Genome-Wide Analysis of Sterol-Lipid Storage and Trafficking in Saccharomyces cerevisiae

Abstract

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...

The Putative Lipid Transporter, Arv1, Is Required for Activating Pheromone-Induced MAP Kinase Signaling in Saccharomyces cerevisiae

“…The lipid microdomain distribution we observed during vegetative growth in wild-type and arv1 cells grown in the absence of a-factor were similar to those previously reported (Fei et al 2008). Thus, we treated cells with a-factor and determined the percentages of the various filipin staining patterns (Figure 9).…”

“…arv1 cells harbor defects in sphingolipid and GPI synthesis, and although their overall sterol content is normal, our data, as well as that of others, strongly suggest these lipids are trafficked improperly and thus are mislocalized (Tinkelenberg et al 2000;Swain et al 2002;Fei et al 2008;Kajiwara et al 2008). Membrane microdomains, also known as lipid rafts, are sphingolipidand sterol-rich regions of the plasma membrane, which are capable of mediating membrane sorting, cell adhesion, and signal transduction (Harder et al 1998).…”

“…Beh and colleagues have used filipin staining to examine the cellular localization of unesterified sterol (Fei et al 2008). The majority was found within lipid microdomains that were associated with the plasma membrane, with cytoplasmic punctate structures, and with membrane strands that may be associated with the ER.…”

“…It is known that Arv1 function is essential at high temperatures and in yeast mutants unable to esterify sterols (Tinkelenberg et al 2000). Moreover, arv1 cells harbor defects in sphingolipid metabolism (Swain et al 2002), glycosylphosphatidylinositol (GPI) biosynthesis (Kajiwara et al 2008), and may harbor defects in sterol trafficking (Tinkelenberg et al 2000;Fei et al 2008). It has been suggested that Arv1 plays a role in cholesterol trafficking from the ER to plasma membrane in mammalian cells (Tong et al 2010).…”

A role for seipin in lipid droplet dynamics and inheritance in yeast

“…The use of model systems such as Drosophila and yeast has provided much insight into the enzymology of lipid-droplet-associated proteins and their regulation (Beller et al, 2008;Fei et al, 2008a;Guo et al, 2009;Guo et al, 2008;Szymanski et al, 2007;Walther and Farese, 2009). In this study, we have taken an imaging-based approach to understand lipid droplet motility and inheritance in yeast.…”