title: Generation and functional in vivo characterization of a lipid kinase defective phosphatidylinositol 3-kinase Vps34p of Candida albicans.
abstract: The phosphatidylinositol (PI) 3-kinase Vps34p of Candida albicans has lipid kinase and autophosphorylation activity and is involved in virulence and vesicular protein transport. In order to characterize the roles of lipid kinase activity, a chimeric Vps34 protein was created which lacks lipid kinase but retains autophosphorylation activity. To this end, six amino acids within the putative lipid-binding site of Vps34p were replaced by the homologous region of the PI 3-kinase-like C. albicans Tor protein. The resulting chimeric Vps34T protein was recombinantly expressed in Escherichia coli and shown to lack lipid kinase activity. The corresponding chimeric VPS34TOR gene was inserted into the genome of C. albicans, and this lipid-kinase-defective strain had a distinctive phenotype compared to those of the wild-type strain SC5314 and the vps34 null mutant. The lipid-kinase-defective strain was non-virulent, and showed altered hyphal growth, reduced adherence, as well as defective vacuole morphology and endosomal vesicle transport. These results demonstrate an important role for the lipid kinase activity of Vps34p in virulence and vesicular protein transport. On the other hand, the lipid-kinase-defective strain and the vps34 null mutant differ in their temperature- and osmotic-stress response. This indicates a possible role for activities different from the lipid kinase function of Vps34p.
authors.affiliation: Hans-Knöll-Institute for Natural Products Research, Department of Infection Biology, Beutenbergstrasse 11, D-07745 Jena, Germany.
authors.forename: Juliane
authors.initials: J
authors.lastname: Günther
authors.affiliation: Hans-Knöll-Institute for Natural Products Research, Department of Infection Biology, Beutenbergstrasse 11, D-07745 Jena, Germany.
authors.forename: Monika
authors.initials: M
authors.lastname: Nguyen
authors.affiliation: Hans-Knöll-Institute for Natural Products Research, Department of Infection Biology, Beutenbergstrasse 11, D-07745 Jena, Germany.
authors.forename: Albert
authors.initials: A
authors.lastname: Härtl
authors.affiliation: University of Applied Sciences, Tatzendpromenade 1b, D-07745 Jena, Germany.
authors.forename: Waldemar
authors.initials: W
authors.lastname: Künkel
authors.affiliation: Hans-Knöll-Institute for Natural Products Research, Department of Infection Biology, Beutenbergstrasse 11, D-07745 Jena, Germany.
authors.forename: Peter F
authors.initials: PF
authors.lastname: Zipfel
authors.affiliation: Hans-Knöll-Institute for Natural Products Research, Department of Infection Biology, Beutenbergstrasse 11, D-07745 Jena, Germany.
authors.forename: Raimund
authors.initials: R
authors.lastname: Eck
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D021461:Animals, Outbred Strains; D001692:Biological Transport; D002176:Candida albicans / Q000201:enzymology* / Q000235:genetics / Q000472:pathogenicity*; D002177:Candidiasis / Q000382:microbiology / Q000503:physiopathology; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D017353:Gene Deletion; D008297:Male; D051379:Mice; D008969:Molecular Sequence Data; D019869:Phosphatidylinositol 3-Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D014617:Vacuoles / Q000378:metabolism; D014774:Virulence
chemical_list: D019869:Phosphatidylinositol 3-Kinases; D017853:Phosphotransferases (Alcohol Group Acceptor); D011994:Recombinant Proteins; C066324:ceramide kinase