Title: Characterization of ARV1-Mediated Sterol Transport in Yeast and Mammalian Systems
Description: Saccharomyces cerevisiae Arv1p (ARE2 required for viability 1) is an endoplasmic reticulum (ER)-localized, functionally conserved protein that was initially observed to mediate subcellular sterol distribution, and has since been implicated in the movement of multiple lipid species. In this thesis, we examined the role of ARV1 in S. cerevisiae and mammalian systems by two approaches. In yeast, we used gene deletion to access loss of Arv1p function. In mammalian cells we utilized antisense oligonucleotides (ASOs) to decrease ARV1 expression in vitro and in vivo. In the yeast model, loss of Arv1p function results in sensitivity to modulators of sphingolipid homeostasis and aberrant accumulation of exogenous sterols. Transcription microarrays demonstrated that ARV1 deletion impacts ER homeostasis and activates the transcription factor HAC1, a component of the unfolded protein response (UPR) signaling cascade in yeast. Moreover, arv1Î” strains exhibited constitutive UPR induction, mediated by the unfolded protein sensor Ire1p.

Genetic interaction studies revealed that the arv1Î” ire1Î” homozygous haploid strain is inviable, suggesting the UPR protects the cell from arv1Î”-mediated stress. In order to assess the stimulus for arv1Î”-mediated UPR induction, arv1Î” ire1Î” heterozygous diploids were transformed with mutated Ire1p core luminal domains (cLDs) that are sufficient to transmit the signal for UPR induction but are defective in sensing unfolded proteins in the ER lumen. The mutant cLDs were able to rescue the lethality of the arv1Î” ire1Î” haploid. These strains exhibited increased UPR induction that was independent and additive with protein misfolding. Furthermore, ARV1 deficiency in murine macrophages activated PERK-mediated UPR induction, particularly an upregulation of the cell death effector, CHOP. ARV1 deficiency also caused apoptosis, likely due to prolonged UPR induction, a phenomenon that was exacerbated by inhibiting cholesterol esterification at the ER. In murine and human models, ARV1 is implicated in intracellular cholesterol homeostasis and bile acid metabolism.

ASO-mediated decreases in ARV1 expression in vivo occurred primarily in the liver and adipose. ARV1 ASO-treated animals did not exhibit UPR activation but were hypercholesterolemic and had increased levels of hepatic and plasma bile acids. Consequently, accumulating bile acids transiently activated FXR-regulatory pathways, including target genes SHP, CYP7Î±1, NTCP and ABCB11. Furthermore, knockdown of ARV1 expression in hepatocytes established a role for human ARV1 in intracellular cholesterol distribution. ARV1 ASO-treated HepG2 cells exhibited accumulation of ER cholesterol, decreased SREBP processing and decreased expression of SREBP targets, suggesting that human ARV1 may mediate cholesterol export from the ER. In summation, loss of ARV1 has a profound impact on lipid homeostasis in yeast and metazoans. Various sterol detoxification pathways are activated in order to offset the loss of ARV1. In a hepatocyte, cholesterol biosynthesis is decreased and bile acid secretion is increased, in response to ARV1 deficiency.

In yeast and macrophage models, where conversion of excess sterols into bile acids is not possible, the UPR is activated in order to compensate for loss of ARV1 function. Taken as a whole, these studies reflect the role of ARV1 in ER sterol distribution and trafficking, and the profound impact of decreased ARV1 expression on intracellular sterol homeostasis.