Analysis of oxysterol binding protein homologue Kes1p function in regulation of Sec14p-dependent protein transport from the yeast Golgi complex

Abstract

Oxysterol binding proteins (OSBPs) comprise a large conserved family of proteins in eukaryotes. Their ubiquity notwithstanding, the functional activities of these proteins remain unknown. Kes1p, one of seven members of the yeast OSBP family, negatively regulates Golgi complex secretory functions that are dependent on the action of the major yeast phosphatidylinositol/phosphatidylcholine Sec14p. We now demonstrate that Kes1p is a peripheral membrane protein of the yeast Golgi complex, that localization to the Golgi complex is required for Kes1p function in vivo, and that targeting of Kes1p to the Golgi complex requires binding to a phosphoinositide pool generated via the action of the Pik1p, but not the Stt4p, PtdIns 4-kinase. Localization of Kes1p to yeast Golgi region also requires function of a conserved motif found in all members of the OSBP family. Finally, we present evidence to suggest that Kes1p may regulate adenosine diphosphate-ribosylation factor (ARF) function in yeast, and that it may be through altered regulation of ARF that Kes1p interfaces with Sec14p in controlling Golgi region secretory function.

The Sac1 Phosphoinositide Phosphatase Regulates Golgi Membrane Morphology and Mitotic Spindle Organization in Mammals

“…Given that catalytic activity of mammalian Sac1 is required for rescue of the Golgi dysmorphology phenotype, one attractive possibility is disorganization of the Golgi system is evoked by mislocalization of specific PIP-binding proteins. Such mislocalization could result from precocious accumulation of PtdIns-4-P pools in inappropriate membrane compartments (as demonstrated in sac1⌬ yeast; Li et al, 2002). Marker localization experiments, however, do not report a significant redistribution of candidate PIP-binding proteins.…”

“…Such an effect could arrive as a consequence of PtdIns-4-P accumulating to high levels on inappropriate membrane surfaces, e.g., ER (Li et al, 2002). [ 3 H]Inositol radiolabeling experiments, coupled with analyses of PIP species, reports a modest but reproducible (ϳ30%) increase in PtdIns-4-P in hSac1-deficient cell populations relative to controls (Supplemental Figure S4B).…”

“…Mutations that compromise ySac1 enzymatic activity phenocopy sac1 null mutations, whereas substitutions outside the ySac1 catalytic motif evoke defects that are likely regulatory in nature (Nemoto et al, 2000;Li et al, 2002). Several lines of evidence demonstrate mSac1 shares a functional relationship to ySac1.…”

“…In keeping with the multiplicity of PIP signaling functions in eukaryotes, Sac1 loss-of-function (LOF) elicits pleitotropic effects in yeast that reflect broad-ranging alterations in multiple aspects of lipid metabolism (Cleves et al, 1989;Novick et al, 1989;Rivas et al, 1999;Tahirovic et al, 2005). Such metabolic alterations contribute to mislocalization of PIP binding proteins from the Golgi complex to inappropriate compartments (Li et al, 2002). ySac1 is an unusual PIP phosphatase in several respects.…”

Roles of Phosphoinositides and of Spo14p (phospholipase D)-generated Phosphatidic Acid during Yeast Sporulation

“…This phenomenon is referred to as "Sec14 bypass" (Cleves et al, 1991); under these circumstances, Spo14p is essential (Sreenivas et al, 1998;Xie et al, 1998;Li et al, 2002). To determine whether the requirement for Sec14p in sporulation could also be relieved by such mutations, we constructed the appropriate double mutants.…”