Cold‐sensitive phenotypes of a yeast null mutant of ARV1 support its role as a GPI flippase

Abstract

Glycosylphosphatidylinositol (GPI) is synthesized in the endoplasmic reticulum (ER) and added onto proteins to form GPI‐anchored proteins. Among the many proteins involved in this process, ACAT‐related enzyme‐2 required for viability 1 (Arv1) is a candidate, functioning as a flippase that translocates GPI intermediates from the cytoplasmic side into the luminal side of the ER membranes. Here, we show that the deletion of the ARV1 gene in yeast leads to cold‐sensitive defects in cell growth and GPI anchor synthesis. Furthermore, complementation assays show that the overexpression of a missense human ARV1‐G189R mutant does not completely restore the cold‐sensitive phenotypes of the yeast arv1 mutant. Our results support the proposed role of Arv1 in GPI anchor synthesis and suggest that ARV1‐linked human diseases result from defective GPI anchor synthesis.

Proteomic identification of the UDP-GlcNAc: PI α1–6 GlcNAc-transferase subunits of the glycosylphosphatidylinositol biosynthetic pathway of Trypanosoma brucei

“…TbArv1 is predicted to contain four transmembrane domains and an Arv1 domain (PF04161). Previous studies in yeast have indicated that Arv1p, although non-essential for growth and therefore GPI-anchoring of proteins at 25˚C [39,40], is required for the efficient synthesis of Man 1 GlcN-acylPI (mannosyl-glucosaminyl-acyl-phosphatidylinositol) [41] It has been postulated to be a GPI flippase [41,42] helping deliver GlcN-acylPI, which is made on the cytoplasmic face of the ER, to the active site of mannosyl-transferase I (MT I) on the luminal face of the ER. The complementation of yeast Arv1 mutants by the human Arv1 [43] and recent findings that human Arv1 mutations lead to deficiencies in GPI anchoring [44,45] strongly suggest a related role in mammalian cells.…”

Epileptic encephalopathy caused by ARV1 deficiency: Refinement of the genotype–phenotype spectrum and functional impact on GPI‐anchored proteins

“…8 It has been proposed that ARV1 functions either by guiding GlcN-acylPI into ER lumen from cytosol or presenting it to mannosyltransferase. 1,12 The results of the flow cytometry analysis of GPI-APs in patient cells and rescue assay confirm the essential role of ARV1 in GPI biosynthesis. The GPI biosynthesis pathway is a multi-step process in which GPI-anchored proteins (GPI-APs) are synthesized in the ER, moved to the Golgi system, and eventually transported to cell surface (Supplementary Figure 4).…”

“…In S. cerevisiae , ARV1 deficiency leads to increased glucosaminyl‐acyl‐PI (GlcN‐acylPI), suggesting that ARV1 is crucial for the first mannosylation of the GPI 8 . It has been proposed that ARV1 functions either by guiding GlcN‐acylPI into ER lumen from cytosol or presenting it to mannosyltransferase 1,12 . The results of the flow cytometry analysis of GPI‐APs in patient cells and rescue assay confirm the essential role of ARV1 in GPI biosynthesis.…”

Proteomic identification of the UDP-GlcNAc : PI α1-6 GlcNAc-transferase subunits of the glycosylphosphatidylinositol biosynthetic pathway ofTrypanosoma brucei

“…TbArv-1 is predicted to contain four transmembrane domains and an Arv1 domain (PF04161). Previous studies in yeast have indicated that Arv1p is required for the efficient synthesis of Man 1 GlcN-acylPI (mannosyl-glucosaminyl-acyl-phosphatidylinositol) [36] and has been postulated to be a GPI flippase [36] [37] helping deliver GlcN-acylPI, which is made on the cytoplasmic face of the ER, to the active site of mannosyl-transferase I (MT I) on the luminal face of the ER. The complementation of yeast Arv1 mutants by the human Arv1 [38] and recent findings that human Arv1 mutations lead to deficiencies in GPI anchoring [39] [40] strongly suggest a related role in mammalian cells and that it is a component of the mammalian UDP-GlcNAc : PI 1-6 GlcNAc-transferase complex.…”