Both cholesterol biosynthesis and storage are controlled in response to levels and localization of regulatory pools of sterols (33
). 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
). 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
), and feedback regulation of cholesterol on its own biosynthesis and storage also controls levels of cellular sterols (16
). 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, appear to be conserved (70
). In yeast, ASAT is encoded by two homologous genes, ARE1
, which together generate steryl esters for lipid storage droplets (84
). Lipid droplets are also comprised of triacylglycerols, which in yeast are produced by the acyl-CoA:diacylglycerol acyltransferase 2 (DGAT2) homologue encoded by DGA1
and by the phospholipid:diacylglycerol acyltransferase (PDAT) homologue encoded by LRO1
). The genes encoding sterol and diacylglycerol acyltransferases are not essential, and a viable strain has been constructed that lacks all genes required for neutral-lipid biosynthesis (61
). These findings indicate that lipid storage is itself not required for yeast growth under normal culture conditions. A likely explanation for why neutral-lipid/sterol storage is dispensable for yeast viability is that it represents only one of several independent mechanisms that contribute to the maintenance of lipid and sterol homeostasis. This leads to the prediction that sterol regulatory pathways are functionally redundant and that growth defects occur only when several of these pathways are disrupted in concert.
In the case of sterol storage and other sterol regulatory pathways, functional redundancy has been successfully exploited to identify novel sterol-associated genes in yeast. ARV1
, which affects the distribution of unesterified sterols, was originally identified as a deletion mutation that is lethal in combination with deletions of both ARE1
). This finding suggests that both sterol storage and trafficking make overlapping contributions to sterol homeostasis. ECM22
encode transcription factors that control another aspect of sterol homeostasis through the coordinate regulation of several sterol biosynthesis genes (79
). Although the combined deletion of ECM22
is not lethal, upc2
Δ cells are inviable with the additional perturbation of sterols caused by the deletion of ERG2
, which encodes the otherwise nonessential enzyme C-8 sterol isomerase (79
). Together these results affirm that the disruption of just one sterol regulatory pathway is not detrimental unless there are additional defects in sterol homeostasis.
In this study, we carried out a functional genomics screen to identify yeast deletion mutants that cannot tolerate drug-induced disruptions in sterol homeostasis. This screen successfully identified 56 known and novel genes that are required for maintenance of sterol homeostasis. The identified deletion mutants were analyzed by cellular and biochemical approaches to establish their specific roles in sterol-lipid biosynthesis, trafficking, and/or storage. In this study, we defined distinct steps required for lipid storage droplet biogenesis and established a link between ASAT/DGAT lipid esterification and secretory protein glycosylation. Our findings provide insights into mechanisms affecting sterol transport, synthesis, and neutral-lipid storage, which together maintain sterol homeostasis and are potentially linked to human lipid disorders.