Developmental Cell 5 (2003) 539-545. doi:10.1016/S1534-5807(03)00296-XReceived by publisher: 0000-01-01Harvest Date: 2016-01-04 12:23:40DOI: 10.1016/S1534-5807(03)00296-XPage Range: 539-54

Quiescence is the most common and, arguably, most poorly understood cell cycle state. This is in part because pure populations of quiescent cells are typically difficult to isolate. We report the isolation and characterization of quiescent and nonquiescent cells from stationary-phase (SP) yeast cultures by density-gradient centrifugation. Quiescent cells are dense, unbudded daughter cells formed after glucose exhaustion. They synchronously reenter the mitotic cell cycle, suggesting that they are in a G0 state. Nonquiescent cells are less dense, heterogeneous, and composed of replicatively older, asynchronous cells that rapidly lose the ability to reproduce. Microscopic and flow cytometric analysis revealed that nonquiescent cells accumulate more reactive oxygen species than quiescent cells, and over 21 d, about half exhibit signs of apoptosis and necrosis. The ability to isolate both quiescent and nonquiescent yeast cells from SP cultures provides a novel, tractable experimental system for studies of quiescence, chronological and replicative aging, apoptosis, and the cell cycle.

The mitochondrial inner membrane consists of two domains, inner boundary membrane and cristae membrane that are connected by crista junctions. Mitofilin/Fcj1 was reported to be involved in formation of crista junctions, however, different views exist on its function and possible partner proteins. We report that mitofilin plays a dual role. Mitofilin is part of a large inner membrane complex, and we identify five partner proteins as constituents of the mitochondrial inner membrane organizing system (MINOS) that is required for keeping cristae membranes connected to the inner boundary membrane. Additionally, mitofilin is coupled to the outer membrane and promotes protein import via the mitochondrial intermembrane space assembly pathway. Our findings indicate that mitofilin is a central component of MINOS and functions as a multifunctional regulator of mitochondrial architecture and protein biogenesis.

No abstract

Protein degradation in the vacuole (lysosome) is an important event in cellular regulation. In yeast, as in mammalian cells, a major route of protein uptake for degradation into the vacuole (lysosome) has been found to be autophagocytosis. The discovery of this process in yeast enables the elucidation of its mechanisms via genetic and molecular biological investigations. Here we report the isolation of yeast mutants defective in autophagocytosis (aut mutants), using a rapid colony screening procedure.

Pex14p, an S. cerevisiae peroxin, is attached to the outer face of the peroxisomal membrane and is a component of the protein import machinery. Pex14p interacts with both the PTS1 and PTS2 receptors. It is the only known peroxisomal membrane protein that binds the PTS2 receptor and might thus mediate the membrane docking event of PTS2-dependent protein import. These results suggest that the two import pathways overlap and, furthermore, that Pex14p represents the point of convergence. Pex14p also interacts with two other membrane-bound peroxins including Pex13p, another binding protein for the PTS1 receptor. The data presented here are consistent with the idea of a common translocation machinery for both PTS-dependent protein import pathways in the peroxisomal membrane.

We find that the peripheral ER in Saccharomyces cerevisiae forms a dynamic network of interconnecting membrane tubules throughout the cell cycle, similar to the ER in higher eukaryotes. Maintenance of this network does not require microtubule or actin filaments, but its dynamic behavior is largely dependent on the actin cytoskeleton. We isolated three conditional mutants that disrupt peripheral ER structure. One has a mutation in a component of the COPI coat complex, which is required for vesicle budding. This mutant has a partial defect in ER segregation into daughter cells and disorganized ER in mother cells. A similar phenotype was found in other mutants with defects in vesicular trafficking between ER and Golgi complex, but not in mutants blocked at later steps in the secretory pathway. The other two mutants found in the screen have defects in the signal recognition particle (SRP) receptor. This receptor, along with SRP, targets ribosome–nascent chain complexes to the ER membrane for protein translocation. A conditional mutation in SRP also disrupts ER structure, but other mutants with translocation defects do not. We also demonstrate that, both in wild-type and mutant cells, the ER and mitochondria partially coalign, and that mutations that disrupt ER structure also affect mitochondrial structure. Our data suggest that both trafficking between the ER and Golgi complex and ribosome targeting are important for maintaining ER structure, and that proper ER structure may be required to maintain mitochondrial structure.

Two mutants of Saccharomyces cerevisiae affected in peroxisomal assembly (pas mutants) have been isolated and characterized. Each strain contains a single mutation that results in (i) the inability to grow on oleic acid, (ii) accumulation of peroxisomal matrix enzymes in the cytosol, and (iu) absence of detectable peroxisomes at the ultrastructural level. These lesions (pasl-l and pas2) are shown to be nonallelic and recessive. Crossing of pasi-l and pas2 strains resulted in diploid cells that had regained the ability to grow on oleic acid as sole carbon source and to form peroxisomes. These pas mutants may provide useful tools for future studies on the molecular mechanisms involved in peroxisomal assembly. Eukaryotic cells maintain a proper level of biochemical compartmentation by means of various distinct subcellular organelles. Peroxisomes represent a class of such organelles. They are widely distributed in eukaryotic organisms, including fungi, and are involved in a variety of metabolic processes (1-4). Their ubiquitous presence indicates that peroxisomes may fulfill a number of essential functions in the cells (2-5). In recent years, the importance of peroxisomes for mammalian cells has also become clear (6,7). This is especially emphasized in a newly recognized class of inborn human diseases, the peroxisomal disorders (8, 9). Dysfunction of peroxisomes in humans usually has profound clinical consequences.Zellweger syndrome was the first of these peroxisomal disorders to be reported (10) and appears to be caused by a single recessive mutation that abolishes the import of proteins into the peroxisomal matrix (11). However, the components ofthe peroxisomal import machinery are still entirely unknown.Yeast mutations have been very successfully used for the dissection of many complex cellular phenomena, such as the cell cycle (12) and the secretory pathway (13). Since it is possible to induce a pronounced peroxisomal proliferation in Saccharomyces cerevisiae (14), this yeast should be useful for the molecular analysis of the biogenesis of peroxisomes.In an approach to analyze sorting of peroxisomal proteins, we have set out to find yeast mutants impaired in peroxisomal functions. Here, we report the occurrence of such mutants among strains of S. cerevisiae that lost the capacity to grow on oleic acid. Emphasis is placed on the isolation and initial characterization of mutants that completely lack detectable peroxisomes. MATERIALS AND METHODSYeast Strains. S. cerevisiae strains were used for mutant isolation and crossing experiments: X2180-1A (MATa, mal, gal2, SUC2, CUP]); XP300-26D (a, ade2, trp5, his6, lysi, gal2); XJB3-1B (MATa, met6, gal2); met6, gall, gal2 (15). The survival rate was -50%. Expression of mutations was allowed by overnight growth on YNB medium. After 12 hr of preincubation on selective YNO medium, a nystatin enrichment was performed for 90 min at an antibiotic concentration of 10 gg/ml (16). Aliquots were plated on YNA-agar plates and subsequently replica plated on YNO-agar p...