Yeast Cells Lacking the ARV1 Gene Harbor Defects in Sphingolipid Metabolism

Abstract

]dihydrosphingosine radiolabeling studies demonstrated that mutant cells had reduced rates of biosynthesis and lower steadystate levels of complex sphingolipids while accumulating certain hydroxylated ceramide species. Phospholipid radiolabeling studies showed that arv1⌬ cells harbored defects in the rates of biosynthesis and steadystate levels of phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and phosphatidylglycerol. Neutral lipid radiolabeling studies indicated that the rate of biosynthesis and steady-state levels of sterol ester were increased in arv1⌬ cells. Moreover, these same studies demonstrated that arv1⌬ cells had decreased rates of biosynthesis and steady-state levels of total fatty acid and fatty acid alcohols. Gas chromatography/mass spectrometry analyses examining different fatty acid species showed that arv1⌬ cells had decreased levels of C18:1 fatty acid. Additional gas chromatography/mass spectrometry analyses determining the levels of various molecular sterol species in arv1⌬ cells showed that mutant cells accumulated early sterol intermediates. Using fluorescence microscopy we found that GFP-Arv1p localizes to the endoplasmic reticulum and Golgi. Interestingly, the heterologous expression of the human ARV1 cDNA suppressed the sphingolipid metabolic defects of arv1⌬ cells. We hypothesize that in eukaryotic cells, Arv1p functions in the sphingolipid metabolic pathway perhaps as a transporter of ceramides between the endoplasmic reticulum and Golgi.Sphingolipids serve as constituents of membrane bilayers (1) and have emerged as potent signaling metabolites (2-5). Sphingolipid synthesis in eukaryotes begins with the condensation of serine and palmitoyl-CoA, a reaction catalyzed by the Lcb1p/ Lcb2p serine palmitoyltransferase subunits (Fig. 1) (6 -10). Ceramide serves as the backbone for all complex sphingolipids, and its biosynthesis is the point where animal and fungal sphingolipid biosynthesis begin to diverge (11). In higher eukaryotes, there are over three hundred different types of complex sphingolipids found in membranes (1), whereas in the yeast S. cerevisiae there are three, inositolphosphorylceramide (IPC), 1 mannose inositolphosphorylceramide (MIPC), and mannose diinositolphosphorylceramide (MIP 2 C). However, IPC, MIPC, and MIP 2 C lipids can differ in their hydroxylation state, giving rise to fifteen possible complex sphingolipids (11).The accumulation of free cholesterol in animal cells causes toxicity (12). One mechanism by which cells relieve this toxic effect is through the use of the membrane-associated SREBP transcription factors, which precisely regulate the genes that are required for sterol biosynthesis and LDL receptor expression (13,14). Worgall et al. (15) recently showed that SREBP activation was regulated by ceramide. Others have shown that animal cells regulate sphingolipid metabolism in response to subtle and transient changes in cholesterol (16,17). In S. cerevisiae, sphingolipid metabolism appears to be coordinately regulated with phospholipid an...

Large-scale identification of yeast integral membrane protein interactions

“…The genes for the two proteins share synthetic genetic and chemical genetic interactions (40): synthetic lethality between erg11 and arv1 strains and hypersensitivity of the arv1 strain to fluconoazole, an antimicrobial compound that targets Erg11. Yeast lacking Arv1 are defective in sphingolipid and sterol metabolism (41) and accumulate lanosterol, the substrate that Erg11 demethylates in the course of ergosterol biosynthesis (42). The identified interaction between these proteins may connect their functions in ergosterol metabolism.…”

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

“…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).…”

“…In addition to having defects in sphingolipid and GPI syntheses, arv1 cells harbor defects in phospholipid biosynthesis and metabolism (Swain et al 2002). Ste5 has an N-terminal amphipathic a-helix, which is important for nuclear localization and membrane binding (Winters et al 2005).…”

“…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).…”

Regulation of the Sphingoid Long-chain Base Kinase Lcb4p by Ergosterol and Heme

“…Previous studies have shown that ergosterol levels affect the amounts of certain sphingolipid species (45). Moreover, ⌬arv1 cells, in which intracellular sterol distribution is altered, also harbor defects in sphingolipid synthesis (46). Thus, there seems to be unknown mechanisms that regulate sphingolipid synthesis in response to cellular ergosterol status.…”