu. NIH Public Access Results Mitochondrial membranes form nets in dnm1 mutant cellsWe previously reported that mitochondrial membranes collapse to one side of the cell in a dnm1Δ mutant strain 11 . Transmission electron microscopy indicated that these collapsed membranes might be organized in an unusual structure 11 . To characterize this structure further, we studied mitochondrial morphology in dnm1Δ cells under several different conditions.As reported previously, dnm1Δ mutants grown at 25 °C lack the highly branched mitochondrial network charac...

The Saccharomyces cerevisiae Dnm1 protein is structurally related to dynamin, a GTPase required for membrane scission during endocytosis. Here we show that Dnm1p is essential for the maintenance of mitochondrial morphology. Disruption of the DNM1 gene causes the wild-type network of tubular mitochondrial membranes to collapse to one side of the cell but does not affect the morphology or distribution of other cytoplasmic organelles. Dnm1 proteins containing point mutations in the predicted GTP-binding domain or completely lacking the GTP-binding domain fail to rescue mitochondrial morphology defects in a dnm1 mutant and induce dominant mitochondrial morphology defects in wild-type cells. Indirect immunofluorescence reveals that Dnm1p is distributed in punctate structures at the cell cortex that colocalize with the mitochondrial compartment. These Dnm1p-containing structures remain associated with the spherical mitochondria found in an mdm10 mutant strain. In addition, a portion of Dnm1p cofractionates with mitochondrial membranes during differential sedimentation and sucrose gradient fractionation of wild-type cells. Our results demonstrate that Dnm1p is required for the cortical distribution of the mitochondrial network in yeast, a novel function for a dynamin-related protein.

Dnm1p belongs to a family of dynamin-related GTPases required to remodel different cellular membranes. In budding yeast, Dnm1p-containing complexes assemble on the cytoplasmic surface of the outer mitochondrial membrane at sites where mitochondrial tubules divide. Our previous genetic studies suggested that Dnm1p's GTPase activity was required for mitochondrial fission and that Dnm1p interacted with itself. In this study, we show that bacterially expressed Dnm1p can bind and hydrolyze GTP in vitro. Coimmunoprecipitation studies and yeast two-hybrid analysis suggest that Dnm1p oligomerizes in vivo. With the use of the yeast two-hybrid system, we show that this Dnm1p oligomerization is mediated, in part, by a C-terminal sequence related to the GTPase effector domain (GED) in dynamin. The Dnm1p interactions characterized here are similar to those reported for dynamin and dynamin-related proteins that form higher order structures in vivo, suggesting that Dnm1p assembles to form rings or collars that surround mitochondrial tubules. Based on previous findings, a K705A mutation in the Dnm1p GED is predicted to interfere with GTP hydrolysis, stabilize active Dnm1p-GTP, and stimulate a ratelimiting step in fission. Here we show that expression of the Dnm1 K705A protein in yeast enhances mitochondrial fission. Our results provide evidence that the GED region of a dynaminrelated protein modulates a rate-limiting step in membrane fission.