Genome-wide lethality screen identifies new PI4,5P2 effectors that regulate the actin cytoskeleton

Abstract

To further understand the roles played by the essential phosphoinositide PI4,5P(2), we have used a synthetic lethal analysis, which systematically combined the mss4(ts) mutation, partially defective in PI4P 5-kinase activity, with each of approximately 4700 deletion mutations. This genomic screening technique uncovered numerous new candidate effectors and regulators of PI4,5P(2) in yeast. In particular, we identified Slm1 (Yil105c), a previously uncharacterized PI4,5P(2) binding protein. Like Mss4, Slm1 and its homolog Slm2 (Ynl047c) were required for actin cytoskeleton polarization and viability. Co-immunoprecipitation experiments revealed that Slm1 interacts with a component of TORC2, a Tor2 kinase-containing complex, which also regulates the actin cytoskeleton. Consistent with these findings, phosphorylation of Slm1 and Slm2 was dependent on TORC2 protein kinase activity, both in vivo and in vitro, and Slm1 localization required both PI4,5P(2) and functional TORC2. Together, these data suggest that Slm1 and Slm2 function downstream of PI4,5P(2) and the TORC2 kinase pathway to control actin cytoskeleton organization.

Regulation of the Transport and Protein Levels of the Inositol Phosphorylceramide Mannosyltransferases Csg1 and Csh1 by the Ca2+-binding Protein Csg2

“…The latter model, which we prefer, is supported by the fact that the Ca 2ϩ -sensitive phenotype of the ⌬csg2 cells was suppressed by the mutations of two genes involved in signal transduction, the protein kinase TOR2 and the phosphatidylinositol-4-phosphate 5-kinase MSS4 (30). Phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P 2 ) generated by Mss4 modulates several cellular events, including actin polarization and cell growth, via the Rho1 GTPase and Tor pathways (31)(32)(33)(34)(35). These signaling events are mediated by PtdIns(4,5)P 2 -binding proteins (33)(34)(35).…”

“…Phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P 2 ) generated by Mss4 modulates several cellular events, including actin polarization and cell growth, via the Rho1 GTPase and Tor pathways (31)(32)(33)(34)(35). These signaling events are mediated by PtdIns(4,5)P 2 -binding proteins (33)(34)(35). A recent study found that the plasma membrane localization of Mss4 is disturbed in ⌬csg2 cells (36), suggesting that accumulation of IPC-C or loss of MIPC causes mislocalization of Mss4.…”

“…All of the supernatants were pooled and then centrifuged (10,000 ϫ g, 10 min), yielding a final supernatant. The cell supernatant (0.4 ml) was loaded onto a step sucrose gradient (22,26,30,34,38,42,46,50,54,60, and 75% (w/v) sucrose, each in 0.35 ml of buffer (10 mM HEPES-NaOH, pH 7.5, 2 mM MgCl 2 , 1ϫ Complete TM , 1 mM PMSF, and 1 mM dithiothreitol). After samples were centrifuged at 250,000 ϫ g for 2.5 h at 4°C, 8 fractions (0.53 ml each) were collected from top to bottom.…”

Determining selectivity of phosphoinositide-binding domains

“…Second, experience has shown that in many cases phosphoinositide binding that appears robust in dot-blots cannot be detected using any of the other commonly used approaches to study lipid binding [26][27][28]. Conversely, the examples of dynamin's PH domain [31][32][33] and the PH domains from S. cerevisiae proteins Ynl047cp and Yil105cp [27,34] illustrate that phosphoinositide binding that is very weak (and only detectable with dot-blots) can nonetheless be physiologically relevant. This can certainly not be assumed, however.…”

TORC2 Signaling Is Antagonized by Protein Phosphatase 2A and the Far Complex in Saccharomyces cerevisiae

“…sac7 mutations suppress TORC2 deficiency by increasing the levels of GTP-bound Rho1. How TORC2 mediates the organization of the actin cytoskeleton is unclear and might involve three TORC2 substrates: Slm1, Slm2, and Ypk2 (Audhya et al 2004;Fadri et al 2005;Kamada et al 2005;Tabuchi et al 2006;Aronova et al 2008).Lst8 is essential for cell viability in Saccharomyces cerevisiae (Roberg et al 1997). It is unknown whether the essential function of Lst8 is linked to TORC1, TORC2, or both.…”