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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.

SUBMITTER: Shechtman CF

PROVIDER: S-EPMC3069397 | biostudies-literature | 2011 Apr

REPOSITORIES: biostudies-literature

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Loss of subcellular lipid transport due to ARV1 deficiency disrupts organelle homeostasis and activates the unfolded protein response.

Shechtman Caryn F CF Henneberry Annette L AL Seimon Tracie A TA Tinkelenberg Arthur H AH Wilcox Lisa J LJ Lee Eunjee E Fazlollahi Mina M Munkacsi Andrew B AB Bussemaker Harmen J HJ Tabas Ira I Sturley Stephen L SL

The Journal of biological chemistry 20110125 14

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 tra ...[more]

PMID: 21266578

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Project description: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 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 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, C/EBP homologous protein (CHOP) 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. Yeast strains were grown to mid-logarithmic stage (A600=0.5-0.6) for RNA extraction and hybridization on Ye6100 or S98 Affymetrix gene chips. The control array (sample name C) was carried out in duplicate. The ARV1 mutant array (sample name A) was carried out in triplicate. Both ARV1 mutants Ye6100 arrays were compared to the same control Ye6100 array. Strains represent different isolates of the same genotype, mating type and genetic background (w303). Sturley lab collection (SCY) strains include SCY328 and SCY2004 for controls and SCY820 and SCY840 for ARV1 mutants.

Project description:Zinc is an essential nutrient because of its role in catalysis and in stabilizing protein structure, but excess zinc can also be deleterious. Four nutritional zinc states have been identified in the alga Chlamydomonas reinhardtii: zinc toxic, zinc replete, zinc deficient and zinc limited. Growth is inhibited in zinc-limited and zinc toxic cells relative to zinc-replete cells, while zinc-deficiency is visually asymptomatic but distinguished by the accumulation of transcripts encoding ZIP family transporters. To identify targets of zinc deficiency and mechanisms of zinc acclimation, we used RNA-seq to probe zinc nutrition responsive changes in gene expression. We identified a subset of genes encoding zinc-handling components, including ZIP family transporters and candidate zinc chaperones. In addition, we noted an impact on two other regulatory pathways, the carbon concentrating mechanism (CCM) and the nutritional copper regulon. Targets of transcription factor Ccm1 and various CAH genes are up-regulated in zinc-deficiency, as a likely consequence of reduced carbonic anhydrase activity, which is validated by mass spectrometry and immunoblot analysis of Cah1, Cah3 and Cah4. Chlamydomonas is therefore not able to grow photoautotrophically in air in zinc limiting conditions, but supplementation with 1% CO2 restores growth to wild-type rates, suggesting that the inability to maintain CCM is a major consequence of zinc limitation. Surprisingly, we noted also that the Crr1 regulon, which responds to Cu limitation, is also turned on in zinc deficiency, and in fact, Crr1 is required for growth in zinc-limiting conditions. Zinc deficient cells are functionally copper deficient, as evidenced by reduced plastocyanin abundance, even though they hyperaccumulate copper up to 50-fold over normal levels. We suggest that zinc-deficient cells sequester Cu in a bio-unavailable form, perhaps to prevent mis-metallation of critical zinc sites. Zn-limited wild-type cells were generated by transfer of cells from the first round of growth in medium with no supplemental Zn into TAP medium supplemented or not with 2.5 ÂµM Zn-EDTA The control samples for this study are represented in GSE25622

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Loss of subcellular lipid transport due to ARV1 deficiency disrupts organelle homeostasis and activates the unfolded protein response.

Publications

Loss of subcellular lipid transport due to ARV1 deficiency disrupts organelle homeostasis and activates the unfolded protein response.

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