A phosphatidylinositol 3-kinase of Candida albicans: molecular cloning andcharacterization

A phosphatidylinositol (PI) 3-kinase gene (CaVPS34) of the human pathogenic yeast Candida albicans was cloned by a PCR-based homology approach. The op en reading frame encodes a 1020 amino acid protein with a calculated molecu lar weight of 118 kDa and a relative isoelectric point of 6.9. It shares 47 % sequence identity with Saccharomyces cerevisiae Vps34p. Southern pattern indicated that CaVPS34 is probably present as a single copy gene per haploi d genome in C. albicans. We localized the CaVPS34 gene on chromosome 1. Und er all conditions tested a major CaVPS34 transcript of approximately 3.5 kb could be detected. CaVPS34 mRNA levels increased during exponential growth up to 12-fold followed by a decline upon entry into stationary phase. The size of a 6x His tag-CaVps34p fusion protein purified from Escherichia call is in agreement with the calculated molecular mass of CaVps34p. It exhibit s bl vine PT 3-kinase activity and produces only phosphatidylinositol 3-pho sphate. The CaVPS34 gene under the control of its own promoter were not abl e to complement the temperature-sensitive growth of S. cerevisiae vps34. Ho wever, overexpression of CaVPS34 was sufficient to rescue the temperature-s ensitive vps34 phenotype, suggesting a functional conservation in C. albica ns. The EMBL Accession No. for the sequence reported in this paper is Y0904 3. Copyright (C) 2000 John Whey & Sons, Ltd.