The ARV1-encoded protein mediates sterol transport from the endoplasmic reticulum (ER) to the plasma membrane. Yeast ARV1 mutants accumulate multiple lipids in the ER and are sensitive to pharmacological modulators of both sterol and sphingolipid metabolism. Using fluorescent and electron microscopy, we demonstrate sterol accumulation, subcellular membrane expansion, elevated lipid droplet formation, and vacuolar fragmentation in ARV1 mutants. Motif-based regression analysis of ARV1 deletion transcription profiles indicates activation of Hac1p, an integral component of the unfolded protein response (UPR). Accordingly, we show constitutive splicing of HAC1 transcripts, induction of a UPR reporter, and elevated expression of UPR targets in ARV1 mutants. IRE1, encoding the unfolded protein sensor in the ER lumen, exhibits a lethal genetic interaction with ARV1, indicating a viability requirement for the UPR in cells lacking ARV1. Surprisingly, ARV1 mutants expressing a variant of Ire1p defective in sensing unfolded proteins are viable. Moreover, these strains also exhibit constitutive HAC1 splicing that interacts with DTT-mediated perturbation of protein folding. These data suggest that a component of UPR induction in arv1⌬ strains is distinct from protein misfolding. Decreased ARV1 expression in murine macrophages also results in UPR induction, particularly up-regulation of activating transcription factor-4, CHOP (C/EBP homologous protein), and apoptosis. Cholesterol loading or inhibition of cholesterol esterification further elevated CHOP expression in ARV1 knockdown cells. Thus, loss or down-regulation of ARV1 disturbs membrane and lipid homeostasis, resulting in a disruption of ER integrity, one consequence of which is induction of the UPR.
Endoplasmic reticulum (ER) membrane cholesterol is maintained at an optimal concentration of ϳ5 mol % by the net impact of sterol synthesis, modification, and export. Arv1p was first identified in the yeast Saccharomyces cerevisiae as a key component of this homeostasis due to its probable role in intracellular sterol transport. Mammalian ARV1, which can fully complement the yeast lesion, encodes a ubiquitously expressed, resident ER protein. Repeated dosing of specific antisense oligonucleotides to ARV1 produced a marked reduction of ARV1 transcripts in liver, adipose, and to a lesser extent, intestine. This resulted in marked hypercholesterolemia, elevated serum bile acids, and activation of the hepatic farnesoid X receptor (FXR) regulatory pathway. Knockdown of ARV1 in murine liver and HepG2 cells was associated with accumulation of cholesterol in the ER at the expense of the plasma membrane and suppression of sterol regulatory element-binding proteins and their targets. These studies indicate a critical role of mammalian Arv1p in sterol movement from the ER and in the ensuing regulation of hepatic cholesterol and bile acid metabolism.The endoplasmic reticulum (ER) 4 is the pivotal organelle with regard to cholesterol homeostasis. It is here that cholesterol is synthesized via the mevalonate pathway, sensed by the sterol regulatory element-binding protein (SREBP) cleavageactivating protein system, and neutralized by esterification (1-3). In addition, in certain cells, ER cholesterol is secreted in lipoprotein particles or hydroxylated to form bile acids. Consequently, sterol levels in the ER of all eukaryotic cells are strikingly low relative to the plasma membrane (PM) (4). Movement of sterol between these organelles is rapid; the majority of endogenously synthesized cholesterol is transported from the ER to the PM within 10 -20 min by an energy-dependent process (5). Vesicular and nonvesicular cholesterol transport pathways have been described (6, 7); however, the molecular components of these events are surprisingly obscure. The net impact of these processes is striking; any variation around the threshold concentration of ϳ5 mol % results in activation or repression of key regulators of lipid homeostasis, including the master regulators, SREBP-1 and -2 (8).In the yeast Saccharomyces cerevisiae, ARV1 encodes a key component of sterol transport from the ER to the PM and was identified by complementation of yeast mutations that confer viability dependence upon sterol esterification (9). This concept of synthetic lethality was based on the hypothesis that loss of one homeostatic pathway (e.g. sterol esterification) might be tolerated; however, removal of multiple "detoxifying" events would be lethal. Yeast with mutations in ARV1 have striking phenotypes, including an elevated ratio of subcellular sterols relative to the PM and abnormal phospholipid, sphingolipid, and glycosylphosphatidylinositol metabolism (9 -11). The yeast ARV1 gene predicts a 322-residue protein with several transmembrane domains. An ARV1...
Proper sterol distribution within the cell is a critical component of membrane homeostasis. The endoplasmic reticulum (ER)‐localized protein Arvl mediates sterol transport from the ER to the plasma membrane. Alterations in ER sterol distribution are a stimulus for unfolded protein response (UPR) activation. We hypothesize that the loss of Arv1p in yeast will increase the free sterol (FS) content of the ER, resulting in UPR induction. The UPR will also be induced in primary murine macrophages where ARV1 expression has been knocked down. Loss of yeast Arv1p function results activation of the UPR, mediated by Ire1p. The yeast arv1Δ ire1Δ homozygous haploid is inviable. Mutant ire1 core luminal domains, defective in sensing unfolded proteins, rescue the lethality of the arv1Δ ire1Δ haploid; these strains exhibited increased UPR induction that was synergistic with protein misfolding. We also demonstrate that decreased ARV1 expression in mammalian cells results in UPR induction, particularly an upregulation of CHOP. As a result of ARV1 knockdown, macrophages undergo apoptosis due to prolonged UPR induction and FS‐induced cytotoxicity. We demonstrate that defects in ER homeostasis, as a result of changes in ARV1 expression, exhibit UPR induction in both yeast and mammalian systems. The UPR appears to be a generalized response, resulting from perturbations in membrane structure, lipid metabolism and protein folding.
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