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David Y Thomas

Publications and source records attributed to David Y Thomas.

25 records · Page 2Linked to original sources
PubMed2003

Phosphorylation of the MAPKKK regulator Ste50p in Saccharomyces cerevisiae: a casein kinase I phosphorylation site is required for proper mating function.

The Ste50 protein of Saccharomyces cerevisiae is a regulator of the Ste11p protein kinase. Ste11p is a member of the MAP3K (or MEKK) family, which is conserved from yeast to mammals. Ste50p is involved in all the signaling pathways that require Ste11p function, yet little is known about the regulation of Ste50p itself. Here, we show that Ste50p is phosphorylated on multiple serine/threonine residues in vivo. Threonine 42 (T42) is phosphorylated both in vivo and in vitro, and the protein kinase responsible has been identified as casein kinase I. Replacement of T42 with alanine (T42A) compromises Ste50p function. This mutation abolishes the ability of overexpressed Ste50p to suppress either the mating defect of a ste20 ste50 deletion mutant or the mating defect of a strain with a Ste11p deleted from its sterile-alpha motif domain. Replacement of T42 with a phosphorylation-mimetic aspartic acid residue (T42D) permits wild-type function in all assays of Ste50p function. These results suggest that phosphorylation of T42 of Ste50p is required for proper signaling in the mating response. However, this phosphorylation does not seem to have a detectable role in modulating the high-osmolarity glycerol synthesis pathway.

Amino Acid Sequence↗
PubMed2002

Correlation between virulence of Candida albicans mutants in mice and Galleria mellonella larvae.

Candida albicans is a dimorphic human pathogen in which the yeast to hyphal switch may be an important factor in virulence in mammals. This pathogen has recently been shown to also kill insects such as the Greater Wax Moth Galleria mellonella when injected into the haemocoel of the insect larvae. We have investigated the effect of previously characterised C. albicans mutations that influence the yeast to hyphal transition on virulence in G. mellonella larvae. There is a good correlation between the virulence of these mutants in the insect host and the virulence measured through systemic infection of mice. Although the predominant cellular species detected in G. mellonella infections is the yeast form of C. albicans, mutations that influence the hyphal transition also reduce pathogenicity in the insect. The correlation with virulence measured in the mouse infection system suggests that Galleria may provide a convenient and inexpensive model for the in vivo screening of mutants of C. albicans.

Animals↗
PubMed2002

Population genomics of drug resistance in Candida albicans.

We followed adaptation in experimental microbial populations to inhibitory concentrations of an antimicrobial drug. The evolution of drug resistance was accompanied in all cases by changes in gene expression that persisted in the absence of the drug; the new patterns of gene expression were constitutive. The changes in gene expression occurred in four replicate populations of the pathogenic fungus Candida albicans during 330 generations of evolution in the presence of the antifungal drug fluconazole. Genome-wide expression profiling of over 5,000 ORFs identified 301 whose expression was significantly modulated. Cluster analysis identified three distinct patterns of gene expression underlying adaptation to the drug. One pattern was unique to one population and included up-regulation of the multidrug ATP-binding cassette transporter gene, CDR2. A second pattern occurred at a late stage of adaptation in three populations; for two of these populations profiled earlier in their evolution, a different pattern was observed at an early stage of adaptation. The succession of early- and late-stage patterns of gene expression, both of which include up-regulation of the multidrug major facilitator transporter gene, MDR1, must represent a common program of adaptation to this antifungal drug. The three patterns of gene expression were also identified in fluconazole-resistant clinical isolates, providing further evidence that these patterns represent common programs of adaptation to fluconazole.

ATP-Binding Cassette Transporters↗
PubMed2002

Localization of the lectin, ERp57 binding, and polypeptide binding sites of calnexin and calreticulin.

Calnexin and calreticulin are membrane-bound and soluble chaperones, respectively, of the endoplasmic reticulum (ER) which interact transiently with a broad spectrum of newly synthesized glycoproteins. In addition to sharing substantial sequence identity, both calnexin and calreticulin bind to monoglucosylated oligosaccharides of the form Glc(1)Man(5-9)GlcNAc(2), interact with the thiol oxidoreductase, ERp57, and are capable of acting as chaperones in vitro to suppress the aggregation of non-native proteins. To understand how these diverse functions are coordinated, we have localized the lectin, ERp57 binding, and polypeptide binding sites of calnexin and calreticulin. Recent structural studies suggest that both proteins consist of a globular domain and an extended arm domain comprised of two sequence motifs repeated in tandem. Our results indicate that the primary lectin site of calnexin and calreticulin resides within the globular domain, but the results also point to a much weaker secondary site within the arm domain which lacks specificity for monoglucosylated oligosaccharides. For both proteins, a site of interaction with ERp57 is centered on the arm domain, which retains approximately 50% of binding compared with full-length controls. This site is in addition to a Zn(2+)-dependent site located within the globular domain of both proteins. Finally, calnexin and calreticulin suppress the aggregation of unfolded proteins via a polypeptide binding site located within their globular domains but require the arm domain for full chaperone function. These findings are integrated into a model that describes the interaction of glycoprotein folding intermediates with calnexin and calreticulin.

Base Sequence↗
PubMed2002

Transcription profiling of Candida albicans cells undergoing the yeast-to-hyphal transition.

The ability of the pathogenic fungus Candida albicans to switch from a yeast to a hyphal morphology in response to external signals is implicated in its pathogenicity. We used glass DNA microarrays to investigate the transcription profiles of 6333 predicted ORFs in cells undergoing this transition and their responses to changes in temperature and culture medium. We have identified several genes whose transcriptional profiles are similar to those of known virulence factors that are modulated by the switch to hyphal growth caused by addition of serum and a 37 degrees C growth temperature. Time course analysis of this transition identified transcripts that are induced before germ tube initiation and shut off later in the developmental process. A strain deleted for the Efg1p and Cph1p transcription factors is defective in hyphae formation, and its response to serum and increased temperature is almost identical to the response of a wild-type strain grown at 37 degrees C in the absence of serum. Thus Efg1p and Cph1p are needed for the activation of the transcriptional program that is induced by the presence of serum.

Candida albicans↗
PubMed2002

CDC42 is required for polarized growth in human pathogen Candida albicans.

Cdc42p is a member of the RAS superfamily of GTPases and plays an essential role in polarized growth in many eukaryotic cells. We cloned the Candida albicans CaCDC42 by functional complementation in Saccharomyces cerevisiae and analyzed its function in C. albicans. A double deletion of CaCDC42 was made in a C. albicans strain containing CaCDC42 under the control of the PCK1 promoter. When expression of the heterologous copy of CaCDC42 was repressed in this strain, the cells ceased proliferation. These arrested cells were large, round, and unbudded and contained predominantly two nuclei. The PCK1-mediated overexpression of wild-type CaCdc42p had no effect on cells. However, in cells overexpressing CaCdc42p containing the dominant-negative D118A substitution, proliferation was blocked and the arrested cells were large, round, unbudded, and multinucleated, similar to the phenotype of the cdc42 double-deletion strain. Cells overexpressing CaCdc42p containing the hyperactive G12V substitution also ceased proliferation in yeast growth medium; in this case the arrested cells were multinucleated and multibudded. An intact CAAX box is essential for the phenotypes associated with either CaCdc42p(G12V) or CaCdc42p(D118A) ectopic expression, suggesting that membrane attachment is involved in CaCdc42p function. In addition, the lethality caused by ectopic expression of CaCdc42p(G12V) was suppressed by deletion of CST20 but not by deletion of CaCLA4. CaCdc42p function was also examined under hypha-inducing conditions. Cdc42p depletion prior to hyphal induction trapped cells in a round, unbudded state, while depletion triggered at the same time as hyphal induction permitted the initiation of germ tubes that failed to be extended. Ectopic expression of either the G12V or D118A substitution protein modified hyphal formation in a CAAX box-dependent manner. Thus, CaCdc42p function appears important for polarized growth of both the yeast and hyphal forms of C. albicans.

Base Sequence↗