Many of the genes required for mating in Saccharomyces cerevisiae are also required for mating in Candida albicans

Candida albicans is the single, most frequently isolated human fungal pathogen. As with most fungal pathogens, the factors which contribute to pathogenesis in C. albicans are not known, despite more than a decade of molecular genetic analysis. Candida albicans was thought to be asexual until the discovery of the MTL loci homologous to the mating type (MAT) loci in Saccharomyces cerevisiae led to the demonstration that mating is possible. Using Candida albicans mutants in genes likely to be involved in mating, we analysed the process to determine its similarity to mating in Saccharomyces cerevisiae. We examined disruptions of three of the genes in the MAPK pathway which is involved in filamentous growth in both S. cerevisiae and C. albicans and is known to control pheromone response in the former fungus. Disruptions in HST7 and CPH1 blocked mating in both MTLa and MTL(alpha) strains, whereas disruptions in STE20 had no effect. A disruption in KEX2, a gene involved in processing the S. cerevisiae pheromone Mf(alpha), prevented mating in MTL(alpha) but not MTLa cells, whereas a disruption in HST6, the orthologue of the STE6 gene which encodes an ABC transporter responsible for secretion of the Mfa pheromone, prevented mating in MTLa but not in MTL(alpha) cells. Disruption of two cell wall genes, ALS1 and INT1, had no effect on mating, even though ALS1 was identified by similarity to the S. cerevisiae sexual agglutinin, SAG1. The results reveal that these two diverged yeasts show a surprising similarity in their mating processes.     

RSC1 and RSC2 are required for expression of mid-late sporulation-specific genes in Saccharomyces cerevisiae

Rsc1 and Rsc2 are alternative bromodomain-containing subunits of the ATP-dependent RSC chromatin remodeling complex in Saccharomyces cerevisiae. Smk1 is a sporulation-specific mitogen-activated protein kinase homolog that is required for the postmeiotic events of spore formation. In this study we show that RSC1 and RSC2 are haploinsufficient for spore formation in a smk1 hypomorph. Moreover, diploids lacking Rsc1 or Rsc2 show a subset of smk1-like phenotypes. High-copy-number RSC1 plasmids do not suppress rsc2-Delta/rsc2-Delta sporulation defects, and high-copy-number RSC2 plasmids do not suppress rsc1-Delta/rsc1-Delta sporulation defects. Mid-late sporulation-specific genes, which are normally expressed while key steps in spore assembly occur and which include genes that are required for spore wall formation, are not expressed in cells lacking Rsc1 or Rsc2. We speculate that the combined action of Rsc1 and Rsc2 at mid-late promoters is specifically required for the proper expression of this uniquely timed set of genes. Our data suggest that Smk1 and Rsc1/2 define parallel pathways that converge to provide signaling information and the expression of gene products, respectively, that are required for spore morphogenesis.     

RAD1 and RAD10, but not other excision repair genes, are required for double-strand break-induced recombination in Saccharomyces cerevisiae.

HO endonuclease-induced double-strand breaks (DSBs) in the yeast Saccharomyces cerevisiae can be repaired by the process of gap repair or, alternatively, by single-strand annealing if the site of the break is flanked by directly repeated homologous sequences. We have shown previously (J. Fishman-Lobell and J. E. Haber, Science 258:480-484, 1992) that during the repair of an HO-induced DSB, the excision repair gene RAD1 is needed to remove regions of nonhomology from the DSB ends. In this report, we present evidence that among nine genes involved in nucleotide excision repair, only RAD1 and RAD10 are required for removal of nonhomologous sequences from the DSB ends. rad1 delta and rad10 delta mutants displayed a 20-fold reduction in the ability to execute both gap repair and single-strand annealing pathways of HO-induced recombination. Mutations in RAD2, RAD3, and RAD14 reduced HO-induced recombination by about twofold. We also show that RAD7 and RAD16, which are required to remove UV photodamage from the silent HML, locus, are not required for MAT switching with HML or HMR as a donor. Our results provide a molecular basis for understanding the role of yeast nucleotide excision repair gene and their human homologs in DSB-induced recombination and repair.     

Saccharomyces cerevisiae mitochondria are required for optimal attractiveness to Drosophila melanogaster

While screening a large collection of wild and laboratory yeast strains for their ability to attract Drosophila melanogaster adults, we noticed a large difference in fly preference for two nearly isogenic strains of Saccharomyces cerevisiae, BY4741 and BY4742. Using standard genetic analyses, we tracked the preference difference to the lack of mitochondria in the BY4742 strain used in the initial experiment. We used gas chromatography coupled with mass spectroscopy to examine the volatile compounds produced by BY4741 and the mitochondria-deficient BY4742, and found that they differed significantly. We observed that several ethyl esters are present at much higher levels in strains with mitochondria, even in fermentative conditions. We found that nitrogen levels in the substrate affect the production of these compounds, and that they are produced at the highest level by strains with mitochondria when fermenting natural fruit substrates. Collectively these observations demonstrate that core metabolic processes mediate the interaction between yeasts and insect vectors, and highlight the importance mitochondrial functions in yeast ecology.     

Two MAPK-signaling pathways are required for mitophagy in Saccharomyces cerevisiae

Macroautophagy (hereafter referred to simply as autophagy) is a catabolic pathway that mediates the degradation of long-lived proteins and organelles in eukaryotic cells. The regulation of mitochondrial degradation through autophagy plays an essential role in the maintenance and quality control of this organelle. Compared with our understanding of the essential function of mitochondria in many aspects of cellular metabolism such as energy production and of the role of dysfunctional mitochondria in cell death, little is known regarding their degradation and especially how upstream signaling pathways control this process. Here, we report that two mitogen-activated protein kinases (MAPKs), Slt2 and Hog1, are required for mitophagy in Saccharomyces cerevisiae. Slt2 is required for the degradation of both mitochondria and peroxisomes (via pexophagy), whereas Hog1 functions specifically in mitophagy. Slt2 also affects the recruitment of mitochondria to the phagophore assembly site (PAS), a critical step in the packaging of cargo for selective degradation.     

DNA polymerases delta and epsilon are required for chromosomal replication in Saccharomyces cerevisiae.

Three DNA polymerases, alpha, delta, and epsilon are required for viability in Saccharomyces cerevisiae. We have investigated whether DNA polymerases epsilon and delta are required for DNA replication. Two temperature-sensitive mutations in the POL2 gene, encoding DNA polymerase epsilon, have been identified by using the plasmid shuffle technique. Alkaline sucrose gradient analysis of DNA synthesis products in the mutant strains shows that no chromosomal-size DNA is formed after shift of an asynchronous culture to the nonpermissive temperature. The only DNA synthesis observed is a reduced quantity of short DNA fragments. The DNA profiles of replication intermediates from these mutants are similar to those observed with DNA synthesized in mutants deficient in DNA polymerase alpha under the same conditions. The finding that DNA replication stops upon shift to the nonpermissive temperature in both DNA polymerase alpha- and DNA polymerase epsilon- deficient strains shows that both DNA polymerases are involved in elongation. By contrast, previous studies on pol3 mutants, deficient in DNA polymerase delta, suggested that there was considerable residual DNA synthesis at the nonpermissive temperature. We have reinvestigated the nature of DNA synthesis in pol3 mutants. We find that pol3 strains are defective in the synthesis of chromosomal-size DNA at the restrictive temperature after release from a hydroxyurea block. These results demonstrate that yeast DNA polymerase delta is also required at the replication fork.     

Three additional genes required for deoxyribonucleic acid synthesis in Saccharomyces cerevisiae

Deoxyribonucleic acid (DNA) synthesis was examined in asynchronous and synchronous cultures of a number of cdc (cell division cycle) temperature-sensitive mutant strains. The kinetics of DNA synthesis after a shift to the restrictive temperature was compared with that obtained after inhibition of protein synthesis at the permissive temperature, a condition that specifically blocks the initiation of new rounds of DNA replication, but does not block those in progress. Mutations in three genes (cdc 4, 7, and 28) appear to block a precondition for DNA synthesis since cells carrying these lesions cannot start new rounds of DNA replication after a shift from permissive to restrictive temperature, but can finish rounds that were in progress. These three genes are classified as having roles in the "initiation" of DNA synthesis. Mutations in two genes (cdc 8 and 21) block DNA synthesis, itself, since cells harboring these lesions that had started DNA synthesis at the permissive temperature arrest synthesis abruptly upon a shift to the restrictive temperature. Mutations in 13 other cdc genes do not impair DNA synthesis in the first cell cycle at the restrictive temperature.