A Complex of Htm1 and the Oxidoreductase Pdi1 Accelerates Degradation of Misfolded Glycoproteins

Abstract

A quality control system in the endoplasmic reticulum (ER) efficiently discriminates polypeptides that are in the process of productive folding from conformers that are trapped in an aberrant state. Only the latter are transported into the cytoplasm and degraded in a process termed ER-associated protein degradation (ERAD). In the ER, an enzymatic cascade generates a specific N-glycan structure of seven mannosyl and two N-acetylglucosamine residues (Man7GlcNAc2) on misfolded glycoproteins to facilitate their disposal. We show that a complex encompassing the yeast lectin-like protein Htm1 and the oxidoreductase Pdi1 converts Man8GlcNAc2 on glycoproteins into the Man7GlcNAc2 signal. In vitro the Htm1-Pdi1 complex processes both unfolded and native proteins albeit with a preference for the former. In vivo, elevated expression of HTM1 causes glycan trimming on misfolded and folded proteins, but only degradation of the non-native species is accelerated. Thus, modification with a Man7GlcNAc2 structure does not inevitably commit a protein for ER-associated protein degradation. The function of Htm1 in ERAD relies on its association with Pdi1, which appears to regulate the access to substrates. Our data support a model in which the balanced activities of Pdi1 and Htm1 are crucial determinants for the efficient removal of misfolded secretory glycoproteins.

Htm1p–Pdi1p is a folding-sensitive mannosidase that marks N-glycoproteins for ER-associated protein degradation

“…Finally, we made a second Htm1 mutant with a truncation of the amino acyl region from residue 572 to residue 657 (Δ572-657), which covers the region necessary for interaction with Pdi1p ( Fig. S2A) (14,23,28). Immunoprecipitation confirmed that Δ572-657 showed little detectable interaction with Pdi1p ( Fig.…”

“…To this point, our findings support two potential models describing how Htm1p-Pdi1p generates the α1,6-linked mannose signal for ERAD-L commitment. In one model, the catalysis by Htm1p-Pdi1p may be intrinsically slow and stochastic, as previously suggested (15,23), and unfolding facilitates demannosylation by increasing access to the glycan. Alternatively, Htm1p-Pdi1p may preferentially target specific folding states of nonnative proteins, and the aggregation-prone nature of misfolded CPY variants prevents them from populating in these states in vitro.…”

“…Finally, both in vivo and in vitro studies have provided evidence for the mannosidase activity of Htm1p in generating Man7 (14,15,23). However, despite the well-documented role of Htm1p in the N-glycan processing for ERAD-L commitment, it is still unclear whether Htm1p targets misfolded proteins specifically and accurately.…”

“…This uncertainty mainly results from the challenges in simultaneously monitoring protein conformations and their N-glycosylation states in the highly heterogeneous pool of glycoproteins in the ER. Additionally, only minimal mannosidase activities of Htm1p were observed in previous in vitro studies (15,23). To advance our understanding of this commitment step, it is critical to develop a reconstituted system for direct investigation of the enzymatic activities of Htm1p against glycoproteins with defined glycosylation and folding states (24).…”

Parallel genome-wide CRISPR analysis identifies a role for heterotypic ubiquitin chains in ER-associated degradation

“…Although A1AT NHK -GFP and GFP-RTA E177Q share a strict dependence on the core membraneintegrated HRD1/SEL1L complex and its associated cytosolic components, they differ strikingly in their dependence on N-glycosylation. A1AT NHK -GFP degradation was strongly dependent on the mannosidase complex composed of EDEM2/3 and the oxidoreductases TXNDC11 and PDI that generates the mannose-trimmed Asn-linked glycan which marks proteins for destruction (Clerc et al, 2009;Gauss et al, 2011;Molinari et al, 2003;Ninagawa et al, 2014;Oda et al, 2003;Pfeiffer et al, 2016;Timms et al, 2016), and the lectin, OS9, that recognizes this glycan signal and delivers proteins to HRD1/SEL1L (Christianson et al, 2008;Hosokawa et al, 2009;Quan et al, 2008;Satoh et al, 2010), as well as nearly every gene involved in the formation or transfer of N-linked glycans ( Figs. 4A and B).…”