Human Endoplasmic Reticulum Mannosidase I Is Subject to Regulated Proteolysis

Abstract

In the early secretory pathway, opportunistic cleavage of asparagine-linked oligosaccharides by endoplasmic reticulum (ER) mannosidase I targets misfolded glycoproteins for dislocation into the cytosol and destruction by 26 S proteasomes. The low basal concentration of the glycosidase is believed to coordinate the glycan cleavage with prolonged conformation-based ER retention, ensuring that terminally misfolded glycoproteins are preferentially targeted for destruction. Herein the intracellular fate of human ER mannosidase I was monitored to determine whether a post-translational process might contribute to the regulation of its intracellular concentration. The transiently expressed recombinant human glycosidase was subject to rapid intracellular turnover in mouse hepatoma cells, as was the endogenous mouse ortholog. Incubation with either chloroquine or leupeptin, but not lactacystin, led to intracellular stabilization, implicating the involvement of lysosomal acid hydrolases. Inhibition of protein synthesis with cycloheximide led to intracellular depletion of the glycosidase and concomitant ablation of asparagine-linked glycoprotein degradation, confirming the physiologic relevance of the destabilization process. Metabolic incorporation of radiolabeled phosphate, detection by anti-phosphoserine antiserum, and the stabilizing effect of general serine kinase inhibition implied that ER mannosidase I is subjected to regulated proteolysis. Stabilization in response to genetically engineered removal of the amino-terminal cytoplasmic tail, a postulated regulatory domain, and colocalization of green fluorescent protein fusion proteins with Lamp1 provided two additional lines of evidence to support the hypothesis. A model is proposed in which proteolytically driven checkpoint control of ER mannosidase I contributes to the establishment of an equitable glycoprotein quality control standard by which the efficiency of asparagine-linked glycoprotein conformational maturation is measured.Genetic information is directly transformed into biological activity in response to the correct conformational maturation and deployment of encoded proteins (1, 2). Arguably, this dual intention is best exemplified in the endoplasmic reticulum (ER) 2 into which nascent secretory and membrane proteins are translocated. The adoption of native protein structure, facilitated by transient physical engagement with one or more molecular chaperones, precedes productive export from the ER (3). Rather than clogging the secretory pathway, molecules incapable of structural maturation are eliminated by a process coined "ER-associated degradation" (ERAD). The associated multiple requisite steps culminate in the dislocation of misfolded proteins into the cytosol for degradation by 26 S proteasomes (4, 5). Polyubiquitination functions as the signal that mediates both dislocation and proteolysis ( Fig. 1) (6, 7).As with glycoprotein folding (8), the earliest events of ERAD are orchestrated by the covalent modification of asparaginelinked oligosaccharides (Fi...

Eeyarestatin I inhibits Sec61-mediated protein translocation at the endoplasmic reticulum

“…Fiebiger et al, 2004). Likewise, several endogenous components of the ERAD machinery are rapidly turned over (Cali et al, 2008;Wu et al, 2007), and any ES I -mediated inhibition of their translocation into the ER would also most probably disrupt the native degradation pathway. Alternatively, the Sec61 complex has been strongly linked to the ERAD process by a number of studies (Romisch, 2005;Scott and Schekman, 2008;Willer et al, 2008), and it is possible that inactivation of the ER translocation machinery simultaneously blocks translocation and retrotranslocation.…”

A novel function of VCP (valosin-containing protein; p97) in the control of N-glycosylation of proteins in the endoplasmic reticulum

“…Therefore, an increase in the level of αTCR with a truncated high-mannose oligosaccharide moiety is expected in a situation, where retrotranslocation of αTCR is blocked. However, it has been recently reported that ERresident mannosidase I is regulated by proteolysis mediated by the lysosomal compartment [52]. Since VCP is required for homotypic vesicle fusion in the ER and Golgi [53,54], VCP depletion likely diminishes the traffic of ER proteins to lysosomes providing an alternative explanation to increased levels of demannosylation of N-glycans in the absence of any direct effect of VCP on ERAD.…”

“…Moreover, RNAi of VCP also induced changes in the levels of different complex glycans synthesized in the post-ER compartments. Those changes may either reflect the different abundance of coreglycosylated precursors trafficked from the ER or -alternatively -they may indicate that VCP controls different aspects of glycosylation, for example the regulated degradation of glycosylation enzymes within a lysosomal compartment [52]. The latter possibility is supported by the fact that VCP is the major ATP-ase associated with transitional ER and the Golgi involved in homotypic membrane fusion within that compartment [53,54].…”

The mammalian UPR boosts glycoprotein ERAD by suppressing the proteolytic downregulation of ER mannosidase I

“…Because the endogenous protein concentration is below the level of immunological detection (Wu et al, 2007), the intracellular fate of the transfected recombinant human homolog was monitored by metabolic pulse-chase radiolabeling and immunoprecipitation in HEK293 cells. As previously reported for cultured hepatoma cells (Wu et al, 2007), an 82 kDa radiolabeled protein was synthesized during a 20-minute pulse with [ 35 S]methionine, and nearly the entire cohort of molecules was degraded during 90 minutes of chase ( Fig. 2A, Co).…”

“…To this end, we recently reported that human ERManI is subject to proteolytic downregulation under basal conditions (Wu et al, 2007) in a manner that is operationally similar to the regulation of checkpoint proteins that monitor DNA damage, except that lysosomal hydrolases contribute to proteolysis. Because many nuclear regulatory proteins are subject to checkpoint activation through impairment of proteolytic downregulation (Zhou and Elledge, 2000), herein we asked whether post-translational stabilization of ERManI might contribute to the coordinated induction between GERAD and the mammalian unfolded protein response.…”