The Saccharomyces cerevisiae LSB6 Gene Encodes Phosphatidylinositol 4-Kinase Activity

• Published in: The Journal of biological chemistry 2002-12, Vol.277 (49), p.47709-47718
• Main Authors: Han, Gil-Soo, Audhya, Anjon, Markley, Daniel J., Emr, Scott D., Carman, George M.
• Format: Article
• Language: English
• Subjects: 1-Phosphatidylinositol 4-Kinase - chemistry; 1-Phosphatidylinositol 4-Kinase - metabolism; Adenosine Triphosphate - pharmacology; Cell Division; Databases as Topic; Detergents - pharmacology; DNA - metabolism; Dose-Response Relationship, Drug; Electrophoresis, Polyacrylamide Gel; Escherichia coli - metabolism; Hot Temperature; Hydrogen-Ion Concentration; Immunoblotting; Intracellular Membranes - metabolism; Magnesium - pharmacology; Manganese - pharmacology; Microscopy, Fluorescence; Mutation; Octoxynol - pharmacology; Phenotype; Polymerase Chain Reaction; Protein Binding; Saccharomyces cerevisiae - genetics; Saccharomyces cerevisiae - metabolism; Sodium Chloride - pharmacology; Subcellular Fractions - metabolism; Temperature; Vacuoles - metabolism
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M207996200

The LSB6 gene product was identified from the Saccharomyces Genome Data Base (locus YJL100W) as a putative member of a novel type II phosphatidylinositol (PI) 4-kinase family. Cell extracts lacking the LSB6 gene had a reduced level of PI 4-kinase activity. In addition, multicopy plasmids containing the LSB6 gene directed the overexpression of PI 4-kinase activity in cell extracts of wild-type cells, in anlsb6Δ mutant, in a pik1tsstt4ts double mutant, and in anpik1tsstt4tslsb6Δ triple mutant. The heterologous expression of theS. cerevisiae LSB6 gene in Escherichia coli resulted in the expression of a protein that possessed PI 4-kinase activity. Although the lsb6Δ mutant did not exhibit a growth phenotype and failed to exhibit a defect in phosphoinositide synthesis in vivo, the overexpression of the LSB6 gene could partially suppress the lethal phenotype of an stt4Δ mutant defective in the type IIISTT4-encoded PI 4-kinase indicating that Lsb6p functions as a PI 4-kinase in vivo. Lsb6p was localized to the membrane fraction of the cell, and when overexpressed, GFP-tagged Lsb6p was observed on both the plasma membrane and the vacuole membrane. The enzymological properties (pH optimum, dependence on magnesium or manganese as a cofactor, the dependence of activity on Triton X-100, the dependence on the PI surface concentration, and temperature sensitivity) of the LSB6-encoded enzyme were very similar to the membrane-associated 55-kDa PI 4-kinase previously purified fromS. cerevisiae.