PT - JOURNAL ARTICLE AU - Yi-Chang Liu AU - Danica Galonić Fujimori AU - Jonathan S. Weissman TI - Htm1p-Pdi1p is a folding sensitive mannosidase that marks N-glycoproteins for ER-associated protein degradation AID - 10.1101/049825 DP - 2016 Jan 01 TA - bioRxiv PG - 049825 4099 - http://biorxiv.org/content/early/2016/04/22/049825.short 4100 - http://biorxiv.org/content/early/2016/04/22/049825.full AB - Our understanding of how the endoplasmic reticulum-associated protein degradation (ERAD) machinery efficiently targets terminally misfolded proteins while avoiding the misidentification of nascent polypeptides and correctly folded proteins is limited. For luminal N-glycoproteins, demannosylation of their N-glycan to expose a terminal α1,6-linked mannose is necessary for their degradation via ERAD, but whether this modification is specific to misfolded proteins is unknown. Here we report that the Htm1p-Pdi1p complex acts as a folding-sensitive mannosidase for catalyzing this first committed step. We reconstitute this step in vitro with Htm1p-Pdi1p and model glycoprotein substrates whose structural states we can manipulate. We find that Htm1p-Pdi1p is a glycoprotein-specific mannosidase, which preferentially targets nonnative glycoproteins trapped in partially structured states. As such, Htm1p-Pdi1p is suited to act as a licensing factor that monitors folding in the ER lumen and preferentially commits glycoproteins trapped in partially structured states for degradation.ERADEndoplasmic reticulum-associated protein degradationHex10Hex10GlcNAc2Man7~9Man7~9GlcNAc2CPYYeast pro-carboxypeptidase YCPY*CPY G255R mutantMALDI-TOF MSMatrix-assisted laser desorption/ionization time-of-flight mass spectrometryDTTDithiothreitolDiamideN,N,N’,N’-tetramethylazodicarboxamideIAAIodoacetamideScr-CPYDisulfide-scrambled CPYCarb-CPYCysteine-carbamidomethylated CPYDMJ1-deoxymannojirimycinE222QHtm1 mutant with putative active site residue Glu222 mutated to GlnΔ572-657Htm1 mutant with truncation of the putative Pdi1p-interacting domainCPY*1110CPY* mutant with an N479Q mutation at the fourth N-glycosylation siteCPY*0001CPY* mutant with the first three N-glycosylation sites mutagenized (N124Q, N198Q and N279Q)CPYm9Man9-carrying CPY purified from mns1Δhtm1ΔScr-CPYm9Disulfide-scrambled CPYm9RNase Bbovine pancreatic ribonuclease BRBspRNase BS proteinRNase BSNon-covalent complex between the S peptide and RBspScr-RBDisulfide-scrambled RNase BCarb-RBCysteine-carbamidomethylated RNase BRed-RBFully reduced RNase BScr-RBspDisulfide-scrambled RBspCarb-RBspCysteine-carbamidomethylated RBsp