The topoisomerase II-associated protein, Pat1p, is required for maintenance of rDNA locus stability in Saccharomyces cerevisiae.

The Pat1 protein of Saccharomyces cerevisiae was identified during a screen for proteins that interact with topoisomerase II. Previously, we have shown that pat1Δ mutants exhibit a slow-growth phenotype and an elevated frequency of both mitotic and meiotic chromosome mis-segregation. Here, we have studied the effects of deleting the PAT1 gene on chromosomal stability, with particular reference to rates of homologous recombination within the rDNA locus. This locus was analyzed because rDNA-specific hyperrecombination is known to occur in conditional top2 mutants. We show that pat1Δ strains mimic top2 mutants in displaying an elevated rate of intrachromosomal excision recombination at the rDNA locus, but not elsewhere in the genome. The elevated rate of recombination is dependent upon Rad52p, but not upon Rad51p or Rad54p. However, pat1Δ strains display additional manifestations of more general genomic instability, in that they show mild sensitivity to UV light and an increased incidence of interchromosomal recombination between heteroalleles. Received: 14 December 1998 / Accepted: 22 February 1999     

Pat1: a topoisomerase II-associated protein required for faithful chromosome transmission in Saccharomyces cerevisiae.

Saccharomyces cerevisiae top2 mutants deficient in topoisomerase II activity are defective in chromosome segregation during both mitotic and meiotic cell divisions. To identify proteins that act in concert with topoisomerase II during chromosome segregation in S.cerevisiae, we have used a two-hybrid cloning approach. We report the isolation of the PAT1 gene (for protein associated with topoisomerase II), which encodes a novel 90 kDa proline- and glutamine-rich protein that interacts with a highly conserved, leucine-rich region of topoisomerase II in vivo. Strains lacking Pat1p exhibit a slow growth rate and a phenotype reminiscent of conditional top2 mutants grown at the semi-permissive temperature; most notably, a reduced fidelity of chromosome segregation during both mitosis and meiosis. These findings indicate that the PAT1 gene is necessary for accurate chromosome transmission during cell division in eukaryotic cells and suggest that the interaction of Pat1p and topoisomerase II is an important component of this function.     

Inp1p is a peroxisomal membrane protein required for peroxisome inheritance in Saccharomyces cerevisiae

Cells have evolved molecular mechanisms for the efficient transmission of organelles during cell division. Little is known about how peroxisomes are inherited. Inp1p is a peripheral membrane protein of peroxisomes of Saccharomyces cerevisiae that affects both the morphology of peroxisomes and their partitioning during cell division. In vivo 4-dimensional video microscopy showed an inability of mother cells to retain a subset of peroxisomes in dividing cells lacking the INP1 gene, whereas cells overexpressing INP1 exhibited immobilized peroxisomes that failed to be partitioned to the bud. Overproduced Inp1p localized to both peroxisomes and the cell cortex, supporting an interaction of Inp1p with specific structures lining the cell periphery. The levels of Inp1p vary with the cell cycle. Inp1p binds Pex25p, Pex30p, and Vps1p, which have been implicated in controlling peroxisome division. Our findings are consistent with Inp1p acting as a factor that retains peroxisomes in cells and controls peroxisome division. Inp1p is the first peroxisomal protein directly implicated in peroxisome inheritance.     

Pag1p, a novel protein associated with protein kinase Cbk1p, is required for cell morphogenesis and proliferation in Saccharomyces cerevisiae

Protein kinases in the Cot-1/Orb6/Ndr/Warts family are important regulators of cell morphogenesis and proliferation. Cbk1p, a member of this family in Saccharomyces cerevisiae, has previously been shown to be required for normal morphogenesis in vegetatively growing cells and in haploid cells responding to mating pheromone. A mutant of PAG1, a novel gene in S. cerevisiae, displayed defects similar to those of cbk1 mutants. pag1 and cbk1 mutants share a common set of suppressors, including the disruption of SSD1, a gene encoding an RNA binding protein, and the overexpression of Sim1p, an extracellular protein. These genetic results suggest that PAG1 and CBK1 act in the same pathway. Furthermore, we found that Pag1p and Cbk1p localize to the same polarized peripheral sites and that they coimmunoprecipitate with each other. Pag1p is a conserved protein. The homologs of Pag1p in other organisms are likely to form complexes with the Cbk1p-related kinases and function with those kinases in the same biological processes.     

Saccharomyces cerevisiae Mob1p is required for cytokinesis and mitotic exit

The Saccharomyces cerevisiae mitotic exit network (MEN) is a conserved set of genes that mediate the transition from mitosis to G(1) by regulating mitotic cyclin degradation and the inactivation of cyclin-dependent kinase (CDK). Here, we demonstrate that, in addition to mitotic exit, S. cerevisiae MEN gene MOB1 is required for cytokinesis and cell separation. The cytokinesis defect was evident in mob1 mutants under conditions in which there was no mitotic-exit defect. Observation of live cells showed that yeast myosin II, Myo1p, was present in the contractile ring at the bud neck but that the ring failed to contract and disassemble. The cytokinesis defect persisted for several mitotic cycles, resulting in chains of cells with correctly segregated nuclei but with uncontracted actomyosin rings. The cytokinesis proteins Cdc3p (a septin), actin, and Iqg1p/ Cyk1p (an IQGAP-like protein) appeared to correctly localize in mob1 mutants, suggesting that MOB1 functions subsequent to actomyosin ring assembly. We also examined the subcellular distribution of Mob1p during the cell cycle and found that Mob1p first localized to the spindle pole bodies during mid-anaphase and then localized to a ring at the bud neck just before and during cytokinesis. Localization of Mob1p to the bud neck required CDC3, MEN genes CDC5, CDC14, CDC15, and DBF2, and spindle pole body gene NUD1 but was independent of MYO1. The localization of Mob1p to both spindle poles was abolished in cdc15 and nud1 mutants and was perturbed in cdc5 and cdc14 mutants. These results suggest that the MEN functions during the mitosis-to-G(1) transition to control cyclin-CDK inactivation and cytokinesis.     

Mitochondrial protein synthesis is required for maintenance of intact mitochondrial genomes in Saccharomyces cerevisiae.

The genes of Saccharomyces cerevisiae coding for the mitochondrial threonine and tryptophan tRNA synthetases and for a putative mitochondrial ribosomal protein have been cloned. These, and the previously cloned gene for a mitochondrial elongation factor, were used to disrupt or partially delete the wild-type chromosomal copies of the genes in the respiratory-competent strain W303. In each case, inactivation of a gene whose product is required for mitochondrial protein synthesis causes an instability in mitochondrial DNA. Although intact mitochondrial genomes are rapidly and quantitatively eliminated in the protein synthesis defective strains, specific rho- genomes can be maintained stably over many generations. These results indicate that mitochondrial protein synthesis is required for the propagation of wild-type mitochondrial DNA in yeast.     

Ded1p, a DEAD-box protein required for translation initiation in Saccharomyces cerevisiae, is an RNA helicase.

The Ded1 protein (Ded1p), a member of the DEAD-box family, has recently been shown to be essential for translation initiation in Saccharomyces cerevisiae. Here, we show that Ded1p purified from Escherichia coli has an ATPase activity, which is stimulated by various RNA substrates. Using an RNA strand-displacement assay, we show that Ded1p has also an ATP-dependent RNA unwinding activity. Hydrolysis of ATP is required for this activity: the replacement of ATP by a nonhydrolyzable analog or a mutation in the DEAD motif abolishing ATPase activity results in loss of RNA unwinding. We find that cells harboring a Ded1 protein with this mutated DEAD motif are nonviable, suggesting that the ATPase and RNA helicase activities of this protein are essential to the cell. Finally, RNA binding measurements indicate that the presence of ATP, but not ADP, increases the affinity of Ded1p for duplex versus single-stranded RNA; we discuss how this differential effect might drive the unwinding reaction.     

The sodium/proton exchanger Nhx1p is required for endosomal protein trafficking in the yeast Saccharomyces cerevisiae

We show that the vacuolar protein sorting gene VPS44 is identical to NHX1, a gene that encodes a sodium/proton exchanger. The Saccharomyces cerevisiae protein Nhx1p shows high homology to mammalian sodium/proton exchangers of the NHE family. Nhx1p is thought to transport sodium ions into the prevacuole compartment in exchange for protons. Pulse-chase experiments show that approximately 35% of the newly synthesized soluble vacuolar protein carboxypeptidase Y is missorted in nhx1 delta cells, and is secreted from the cell. nhx1 delta cells accumulate late Golgi, prevacuole, and lysosome markers in an aberrant structure next to the vacuole, and late Golgi proteins are proteolytically cleaved more rapidly than in wild-type cells. Our results show that efficient transport out of the prevacuolar compartment requires Nhx1p, and that nhx1 delta cells exhibit phenotypes characteristic of the "class E" group of vps mutants. In addition, we show that Nhx1p is required for protein trafficking even in the absence of the vacuolar ATPase. Our analysis of Nhx1p provides the first evidence that a sodium/proton exchange protein is important for correct protein sorting, and that intraorganellar ion balance may be important for endosomal function in yeast.     

The Saccharomyces cerevisiae centromere protein Slk19p is required for two successive divisions during meiosis

Meiotic cell division includes two separate and distinct types of chromosome segregation. In the first segregational event the sister chromatids remain attached at the centromere; in the second the chromatids are separated. The factors that control the order of chromosome segregation during meiosis have not yet been identified but are thought to be confined to the centromere region. We showed that the centromere protein Slk19p is required for the proper execution of meiosis in Saccharomyces cerevisiae. In its absence diploid cells skip meiosis I and execute meiosis II division. Inhibiting recombination does not correct this phenotype. Surprisingly, the initiation of recombination is apparently required for meiosis II division. Thus Slk19p appears to be part of the mechanism by which the centromere controls the order of meiotic divisions.     

A cytoskeleton-related gene, uso1, is required for intracellular protein transport in Saccharomyces cerevisiae

The Saccharomyces cerevisiae mutant strains blocked in the protein secretion pathway are not able to induce sexual aggregation. We have utilized the defect of aggregation to concentrate the secretion-deficient cells and identified a new gene which functions in the process of intracellular protein transport. The new mutant, uso1, is temperature sensitive for growth and protein secretion. At the restrictive temperature (37 degrees C), uso1 mutant accumulated the core-glycosylated precursor form of the exported protein invertase in the cells. Ultrastructural study of the mutant fixed by the freeze-substitution method revealed expansion of the nuclear envelope lumen and accumulation of the ER at the restrictive temperature. Abnormally oriented bundles of microtubules were often found in the nucleus. The USO1 gene was cloned by complementation of the uso1 temperature-sensitive growth defect. DNA sequence analysis revealed a hydrophilic protein of 1790 amino acids with a COOH-terminal 1,100-amino acid-long alpha-helical structure characteristic of the coiled-coil rod region of the cytoskeleton-related proteins. These observations suggest that Uso1 protein plays a role as a cytoskeletal component in the protein transport from the ER to the later secretory compartments.     

Mnl1p, an alpha -mannosidase-like protein in yeast Saccharomyces cerevisiae, is required for endoplasmic reticulum-associated degradation of glycoproteins

The endoplasmic reticulum (ER) has a mechanism to block the exit of misfolded or unassembled proteins from the ER for the downstream organelles in the secretory pathway. Misfolded proteins retained in the ER are subjected to proteasome-dependent degradation in the cytosol when they cannot achieve correct folding and/or assembly within an appropriate time window. Although specific mannose trimming of the protein-bound oligosaccharide is essential for the degradation of misfolded glycoproteins, the precise mechanism for this recognition remains obscure. Here we report a new alpha-mannosidase-like protein, Mnl1p (mannosidase-like protein), in the yeast ER. Mnl1p is unlikely to exhibit alpha1,2-mannosidase activity, because it lacks cysteine residues that are essential for alpha1,2-mannosidase. However deletion of the MNL1 gene causes retardation of the degradation of misfolded carboxypeptidase Y, but not of the unglycosylated mutant form of the yeast alpha-mating pheromone. Possible roles of Mnl1p in the degradation and in the ER-retention of misfolded glycoproteins are discussed.     

The ubiquitin ligase Rsp5 is required for ribosome stability in Saccharomyces cerevisiae

Rsp5p is a conserved HECT-domain ubiquitin ligase with diverse roles in cellular physiology. Here we report a previously unknown role of Rsp5p in facilitating the stability of the cytoplasmic ribosome pool in budding yeast. Yeast strains carrying temperature-sensitive mutations in RSP5 showed a progressive decline in levels of 18S and 25S rRNAs and accumulation of rRNA decay fragments when cells grown in rich medium were shifted to restrictive temperature. This was accompanied by a decreased number of translating ribosomes and the appearance of ribosomal subunits with an abnormally low sedimentation rate in polysome analysis. Abrogating Rsp5p function affected stability of other tested noncoding RNA species (tRNA and snoRNA), but to a lower extent than that of rRNA, and also inhibited processing of rRNA and tRNA precursors, in agreement with previous studies. The breakdown of cellular ribosomes was not affected by deletion of key genes involved in autophagy, previously implicated in ribosome turnover upon starvation. Our results suggest that functional Rsp5p is required to maintain the integrity of cytoplasmic ribosomes under rich nutrient conditions.     

The kinesin-related protein Kip1p of Saccharomyces cerevisiae is bipolar.

Kip1p is a mitotic spindle-associated kinesin-related protein in Saccharomyces cerevisiae that participates in spindle pole separation. Here, we define the domain arrangement and polypeptide composition of the Kip1p holoenzyme. Electron microscopy of rotary shadowed Kip1p molecules revealed two globular domains 14 nm in diameter connected by a 73-nm long stalk. When the Kip1p domain homologous to the kinesin motor domain was decorated with an unrelated protein, the diameter of the globular domains at both ends of the stalk increased, indicating that Kip1p is bipolar. Soluble Kip1p isolated from S. cerevisiae cells was homomeric, based on the similarity of the sedimentation coefficients of native Kip1p from S. cerevisiae and Kip1p which was purified after expression in insect cells. The holoenzyme molecular weight was estimated using the sedimentation coefficient and Stokes radius, and was most consistent with a tetrameric composition. Kip1p exhibited an ionic strength-dependent transition in its sedimentation coefficient, revealing a potential regulatory mechanism. The position of kinesin motor-related domains at each end of the stalk may allow Kip1p to cross-link either parallel or antiparallel microtubules during mitotic spindle assembly and pole separation.     

The GLC7 type 1 protein phosphatase is required for glucose repression in Saccharomyces cerevisiae.

We cloned the GLC7/DIS2S1 gene by complementation of the cid1-226 mutation, which relieves glucose repression in Saccharomyces cerevisiae. GLC7 encodes the catalytic subunit of type 1 protein phosphatase (PP1). Genetic analysis and sequencing showed that cid1-226 is an allele of GLC7, now designated glc7-T152K, which alters threonine 152 to lysine. We also show that the glc7-1 and glc7-T152K alleles cause distinct phenotypes: glc7-1 causes a severe defect in glycogen accumulation but does not relieve glucose repression, whereas glc7-T152K does not prevent glycogen accumulation. These findings are discussed in light of evidence that interaction with different regulatory or targeting subunits directs the participation of PP1 in diverse cellular regulatory mechanisms. Finally, genetic studies suggest that PP1 functions antagonistically to the SNF1 protein kinase in the regulatory response to glucose.     

Replication protein A is required for meiotic recombination in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, meiotic recombination is initiated by transient DNA double-stranded breaks (DSBs). These DSBs undergo a 5' --> 3' resection to produce 3' single-stranded DNA ends that serve to channel DSBs into the RAD52 recombinational repair pathway. In vitro studies strongly suggest that several proteins of this pathway--Rad51, Rad52, Rad54, Rad55, Rad57, and replication protein A (RPA)--play a role in the strand exchange reaction. Here, we report a study of the meiotic phenotypes conferred by two missense mutations affecting the largest subunit of RPA, which are localized in the protein interaction domain (rfa1-t11) and in the DNA-binding domain (rfa1-t48). We find that both mutant diploids exhibit reduced sporulation efficiency, very poor spore viability, and a 10- to 100-fold decrease in meiotic recombination. Physical analyses indicate that both mutants form normal levels of meiosis-specific DSBs and that the broken ends are processed into 3'-OH single-stranded tails, indicating that the RPA complex present in these rfa1 mutants is functional in the initial steps of meiotic recombination. However, the 5' ends of the broken fragments undergo extensive resection, similar to what is observed in rad51, rad52, rad55, and rad57 mutants, indicating that these RPA mutants are defective in the repair of the Spo11-dependent DSBs that initiate homologous recombination during meiosis.     

Aux1p/Swa2p is required for cortical endoplasmic reticulum inheritance in Saccharomyces cerevisiae

In the yeast Saccharomyces cerevisiae, the endoplasmic reticulum (ER) is found at the periphery of the cell and around the nucleus. The segregation of ER through the mother-bud neck may occur by more than one mechanism because perinuclear, but not peripheral ER, requires microtubules for this event. To identify genes whose products are required for cortical ER inheritance, we have used a Tn3-based transposon library to mutagenize cells expressing a green fluorescent protein-tagged ER marker protein (Hmg1p). This approach has revealed that AUX1/SWA2 plays a role in ER inheritance. The COOH terminus of Aux1p/Swa2p contains a J-domain that is highly related to the J-domain of auxilin, which stimulates the uncoating of clathrin-coated vesicles. Deletion of the J-domain of Aux1p/Swa2p leads to vacuole fragmentation and membrane accumulation but does not affect the migration of peripheral ER into daughter cells. These findings suggest that Aux1p/Swa2p may be a bifunctional protein with roles in membrane traffic and cortical ER inheritance. In support of this hypothesis, we find that Aux1p/Swa2p localizes to ER membranes.