Mutations in Yeast ARV1 Alter Intracellular Sterol Distribution and Are Complemented by Human ARV1

Abstract

Intracellular cholesterol redistribution between membranes and its subsequent esterification are critical aspects of lipid homeostasis that prevent free sterol toxicity. To identify genes that mediate sterol trafficking, we screened for yeast mutants that were inviable in the absence of sterol esterification. Mutations in the novel gene, ARV1, render cells dependent on sterol esterification for growth, nystatin-sensitive, temperaturesensitive, and anaerobically inviable. Cells lacking Arv1p display altered intracellular sterol distribution and are defective in sterol uptake, consistent with a role for Arv1p in trafficking sterol into the plasma membrane. Human ARV1, a predicted sequence ortholog of yeast ARV1, complements the defects associated with deletion of the yeast gene. The genes are predicted to encode transmembrane proteins with potential zincbinding motifs. We propose that ARV1 is a novel mediator of eukaryotic sterol homeostasis.Sterols are essential structural and regulatory components of eukaryotic cellular membranes (1, 2). However, cholesterol over-accumulation is cytotoxic (3), necessitating mechanisms to maintain this metabolite at appropriate levels. A pivotal component of this homeostasis is the esterification of free sterol by acyl-coenzyme A:cholesterol O-acyltransferase (ACAT) 1 (4, 5).Indeed, the inhibition of ACAT in sterol-loaded cells induces cell death when extracellular sterol acceptors such as high density lipoproteins are absent (6, 7). Intracellular cholesterol redistribution mediates a number of responses to elevated free sterol levels. These include elevated ACAT activity, down-regulated sterol and fatty acid biosynthesis, and reduced lipoprotein uptake via LDL receptors (8, 9). The latter two events reflect changes in transcriptional activation by sterol regulatory element-binding proteins (SREBPs) in response to sterol accumulation in regulatory pools (9), whereas ACAT activity is allosterically regulated by substrate supply (10). Sterols are maintained at a high concentration in the plasma membrane (PM) relative to the endoplasmic reticulum (ER) (1, 2), where SREBP and ACAT reside. Thus trafficking of sterol to and from the ER is a critical component of sterol homeostasis.The process of sterol trafficking is poorly understood at the molecular level. In certain cell types, caveolin influences what has been termed "fast" movement of cholesterol to plasma membrane cholesterol-rich microdomains (caveolae) (11,12). Mutations in the Niemann Pick type C (NPC1) gene result in accumulation of LDL-derived cholesterol in the lysosome (13, 14). However, not all cells express caveolin, and the movement of endogenously synthesized cholesterol to the plasma membrane in NPC1-deficient cells is normal (15).To identify novel genes that mediate sterol trafficking in all higher cells, we utilized the genetically tractable model eukaryote, Saccharomyces cerevisiae (budding yeast). We reasoned that dependence on sterol esterification for viability would be one criterion for identifying novel sterol...

A role for yeast oxysterol-binding protein homologs in endocytosis and in the maintenance of intracellular sterol-lipid distribution

“…In arv1∆ cells, levels of plasma membrane sterols are reduced and sterols accumulate in intracellular membranes (Fig. 9B) (Tinkelenberg et al, 2000). We also observed endocytosis defects by lucifer yellow and FM4-64 uptake assays.…”

“…In arv1∆ and wild-type cells, most ergosterol is present at the plasma membrane. However, in arv1∆ cells some ergosterol can be found in fractions corresponding to the endoplasmic reticulum (ER) and the vacuole (Tinkelenberg et al, 2000), organelles that are normally sterol deficient (Zinser et al, 1991). In arv1∆ cells filipin fluorescence was always observed at the cell periphery but in some cells fluorescence was also observed within the cell (Fig.…”

“…To date, ARV1 is the only gene reported other than the OSHs that affects sterol levels and distribution within yeast cells, without simply blocking sterol synthesis (Tinkelenberg et al, 2000). To determine if, like the OSHs, ARV1 also plays a role in endocytosis, we evaluated lucifer yellow and FM4-64 uptake in arv1∆ cells.…”

“…ARV1 encodes a predicted zinc-binding integral membrane protein involved in the maintenance of normal ergosterol distribution in yeast cells (Tinkelenberg et al, 2000). In arv1∆ and wild-type cells, most ergosterol is present at the plasma membrane.…”

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

“…How the AHD functions within various Arv1-dependent events is not known. 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).…”