RESUMEN
Yeast phosphatidylinositol transfer protein (Sec14p) coordinates lipid metabolism with protein-trafficking events. This essential Sec14p requirement for Golgi function is bypassed by mutations in any one of seven genes that control phosphatidylcholine or phosphoinositide metabolism. In addition to these "bypass Sec14p" mutations, Sec14p-independent Golgi function requires phospholipase D activity. The identities of lipids that mediate Sec14p-dependent Golgi function, and the identity of the proteins that respond to Sec14p-mediated regulation of lipid metabolism, remain elusive. We now report genetic evidence to suggest that two ADP ribosylation factor-GTPase-activating proteins (ARFGAPs), Gcs1p and Age2p, may represent these lipid-responsive elements, and that Gcs1p/Age2p act downstream of Sec14p and phospholipase D in both Sec14p-dependent and Sec14p-independent pathways for yeast Golgi function. In support, biochemical data indicate that Gcs1p and Age2p ARFGAP activities are both modulated by lipids implicated in regulation of Sec14p pathway function. These results suggest ARFGAPs are stimulatory factors required for regulation of Golgi function by the Sec14p pathway, and that Sec14p-mediated regulation of lipid metabolism interfaces with the activity of proteins involved in control of the ARF cycle.
Asunto(s)
Factores de Ribosilacion-ADP/metabolismo , Proteínas Portadoras/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas Activadoras de GTPasa/metabolismo , Aparato de Golgi/metabolismo , Proteínas de la Membrana/metabolismo , Fosfolípidos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/fisiología , Factores de Ribosilacion-ADP/genética , Secuencia de Aminoácidos , Transporte Biológico/fisiología , Proteínas Sanguíneas/genética , Proteínas Portadoras/genética , Proteínas de Unión al ADN/genética , Proteínas Activadoras de GTPasa/genética , Proteínas de la Membrana/genética , Modelos Biológicos , Datos de Secuencia Molecular , Mutación , Fosfolipasa D/genética , Fosfolipasa D/metabolismo , Proteínas de Transferencia de Fosfolípidos , Fosfoproteínas/genética , Estructura Terciaria de Proteína , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/ultraestructura , Proteínas de Saccharomyces cerevisiae/genética , Alineación de Secuencia , Vacuolas/metabolismo , Vacuolas/ultraestructuraRESUMEN
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.