Oxysterol-binding protein (OSBP)-related protein Kes1/Osh4p is implicated in nonvesicular sterol transfer between membranes in Saccharomyces cerevisiae. However, we found that Osh4p associated with exocytic vesicles that move from the mother cell into the bud, where Osh4p facilitated vesicle docking by the exocyst tethering complex at sites of polarized growth on the plasma membrane. Osh4p formed complexes with the small GTPases Cdc42p, Rho1p and Sec4p, and the exocyst complex subunit Sec6p, which was also required for Osh4p association with vesicles. Although Osh4p directly affected polarized exocytosis, its role in sterol trafficking was less clear. Contrary to what is predicted for a sterol-transfer protein, inhibition of sterol binding by the Osh4p Y97F mutation did not cause its inactivation. Rather, OSH4 Y97F is a gain-of-function mutation that causes dominant lethality. We propose that in response to sterol binding and release Osh4p promotes efficient exocytosis through the co-ordinate regulation of Sac1p, a phosphoinositide 4-phosphate (PI4P) phosphatase, and the exocyst complex. These results support a model in which Osh4p acts as a sterol-dependent regulator of polarized vesicle transport, as opposed to being a sterol-transfer protein.
Polarized cell growth requires the establishment of an axis of growth along which secretion can be targeted to a specific site on the cell cortex. How polarity establishment and secretion are choreographed is not fully understood, though Rho GTPase-and Rab GTPase-mediated signaling is required. Superimposed on this regulation are the functions of specific lipids and their cognate binding proteins. In a screen for Saccharomyces cerevisiae genes that interact with Rho family CDC42 to promote polarity establishment, we identified KES1/OSH4, which encodes a homologue of mammalian oxysterol-binding protein (OSBP). Other yeast OSH genes (OSBP homologues) had comparable genetic interactions with CDC42, implicating OSH genes in the regulation of CDC42-dependent polarity establishment. We found that the OSH gene family (OSH1-OSH7) promotes cell polarization by maintaining the proper localization of septins, the Rho GTPases Cdc42p and Rho1p, and the Rab GTPase Sec4p. Disruption of all OSH gene function caused specific defects in polarized exocytosis, indicating that the Osh proteins are collectively required for a secretory pathway implicated in the maintenance of polarized growth.
Many ATP-dependent molecular chaperones, including Hsp70, Hsp90, and the chaperonins GroEL/Hsp60, require cofactor proteins to regulate their ATPase activities and thus folding functions in vivo. One conspicuous exception has been the eukaryotic chaperonin CCT, for which no regulator of its ATPase activity, other than non-native substrate proteins, is known. We identify the evolutionarily conserved PhLP3 (phosducin-like protein 3) as a modulator of CCT function in vitro and in vivo. PhLP3 binds CCT, spanning the cylindrical chaperonin cavity and contacting at least two subunits. When present in a ternary complex with CCT and an actin or tubulin substrate, PhLP3 significantly diminishes the chaperonin ATPase activity, and accordingly, excess PhLP3 perturbs actin or tubulin folding in vitro. Most interestingly, however, the Saccharomyces cerevisiae PhLP3 homologue is required for proper actin and tubulin function. This cellular role of PhLP3 is most apparent in a strain that also lacks prefoldin, a chaperone that facilitates CCT-mediated actin and tubulin folding. We propose that the antagonistic actions of PhLP3 and prefoldin serve to modulate CCT activity and play a key role in establishing a functional cytoskeleton in vivo.
The pandemic of lipid-related disease necessitates a determination of how cholesterol and other lipids are transported and stored within cells. The first step in this determination is the identification of the genes involved in these transport and storage processes. Using genome-wide screens, we identified 56 yeast (Saccharomyces cerevisiae) genes involved in sterol-lipid biosynthesis, intracellular trafficking, and/or neutral-lipid storage. Direct biochemical and cytological examination of mutant cells revealed an unanticipated link between secretory protein glycosylation and triacylglycerol (TAG)/steryl ester (SE) synthesis for the storage of lipids. Together with the analysis of other deletion mutants, these results suggested at least two distinct events for the biogenesis of lipid storage particles: a step affecting neutral-lipid synthesis, generating the lipid core of storage particles, and another step for particle assembly. In addition to the lipid storage mutants, we identified mutations that affect the localization of unesterified sterols, which are normally concentrated in the plasma membrane. These findings implicated phospholipase C and the protein phosphatase Ptc1p in the regulation of sterol distribution within cells. This study identified novel sterol-related genes that define several distinct processes maintaining sterol homeostasis.Both cholesterol biosynthesis and storage are controlled in response to levels and localization of regulatory pools of sterols (33,37,54,65). In response to high cholesterol levels in the endoplasmic reticulum (ER) membrane, the enzyme acyl coenzyme A (CoA):sterol O-acyltransferase (ASAT) initiates sterol esterification and storage by covalently coupling fatty acids to cholesterol. Through an active process, the esterified cholesterol is amalgamated with other neutral lipids into lipid storage droplets that are released from the ER membrane (42, 88). The trafficking of unesterified sterols also affects the sterol distribution in regulatory pools. Although cholesterol is synthesized in the ER, the highest level of unesterified cholesterol is found in the plasma membrane (33) and maintenance of normal sterol levels requires the efficient transport of cholesterol from the ER membrane to the plasma membrane. The maintenance of cholesterol levels in the plasma membrane is affected by sorting from endosomal compartments and recycling back to the cell surface (33, 54), and feedback regulation of cholesterol on its own biosynthesis and storage also controls levels of cellular sterols (16, 68). These findings suggest that the maintenance of cellular cholesterol homeostasis requires the regulatory integration of cholesterol synthesis, storage, and transport pathways.As in mammalian cells, the budding yeast Saccharomyces cerevisiae synthesizes its own cholesterol-like lipids but, under normal aerobic conditions, yeast does not internalize exogenous sterol lipids. Apart from this difference, other elements of sterol homeostasis, including lipid storage and transport pathways, app...
Polarized growth is maintained by both polarized exocytosis, which transports membrane components to specific locations on the cell cortex, and endocytosis, which retrieves these components before they can diffuse away. Despite functional links between these two transport pathways, they are generally considered to be separate events. Using live cell imaging, in vivo and in vitro protein binding assays, and in vitro pyrene-actin polymerization assays, we show that the yeast Rab GTPase Sec4p couples polarized exocytosis with cortical actin polymerization, which induces endocytosis. After polarized exocytosis to the plasma membrane, Sec4p binds Las17/Bee1p (yeast Wiskott—Aldrich Syndrome protein [WASp]) in a complex with Sla1p and Sla2p during actin patch assembly. Mutations that inactivate Sec4p, or its guanine nucleotide exchange factor (GEF) Sec2p, inhibit actin patch formation, whereas the activating sec4-Q79L mutation accelerates patch assembly. In vitro assays of Arp2/3-dependent actin polymerization established that GTPγS-Sec4p overrides Sla1p inhibition of Las17p-dependent actin nucleation. These results support a model in which Sec4p relocates along the plasma membrane from polarized sites of exocytic vesicle fusion to nascent sites of endocytosis. Activated Sec4p then promotes actin polymerization and triggers compensatory endocytosis, which controls surface expansion and kinetically refines cell polarization.
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