Y. Koltin

Dominant missense mutations in a novel yeast protein related to mammalian phosphatidylinositol 3-kinase and VPS34 abrogate rapamycin cytotoxicity.

Rapamycin is a macrolide antifungal agent that exhibits potent immunosuppressive properties. In Saccharomyces cerevisiae, rapamycin sensitivity is mediated by a specific cytoplasmic receptor which is a homolog of human FKBP12 (hFKBP12). Deletion of the gene for yeast FKBP12 (RBP1) results in recessive drug resistance, and expression of hFKBP12 restores rapamycin sensitivity. These data support the idea that FKBP12 and rapamycin form a toxic complex that corrupts the function of other cellular proteins. To identify such proteins, we isolated dominant rapamycin-resistant mutants both in wild-type haploid and diploid cells and in haploid rbp1::URA3 cells engineered to express hFKBP12. Genetic analysis indicated that the dominant mutations are nonallelic to mutations in RBP1 and define two genes, designated DRR1 and DRR2 (for dominant rapamycin resistance). Mutant copies of DRR1 and DRR2 were cloned from genomic YCp50 libraries by their ability to confer drug resistance in wild-type cells. DNA sequence analysis of a mutant drr1 allele revealed a long open reading frame predicting a novel 2470-amino-acid protein with several motifs suggesting an involvement in intracellular signal transduction, including a leucine zipper near the N terminus, two putative DNA-binding sequences, and a domain that exhibits significant sequence similarity to the 110-kDa catalytic subunit of both yeast (VPS34) and bovine phosphatidylinositol 3-kinases. Genomic disruption of DRR1 in a mutant haploid strain restored drug sensitivity and demonstrated that the gene encodes a nonessential function. DNA sequence comparison of seven independent drr1dom alleles identified single base pair substitutions in the same codon within the phosphatidylinositol 3-kinase domain, resulting in a change of Ser-1972 to Arg or Asn. We conclude either that DRR1 (alone or in combination with DRR2) acts as a target of FKBP12-rapamycin complexes or that a missense mutation in DRR1 allows it to compensate for the function of the normal drug target.

Analysis of a Candida albicans gene that encodes a novel mechanism for resistance to benomyl and methotrexate

SummaryThe pathogenic yeast, Candida albicans, is insensitive to the anti-mitotic drug, benomyl, and to the dihydrofolate reductase inhibitor, methotrexate. Genes responsible for the intrinsic drug resistance were sought by transforming Saccharomyces cerevisiae, a yeast sensitive to both drugs, with genomic C. albicans libraries and screening on benomyl or methotrexate. Restriction analysis of plasmids isolated from benomyl- and methotrexate-resistant colonies indicated that both phenotypes were encoded by the same DNA fragment. Sequence analysis showed that the fragments were nearly identical and contained a long open reading frame of 1694 bp (ORF1) and a small ORF of 446 bp (ORF2) within ORF1 on the opposite strand. By site-directed mutagenesis, it was shown that ORF1 encoded both phenotypes. The protein had no sequence similarity to any known proteins, including β-tubulin, dihydrofolate reductase, and the P-glycoprotein of the multi-drug resistance family. The resistance gene was detected in several C. albicans strains and in C. stellatoidea by DNA hybridization and by the polymerase chain reaction.

The yeast FKS1 gene encodes a novel membrane protein, mutations in which confer FK506 and cyclosporin A hypersensitivity and calcineurin-dependent growth

FK506 and cyclosporin A (CsA) are potent immunosuppressive agents that display antifungal activity. They act by blocking a Ca(2+)-dependent signal transduction pathway leading to interleukin-2 transcription. Each drug forms a complex with its cognate cytosolic immunophilin receptor (i.e., FKBP12-FK506 and cyclophilin-CsA) which acts to inhibit the Ca2+/calmodulin-dependent protein phosphatase 2B, or calcineurin (CN). We and others have defined the Saccharomyces cerevisiae FKS1 gene by recessive mutations resulting in 100-1000-fold hypersensitivity to FK506 and CsA (as compared to wild type), but which do not affect sensitivity to a variety of other antifungal drugs. The fks1 mutant also exhibits a slow-growth phenotype that can be partially alleviated by exogenously added Ca2+ [Parent et al., J. Gen. Microbiol. 139 (1993) 2973-2984]. We have cloned FKS1 by complementation of the drug-hypersensitive phenotype. It contains a long open reading frame encoding a novel 1876-amino-acid (215 kDa) protein which shows no similarity to CN or to other protein phosphatases. The FKS1 protein is predicted to contain 10 to 12 transmembrane domains with a structure resembling integral membrane transporter proteins. Genomic disruption experiments indicate that FKS1 encodes a nonessential function; fks1::LEU2 cells exhibit the same growth and recessive drug-hypersensitive phenotypes observed in the original fks1 mutants. Furthermore, the fks1::LEU2 allele is synthetically lethal in combination with disruptions of both of the nonessential genes encoding the alternative forms of the catalytic A subunit of CN (CNA1 and CNA2). These data suggest that FKS1 provides a unique cellular function which, when absent, increases FK506 and CsA sensitivity by making the CNs (or a CN-dependent function) essential.

A Candida albicans RAS-related gene (CaRSR1) is involved in budding, cell morphogenesis and hypha development

Candida albicans, the most important human fungal pathogen, is a dimorphic fungus that can grow either as a yeast or as a hyphal form in response to medium conditions. A RAS-related C. albicans gene (CaRSR1) was isolated as a suppressor of a cdc24ts bud-emergence mutation of the bakers yeast, Saccharomyces cerevisiae. The deduced protein encoded by CaRSR1 is 248 amino acids long and 56% identical to that encoded by the S. cerevisiae RSR1 (BUD1) gene. Disruption of CaRSR1 in C. albicans indicated that CaRSR1 is involved in both yeast and hypha development. In the yeast phase, CaRSR1 is required for normal (polar) bud site selection and is involved in cell morphogenesis; in the yeast-mycelial transition it is involved in germ tube emergence; and in the development of the hyphae it is involved in cell elongation. The disruption of CaRSR1 leads to reduced virulence in both heterozygote and homozygote disruptants in a dose-dependent manner. The reduced virulence can be attributed to the reduced germination and shorter hyphae resulting from the disruption of CaRSR1.

Purification and characterization of pectolytic enzymes produced by virulent and hypovirulent isolates of Rhizoctonia solani Kuhn

Four pectolytic enzymes were purified from an isolate of Rhizoctonia solani (No. 82, AG-4) virulent on a wide range of hosts (Ichielvich-Auster et al. 1985, Phyloparasilica vol. 13, 103–112). The enzymes designated as endopolygalacturonase I and II (endoPG-I and endoPG-II), pectinesterase (PE) and endopectinlyase (endoPL) have been purified to homogeneity by a single chromatographic step on a cross linked polypectate column. These enzymes were identified also in two virulent isolates of R. zeae and two virulent binucleate Rhizoctonia spp. The endoPG-I, endoPG-II and PE but not endoPL were identified in three hypovirulent isolates of R. solani and two of R. zeae. These enzymes were purified to homogeneity from R. solani (No. 521, AG-4). The molecular weight (mol.wt), pH optimum, isoelectric point (pI) and optimal temperature (T) for each enzyme were endoPG-I, mol.wt 34 000, pH 4·8, pI 6·8, T 50°C); endoPG-II, mol.wt 37 000, pH 5·4, pI 7·4, T 42°C; PE, mol.wt 26 000, pH 7·7, pI 6·2, T 48°C; and endoPL (mol.wt. 45 500, pH 8·4, pI 8·1, T 53°C.

Isolation and characterization of a β-tubulin gene from Candida albicans

We report the isolation and nucleotide sequence determination of a beta-tubulin gene (TUB2) from the pathogenic dimorphic fungus Candida albicans. Nucleotide sequence analysis revealed that TUB2 encodes a protein of 449 amino acids (aa) with considerable sequence homology to beta-tubulins isolated from other fungal species. The nucleotide sequence of the C. albicans gene is 70% homologous to that of the Saccharomyces cerevisiae gene. The coding region for the C. albicans beta-tubulin gene is interrupted by two introns. The first intron occurs after the 4th aa and the second intron occurs after the 13th aa. A comparison with other fungal beta-tubulin genes indicates that the intron locations are highly conserved. Codon usage in the C. albicans TUB2 gene is nonrandom, as has been observed for other fungal beta-tubulin genes. The C. albicans TUB2 gene is transcribed to yield a 1.8-kb mRNA species. On the basis of genomic Southern-blot analysis, we conclude that C. albicans most likely possesses a single beta-tubulin gene.

Isolation of a Virus from Virulent Strains of Rhizoctonia solani

Summary Double-stranded RNA viruses were detected in virulent strains of Rhizoctonia solani. The virus particles were 33 nm in diameter, contained two or three major segments of dsRNA (mol. wt. 1.6 × 106, 1.45 × 106 and 1.25 × 106), had a density of 1.34 g/ml and had an estimated sedimentation coefficient of 161S. The major coat protein had a mol. wt. of 46000. An RNA-dependent RNA polymerase activity was associated with the viral capsids. Native hypovirulent strains of R. solani were devoid of these viruses and attenuation of virulent strains to hypovirulence appeared to be correlated with the loss of some or all of the segments of dsRNA. Transmission of dsRNA from a virulent to a hypovirulent strain by heterokaryon formation was associated with the transmission of virulence.

Structure and heterologous expression of the Ustilago maydis viral toxin KP4

Killer toxins are polypeptides secreted by some fungal species that kill sensitive cells of the same or related species, in the best‐characterized cases, they function by creating new pores in the ceil membrane and disrupting ion fluxes. Immunity or resistance to the toxins is conferred by the preprotoxins (or products thereof) or by nuclear resistance genes. In several cases, the toxins are encoded by one or more genomic segments of resident double‐stranded RNA viruses. The known toxins are composed of one to three polypeptides, usually present as multimers. We have further characterized the KP4 killer toxin from the maize smut fungus Ustilago maydis. This toxin is also encoded by a single viral double‐stranded RNA but differs from other known killer toxins in several respects: it has no N‐linked glycosylation either in the precursor or in the mature polypeptide, it is the first killer toxin demonstrated to be a single polypeptide, and h Is not processed by any of the known secretory protelnases (other than the signal peptidase). It is efficiently expressed in a heterologous fungal system.

Isolation of genes from Candida albicans by complementation in Saccharomyces cerevisiae

SummaryA genomic library of the asexual pathogenic yeast Candida albicans was constructed in the S. cerevisiae vector YEp13. The library contains a representation of the entire genome with a probability of 99%. The expression of the genes of C. albicans in S. cerevisiae was examined and two mutations his3-1 and trp1-289 of S. cerevisiae were complemented by the cloned genes of C. albicans. The hybridization data indicates that the plasmids complementing the mutations of S. cerevisiae contain sequences from C. albicans.

Homologs of the yeast neck filament associated genes : isolation and sequence analysis of Candida albicans CDC3 and CDC10

Morphogenesis in the yeast Saccharomyes cerevisiae consists primarily of bud formation. Certain cell division cycle (CDC) genes, CDC3, CDC10, CDC11, CDC12, are known to be involved in events critical to the pattern of bud growth and the completion of cytokinesis. Their products are associated with the formation of a ring of neck filaments that forms at the region of the mother cell-bud junction during mitosis. Morphogenesis in Candida albicans, a major fungal pathogen of humans, consists of both budding and the formation of hyphae. The latter is thought to be related to the pathogenesis and invasiveness of C. albicans. We have isolated and characterized C. albicans homologs of the S. cerevisiae CDC3 and CDC10 genes. Both C. albicans genes are capable of complementing defects in the respective S. cerevisiae genes. RNA analysis of one of the genes suggests that it is a regulated gene, with higher overall expression levels during the hyphal phase than in the yeast phase. Not surprisingly, DNA sequence analysis reveals that the proteins share extensive homology at the amino acid level with their respective S. cerevisiae counterparts. Related genes are also found in other species of Candida and, more importantly, in filamentous fungi such as Aspergillus nidulans and Neurospora crassa. A database search revealed significant sequence similarity with two peptides, one from Drosophila and one from mouse, suggesting strong evolutionary conservation of function.