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. 151:353–365), 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.

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...

Telomerase is an RNA-dependent reverse transcriptase that maintains telomeric DNA at a species-specific equilibrium length. To determine an upper limit for the number of telomerase molecules in a Saccharomyces cerevisiae cell, we have established real-time RT-PCR assays to quantify the noncoding telomerase RNA, TLC1. We find that the number of TLC1 molecules in a haploid yeast cell is ;29, less than the number of chromosome ends (64) in late S-phase. Wild-type diploid cells contain ;37 telomerase RNAs, while diploids heterozygous for a null tlc1 allele have half the wild-type amount, ;19 TLC1 molecules. For comparison, there are ;480 molecules of the U2 snRNA per haploid cell. We show that a biological consequence of this low level of telomerase is haploinsufficiency: A TLC1/tlc1D heterozygote maintains shorter telomeres. A dominant-negative telomerase RNA, with a deletion of the template for telomeric DNA synthesis, further demonstrates that yeast telomere length is sensitive to telomerase dosage. Sixfold overexpression of tlc1Dtemplate establishes a new telomere length set point, ;160 bp shorter than wild type. Removing telomerase protein-interaction sites from the tlc1Dtemplate RNA mitigates the dominantnegative effect, suggesting that the tlc1Dtemplate RNA competes with wild-type TLC1 for a limited supply of telomerase proteins or for telomeres. Because yeast telomerase is tethered at chromosome ends, the finding that it may be outnumbered by its telomeric DNA substrates provides a new perspective for interpreting the results of telomere maintenance studies.

Membrane fusion is fundamental to eukaryotic life. Unlike the predominant intracellular fusion machineries that fuse compartments bounded by a single membrane, the mitochondrial fusion machinery must sequentially fuse the outer and inner mitochondrial membranes. These coordinated fusion events rely on a transmembrane GTPase that is known as fuzzy onions or Fzo. Recent studies have revealed that Fzo has an evolutionarily conserved role in mitochondrial fusion, and they take the first strides in determining the molecular nature of such a role.