Loss of Subcellular Lipid Transport Due to ARV1 Deficiency Disrupts Organelle Homeostasis and Activates the Unfolded Protein Response

Abstract

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.

Sphingolipids regulate telomere clustering by affecting transcriptional levels of genes involved in telomere homeostasis

“…RTA1 encodes a protein involved in 7-aminocholesterol resistance, and SIL1 encodes a nucleotide exchange factor for the ER luminal chaperone Kar2, which has a protective role in response to ER stress. The increased expression levels of these genes are in good agreement with the functions of Arv1p in GPI anchor synthesis, sterol and ER homeostasis (Kajiwara et al, 2012(Kajiwara et al, , 2008Shechtman et al, 2011;Tinkelenberg et al, 2000), and the increased MCD4 levels in the arv1Δ mutant suggest that GPI anchor synthesis might signal in a feedback loop to Mcd4. By contrast, the overlapping genes with more than a threefold decrease in transcription included RPI1 and SST2.…”

“…Although the experiments were carried out under different culture conditions (SD versus YPD medium) and with different genetic backgrounds (RH6082 versus W303), we compared our data with the previously reported microarray data from the arv1Δ mutant (Shechtman et al, 2011). As shown in supplementary material Fig.…”

Regulation of sphingolipid synthesis via Orm1 and Orm2 in yeast

“…6) (Han et al, 2010). Other reports have linked ER stress and UPR activation with the perturbation of lipid homeostasis (Brookheart et al, 2009;Erbay et al, 2009;Pineau et al, 2009;Shechtman et al, 2011). If UPR is solely a response to protein misfolding in the ER, a possible explanation for activated UPR in orm1D orm2D cells is that lipid imbalance causes misfolding of membrane proteins that have a conformational requirement for lipids.…”

Mice lacking have reduced signs of metabolic syndrome and non-alcoholic fatty liver disease

“…Our results strongly suggest that complete loss of Arv1 protects mice from acquiring MetS and NAFLD in response to a HFD challenge. While it is still up for debate as to what is the function of ARV1 (13)(14)(15)18,19,41), evidence is accumulating that suggests it's involvement in maintaining proper sterol distribution (16,20,42,43).…”

“…arv1 deficient cells are hypersensitive to the PS (phosphatidylserine) binding agent, papuamide-B, suggesting a mislocalization of PS to the plasma membrane (17). They contain fragmented vacuoles and a disrupted organelle phenotype (17), which most likely activates the unfolded protein response (18). Null cells are hypersensitive to fatty acid supplementation (19), suggesting that Arv1 also regulates fatty acid metabolism.…”