Inositol Deacylation by Bst1p Is Required for the Quality Control of Glycosylphosphatidylinositol-anchored Proteins

Abstract

Misfolded proteins are recognized in the endoplasmic reticulum (ER), transported back to the cytosol, and degraded by the proteasome. A number of proteins are processed and modified by a glycosylphosphatidylinositol (GPI) anchor in the ER, but the quality control mechanisms of GPI-anchored proteins remain unclear. Here, we report on the quality control mechanism of misfolded GPI-anchored proteins. We have constructed a mutant form of the ␤-1,3-glucanosyltransferase Gas1p (Gas1*p) as a model misfolded GPI-anchored protein. Gas1*p was modified with a GPI anchor but retained in the ER and was degraded rapidly via the proteasome. Disruption of BST1, which encodes GPI inositol deacylase, caused a delay in the degradation of Gas1*p. This delay was because of an effect on the deacylation activity of Bst1p. Disruption of genes involved in GPI-anchored protein concentration and N-glycan processing caused different effects on the degradation of Gas1*p and a soluble misfolded version of carboxypeptidase Y. Furthermore, Gas1*p associated with both Bst1p and BiP/Kar2p, a molecular chaperone, in vivo. Our data suggest that GPI inositol deacylation plays important roles in the quality control and ER-associated degradation of GPI-anchored proteins. INTRODUCTIONCells possess several quality control mechanisms for the maintenance of proper protein folding and function. On synthesis, membrane and secretory proteins are inserted into the lumen of the endoplasmic reticulum (ER), where they are folded and undergo oligomerization. There are a number of chaperones and enzymes in the ER required for proper protein folding (Ellgaard et al., 1999). Before exiting from the ER, proteins are monitored by a quality control system that ensures correct folding. Misfolded proteins that fail to pass the quality control checkpoint are transported back to the cytosol and degraded by an ER-associated degradation (ERAD) mechanism that involves the ubiquitinproteasome pathway (Kopito, 1997;Kostova and Wolf, 2003). N-linked oligosaccharide, one of the major posttranslational modifications in the ER, is trimmed and processed by glucosidase I, glucosidase II, and mannosidase I (Jakob et al., 1998;Helenius and Aebi, 2001). The processing of Nlinked oligosaccharides plays important roles in the quality control of glycoprotein folding in the ER Aebi, 2001, 2004).A number of cell surface proteins are posttranslationally modified in the ER with glycosylphosphatidylinositol (GPI). Mechanisms for quality control and degradation of GPIanchored proteins are important in folding diseases, including prion diseases and transmissible spongiform encephalopathies, which are caused by a conformational modification of prion, a GPI-anchored protein (Prusiner, 1998). There have been several biochemical studies on both the degradation of mutated prions and the quality control of proteins with mutated GPI attachment signals (Field et al., 1994;Oda et al., 1996;Wainwright and Field, 1997;Jin et al., 2000;Ito et al., 2002;Ishida et al., 2003). In contrast, the molecular mech...

Protein O-mannosyltransferases participate in ER protein quality control

“…These results show that Gas1*p is indeed targeted for degradation by the ERAD-L pathway. Our results are different from those published previously reporting that deleting Hrd1p had no effect on Gas1*p turnover (Fujita et al, 2006).…”

“…As can be seen in Fig. 2B, lanes 1-4 and graph, when expressed in wild-type cells Gas1*p is degraded with a half-life of roughly 1.5 hours, consistent with previous data (Fujita et al, 2006). When we tested deletion mutants of all membrane-bound Hrd1p complex components (Hrd1p, Hrd3p, Der1p,and Usa1p), we found that they stabilized Gas1*p whereas Pmt1p-Pmt2p complex in ER quality control deletion of Doa10p (the central component of the ERAD-C pathway) did not (Fig.…”

“…In its wild-type form, the protein leaves the ER depending on the p24 protein complex and is targeted to the plasma membrane (Muniz et al, 2001;Muniz et al, 2000;Schimmoller et al, 1995). Importantly, a mutant version of Gas1p, Gas1*p, is unstable and degraded by an unidentified proteasomedependent pathway (Fujita et al, 2006). Interestingly, whereas Gas1p is O-glycosylated by Pmt4p and Pmt6p alone, Gas1*p is further O-glycosylated by Pmt1p and Pmt2p (Hirayama et al, 2008).…”

Architecture and Biosynthesis of the Saccharomyces cerevisiae Cell Wall

“…Roles for the noncatalytic subunits include recognition of the peptide and glycolipid substrates (Signorell and Menon 2009), and, in the case of Gab1 and Gpi8, possible interactions with the actin cytoskeleton (Grimme et al 2004;File S4) Remodeling of protein-bound GPIs: Following GPI transfer to protein, both the anchor's lipid and glycan remodeled (Figure 6;Fujita and Kinoshita 2010). The earliest event, which occurs in the ER, is removal of the inositol acyl moiety by lipase-related Bst1 (Tanaka et al 2004;Fujita et al 2006a). Next, the sn-2 acyl chain of the diacylglycerol is removed by the ER membrane protein Per1 to generate a lyso-GPI , whereupon a C 26:0 acyl chain is transferred to the sn-2 position by Gup1 in the ER membrane .…”

Potential Roles of GPI-Anchor Remodeling in Protein Trafficking and Raft Association in Mammalian Cells

“…PGAP1 is a multiple membrane-spanning protein localized in the ER that contains a lipase motif (GxSxG) in the luminal portion. Inositol deacylation by PGAP1 is required for the efficient transport of GPI-anchored proteins from the ER to the Golgi, as described in Section D. In yeast, the reaction is also needed for the efficient degradation of misfolded GPIanchored proteins (38). In addition, inositol deacylation is indispensable for the following fatty acid remodeling described in Section C-4 (39).…”