The Saccharomyces cerevisiae LSB6 Gene Encodes Phosphatidylinositol 4-Kinase Activity

Abstract

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 an lsb6⌬ mutant, in a pik1 ts stt4 ts double mutant, and in an pik1 ts stt4 ts lsb6⌬ triple mutant. The heterologous expression of the S. cerevisiae LSB6 gene in Escherichia coli resulted in the expression of a protein that possessed PI 4-kinase activity. Although the lsb6⌬ 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 stt4⌬ mutant defective in the type III STT4-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 from S. cerevisiae. PI1 4-kinase (ATP:phosphatidylinositol-4-phosphotransferase, EC 2.7.1.67) catalyzes the formation of PI 4-phosphate and ADP from PI and ATP (1). The PI 4-phosphate product of the reaction serves as the precursor to the polyphosphoinositides PI 4,5-bisphosphate, PI 3,4-bisphosphate, and PI 3,4,5-trisphosphate (2-4). The synthesis and turnover of these polyphosphoinositides have received a great deal of attention because of their roles in receptor-mediated signal transduction, vesicle trafficking, endocytosis, and cytoskeletal reorganization (2-5). Two types of PI 4-kinase enzymes (types II and III) have been identified in mammalian cells and in the yeast Saccharomyces cerevisiae based on their biochemical properties (3,4,6). Genes (or cDNAs) encoding the type III PI 4-kinase enzymes have been identified from mammalian cells and yeast (2, 3, 7). They all contain a common catalytic kinase domain, which is also found in the type I PI 3-kinase family of enzymes (2, 3, 7).Until recently (8, 9), a cDNA encoding a type II PI 4-kinase had not been identified. In fact, the elusive nature of this identification has led to the assumption that type II PI 4-kinase enzymes may be proteolytic fragments or splice variants of the type III PI 4-kinase enzymes (8, 10). However, advances in the sensitivity of protein sequence methodology have facilitated the identification and the cloning of human (8) and rat brain (9) cDNAs encoding type II PI 4-kinase enzymes. The predicted protein sequences of the type II PI 4-kinase enzymes lack the characteri...

Phosphatidylinositol 4-kinase type IIα is responsible for the phosphatidylinositol 4-kinase activity associated with synaptic vesicles

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The multiple roles of PtdIns(4)P – not just the precursor of PtdIns(4,5)P2

“…Finally, Lsb6p (Las17-binding 6) is the most recently identified of the yeast PI4Ks, and is encoded by a non-essential gene. It localises to the PM and the vacuole, behaving similar to an integral membrane protein, possibly due to S-palmitoylation (Han et al, 2002). It has been proposed that, because Lsb6p is the only PI4K that localises to the vacuole (Shelton et al, 2003), it has a role in vacuole fusion by providing the substrate for localised PtdIns(4,5)P 2 production.…”

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