A phosphatidylinositol 3‐kinase of Candida albicans: molecular cloning and characterization

• Published in: Yeast (Chichester, England) England), 2000-07, Vol.16 (10), p.933-944
• Main Authors: Eck, Raimund, Bruckmann, Astrid, Wetzker, Reinhard, Künkel, Waldemar
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Blotting, Northern; Blotting, Southern; Candida albicans; Candida albicans - enzymology; Candida albicans - genetics; Candida albicans - growth & development; Candidiasis - microbiology; CaVPS34 gene; CaVps34 protein; chromosome 1; Cloning, Molecular; Gene Expression Regulation, Fungal; Genetic Complementation Test; growth regulated; Humans; Molecular Sequence Data; Phosphatidylinositol 3-Kinases - chemistry; Phosphatidylinositol 3-Kinases - genetics; Phosphatidylinositol 3-Kinases - isolation & purification; Phosphatidylinositol 3-Kinases - metabolism; phosphatidylinositol 3‐kinase; RNA, Fungal - isolation & purification; Sequence Analysis, DNA; Vps34 protein
• ISSN: 0749-503X; 1097-0061
• DOI: 10.1002/1097-0061(200007)16:10<933::AID-YEA591>3.0.CO;2-C

A phosphatidylinositol (PI) 3‐kinase gene (CaVPS34) of the human pathogenic yeast Candida albicans was cloned by a PCR‐based homology approach. The open reading frame encodes a 1020 amino acid protein with a calculated molecular 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 haploid genome in C. albicans. We localized the CaVPS34 gene on chromosome 1. Under 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 6×His tag–CaVps34p fusion protein purified from Escherichia coli is in agreement with the calculated molecular mass of CaVps34p. It exhibits in vitro PI 3‐kinase activity and produces only phosphatidylinositol 3‐phosphate. The CaVPS34 gene under the control of its own promoter were not able to complement the temperature‐sensitive growth of S. cerevisiae vps34. However, overexpression of CaVPS34 was sufficient to rescue the temperature‐sensitive vps34 phenotype, suggesting a functional conservation in C. albicans. The EMBL Accession No. for the sequence reported in this paper is Y09043. Copyright © 2000 John Wiley & Sons, Ltd.