The biogenesis of mitochondrial outer membrane proteins involves the general translocase of the outer membrane (TOM complex) and the sorting and assembly machinery (SAM complex). The two known subunits of the SAM complex, Mas37 and Sam50, are required for assembly of the abundant outer membrane proteins porin and Tom40. We have identified an unexpected subunit of the SAM complex, Mdm10, which is involved in maintenance of mitochondrial morphology. Mitochondria lacking Mdm10 are selectively impaired in the final steps of the assembly pathway of Tom40, including the association of Tom40 with the receptor Tom22 and small Tom proteins, while the biogenesis of porin is not affected. Yeast mutants of TOM40, MAS37, and SAM50 also show aberrant mitochondrial morphology. We conclude that Mdm10 plays a specific role in the biogenesis of the TOM complex, indicating a connection between the mitochondrial protein assembly apparatus and the machinery for maintenance of mitochondrial morphology.

Presenilin-1 (PS1) and -2 (PS2), which when mutated cause familial Alzheimer disease, have been localized to numerous compartments of the cell, including the endoplasmic reticulum, Golgi, nuclear envelope, endosomes, lysosomes, the plasma membrane, and mitochondria. Using three complementary approaches, subcellular fractionation, ␥-secretase activity assays, and immunocytochemistry, we show that presenilins are highly enriched in a subcompartment of the endoplasmic reticulum that is associated with mitochondria and that forms a physical bridge between the two organelles, called endoplasmic reticulum-mitochondria-associated membranes. A localization of PS1 and PS2 in mitochondria-associated membranes may help reconcile the disparate hypotheses regarding the pathogenesis of Alzheimer disease and may explain many seemingly unrelated features of this devastating neurodegenerative disorder. Alzheimer disease (AD) is a late onset neurodegenerative disorder characterized by progressive neuronal loss, especially in the cortex and the hippocampus. 1 The two main histopathological hallmarks of AD are the accumulation of extracellular neuritic plaques, consisting predominantly of ␤-amyloid (A␤), and of neurofibrillary tangles, consisting mainly of hyperphosphorylated forms of the microtubule-associated protein tau. 1The vast majority of AD is sporadic, but mutations in amyloid precursor protein (APP), presenilin-1 (PS1), and presenilin-2 (PS2) have been identified in the rarer familial form, which is similar to sporadic AD but has an earlier age of onset.PS1 and PS2 are aspartyl proteases that cleave their substrates within transmembrane regions. The active forms of PS1 and PS2 are N-and C-terminal fragments, which are produced by cleavage of full-length presenilin in its "loop" domain.2 PS1 and PS2 are components of the ␥-secretase complex that processes a number of plasma-membrane proteins, including Notch, Jagged, E-cadherin, and, most relevant to AD, APP. The ␥-secretase complex also contains three other structural subunits: APH1, nicastrin (also called APH2), and presenilin enhancer protein 2.2 Following cleavage of APP by ␤-secretase, ␥-secretase cleaves the ϳ100-aa C-terminal "␤-stub" to release small amyloidogenic fragments, 40-and 42-aa in length (A␤40 and A␤42), that have been implicated in the pathogenesis of AD, as well as a ϳ60-aa APP intracellular domain.

No abstract

We isolated two yeast mutants that are temperature-sensitive for import of mitochondrial proteins. Each strain contains a single mutation that results in arrest of growth and accumulation of precursor to the f8 subunit of the mitochondrial Fl-ATPase after incubation at 37°C. These lesions (masi and mas2) are nonallelic and recessive. Cells harboring either mutation stop growing only after 2-3 generations at 37°C. Import of the F1 fl subunit at 37C is more than 250 times slower in masl and 15 times slower in mas2 than in wildtype cells. At 23°C, import occurs with si~ihar rates in mutant and wild-type cells. The two mutations also reduce the rate of import of other proteins; however, import of different precursors is affected to different degrees in the two strains. The temperature-sensitive step in import in both masi and mas2 occurs before arrival of precursors in the mitochondrial matrix.A central feature of mitochondrial assembly is the import of mitochondrial proteins from their site of synthesis in the extramitochondrial cytoplasm (1). Key steps in this import process have been identified by isotope-tracer studies with intact cells and by incubation of isolated mitochondria or mitochondrial subfractions with radiolabeled protein precursors synthesized in vitro (2, 3). These approaches have revealed that most mitochondrial proteins are synthesized as precursors of larger molecular weight (2, 4) and that import involves: (i) specific interaction of precursors with the mitochondrial outer membrane (5, 6), (ii) energy-dependent translocation of polypeptides across one or both mitochondrial membranes (7,8), and (iii) processing of the precursors by a matrix-localized protease (9). Thus far, little is known about how this import pathway is regulated, and most of its molecular components remain to be identified or isolated.Conditional yeast mutants have been an important tool for the dissection of many complex cellular phenomena such as the cell cycle (10) days at 23TC. Tdmperature-sensitive mutants were identified by replica-plating the colonies onto two sets of YPD-agar plates and incubating these at 370C and 23TC, respectively. After 2-3 days, a second replica was prepared from the 370C plate, and the replica was again incubated at 37°C. Strains which grew on the 230C plate but not on the second 37°C replica were scored as temperature-sensitive mutants. Import mutants were identified by screening temperaturesensitive mutants for accumulation of the precursor to the 83 subunit of the Fl-ATPase after incubation at 37°C. Cells were picked from agar slants to 250 ,l of YPD-10 medium in 2.2-ml Eppendorf tubes and grown overnight with shaking at 23°C. One milliliter of YPD medium was then added, and the cultures were incubated for 7 hr at 37°C. Cells were lysed by addition of 160 ,ul of fresh 1.85 M NaOH/7.4% 2-mercaptoethanol to the total culture. This mixture was held on ice for 10 min, and protein was then precipitated by addition of 160 Al of 50% (wt/vol) trichloroacetic acid. After a further 10 min on i...

In Saccharomyces cerevisiae, mitochondrial fusion requires at least two outer membrane proteins, Fzo1p and Ugo1p. We provide direct evidence that the dynamin-related Mgm1 protein is also required for mitochondrial fusion. Like fzo1 and ugo1 mutants, cells disrupted for the MGM1 gene contain numerous mitochondrial fragments instead of the few long, tubular organelles seen in wild-type cells. Fragmentation of mitochondria in mgm1 mutants is rescued by disrupting DNM1, a gene required for mitochondrial division. In zygotes formed by mating mgm1 mutants, mitochondria do not fuse and mix their contents. Introducing mutations in the GTPase domain of Mgm1p completely block mitochondrial fusion. Furthermore, we show that mgm1 mutants fail to fuse both their mitochondrial outer and inner membranes. Electron microscopy demonstrates that although mgm1 mutants display aberrant mitochondrial inner membrane cristae, mgm1 dnm1 double mutants restore normal inner membrane structures. However, mgm1 dnm1 mutants remain defective in mitochondrial fusion, indicating that mitochondrial fusion requires Mgm1p regardless of the morphology of mitochondria. Finally, we find that Mgm1p, Fzo1p, and Ugo1p physically interact in the mitochondrial outer membrane. Our results raise the possibility that Mgm1p regulates fusion of the mitochondrial outer membrane through its interactions with Fzo1p and Ugo1p.

Abstract. Yeast cells with the mdm/0 mutation possess giant spherical mitochondria and are defective for mitochondrial inheritance. The giant mitochondria display classical features of mitochondrial ultrastructure, yet they appear incapable of movement or division. Genetic analysis indicated that the mutant phenotypes resulted from a single nuclear mutation, and the isolated MDMIO gene restored wild-type mitochondrial distribution and morphology when introduced into mutant cells. MDMIO encodes a protein of 56.2 kD located in the mitochondrial outer membrane. Depletion of Mdml0p from cells led to a condensation of normally extended, tubular mitochondria into giant spheres, and reexpression of the protein resulted in a rapid restoration of normal mitochondrial morphology. These results demonstrate that Mdml0p can control mitochondrial morphology, and that it plays a role in the inheritance of mitochondria. MITOCHONDRIAL inheritance is an essential component of cell proliferation (Yaffe, 1991b). This inheritance requires the growth and division of preexisting mitochondria and the distribution of mitochondria between daughter cells before cell division (Attardi and Schatz, 1988). Cytoskeletal elements have been implicated in the positioning and movement of mitochondria (Heggeness et al., 1978;Chen, 1988;McConnell and Yaffe, 1992;Drubin et al., 1993), but mechanisms underlying mitochondrial inheritance are poorly understood.Mitochondria are usually found as snakelike tubules, widely distributed in the eukaryotic cytoplasm (Tzagoloff, 1982;Bereiter-Hahn, 1990). Often, these tubules are interconnected into extended mitochondrial reticula (Stevens, 1981;Chen, 1988). Microscopic studies of live cells have revealed that these mitochondrial networks are extremely dynamic, with tubular processes undergoing frequent fragmentation, branching, and fusion, as well as redistribution within the cytoplasm (Bereiter-Hahn, 1990; Koning et ai., 1993). In addition to the dynamic properties of mitochondria found in most types of cells, certain pathological conditions lead to gross changes in mitochondrial morphology, including the development of giant mitochondria (Tandler and Hoppel, 1986;Inagaki et al., 1992). The molecular bases for these changes in mitochondrial morphology and distribution in both normal and diseased cells are unknown.We recently described several Saccharomyces cerevisiae mutants defective for mitochondrial inheritance (McConnell et al., 1990). These mitochondrial distribution and morphology (mdm) I mutants were identified by screening collections of temperature-sensitive strains by fluorescence microscopy for cells that failed to transfer mitochondria into daughter buds during incubation at the nonpermissive temperature. Analysis of two of these mutant strains led to the identification of a novel cytoskeletal component that mediates mitochondrial inheritance Yaffe, 1992, 1993), and it indicated a role for unsaturated fatty acids in mitochondrial movement (Stewart and Yaffe, 1991). Here, we describe a new mdm m...

Phb2p, a homolog of the tumor suppressor protein prohibitin, was identified in a genetic screen for suppressors of the loss of Mdm12p, a mitochondrial outer membrane protein required for normal mitochondrial morphology and inheritance in Saccharomyces cerevisiae. Phb2p and its homolog, prohibitin (Phb1p), were localized to the mitochondrial inner membrane and characterized as integral membrane proteins which depend on each other for their stability. In otherwise wild-type genetic backgrounds, null mutations in PHB1 and PHB2 did not confer any obvious phenotypes. However, loss of function of either PHB1 or PHB2 in cells with mitochondrial DNA deleted led to altered mitochondrial morphology, and phb1 or phb2 mutations were synthetically lethal when combined with a mutation in any of three mitochondrial inheritance components of the mitochondrial outer membrane, Mdm12p, Mdm10p, and Mmm1p. These results provide the first evidence of a role for prohibitin in mitochondrial inheritance and in the regulation of mitochondrial morphology.Mitochondrial inheritance is an essential and active process by which daughter cells receive mitochondria prior to the completion of cytokinesis. In budding yeast, factors specifically required for mitochondrial inheritance have been identified and characterized through the analysis of conditional mutants (7,25). Three distinct proteins of the mitochondrial outer membrane, Mdm10p, Mmm1p, and Mdm12p, have been found to constitute one class of mitochondrial inheritance factors. Each protein is required for normal mitochondrial morphology and inheritance, and mdm10, mmm1, and mdm12 loss-of-function mutants exhibit similar phenotypes of temperature-sensitive growth and enlarged, round mitochondria (6, 9, 39). At least one of these proteins, Mdm12p, has been evolutionarily conserved and possesses a homolog in the fission yeast Schizosaccharomyces pombe (6). While the location of these proteins in the mitochondrial outer membrane suggests that they may interact with cytoskeletal elements to mediate normal mitochondrial distribution, their molecular activity remains to be defined.To explore Mdm12p function, high-copy-number plasmidborne suppressors able to bypass the cellular requirement for Mdm12p were identified. This paper describes the characterization of a plasmid-borne suppressor encoding a prohibitinrelated protein localized to the mitochondrial inner membrane and exhibiting genetic interactions with mitochondrial outer membrane inheritance components. Prohibitins are a family of conserved proteins whose first member was identified as a negative regulator of cell division in cultured animal cells (29). Prohibitin homologs have been identified in diverse organisms and cell types and have been localized to mitochondria in animal and plant cells (20,38). The function of prohibitin at the molecular level is unknown. MATERIALS AND METHODSStrains and media. The Saccharomyces cerevisiae strains used in this work are listed in Table 1. All strains were derived from MYY290 or MYY291 (37). Media fo...

Saccharomyces cerevisiae cells lacking the MDM12 gene product display temperature-sensitive growth and possess abnormally large, round mitochondria that are defective for inheritance by daughter buds. Analysis of the wild-type MDM12 gene revealed its product to be a 31-kD polypeptide that is homologous to a protein of the fission yeast Schizosaccharomyces pombe. When expressed in S. cerevisiae, the S. pombe Mdm12p homolog conferred a dominant-negative phenotype of giant mitochondria and aberrant mitochondrial distribution, suggesting partial functional conservation of Mdm12p activity between budding and fission yeast. The S. cerevisiae Mdm12p was localized by indirect immunofluorescence microscopy and by subcellular fractionation and immunodetection to the mitochondrial outer membrane and displayed biochemical properties of an integral membrane protein. Mdm12p is the third mitochondrial outer membrane protein required for normal mitochondrial morphology and distribution to be identified in S. cerevisiae and the first such mitochondrial component that is conserved between two different species.