Abstract
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.
Abbreviations List
- ERAD
- Endoplasmic reticulum-associated protein degradation
- Hex10
- Hex10GlcNAc2
- Man7~9
- Man7~9GlcNAc2
- CPY
- Yeast pro-carboxypeptidase Y
- CPY*
- CPY G255R mutant
- MALDI-TOF MS
- Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry
- DTT
- Dithiothreitol
- Diamide
- N,N,N’,N’-tetramethylazodicarboxamide
- IAA
- Iodoacetamide
- Scr-CPY
- Disulfide-scrambled CPY
- Carb-CPY
- Cysteine-carbamidomethylated CPY
- DMJ
- 1-deoxymannojirimycin
- E222Q
- Htm1 mutant with putative active site residue Glu222 mutated to Gln
- Δ572-657
- Htm1 mutant with truncation of the putative Pdi1p-interacting domain
- CPY*1110
- CPY* mutant with an N479Q mutation at the fourth N-glycosylation site
- CPY*0001
- CPY* mutant with the first three N-glycosylation sites mutagenized (N124Q, N198Q and N279Q)
- CPYm9
- Man9-carrying CPY purified from mns1Δhtm1Δ
- Scr-CPYm9
- Disulfide-scrambled CPYm9
- RNase B
- bovine pancreatic ribonuclease B
- RBsp
- RNase BS protein
- RNase BS
- Non-covalent complex between the S peptide and RBsp
- Scr-RB
- Disulfide-scrambled RNase B
- Carb-RB
- Cysteine-carbamidomethylated RNase B
- Red-RB
- Fully reduced RNase B
- Scr-RBsp
- Disulfide-scrambled RBsp
- Carb-RBsp
- Cysteine-carbamidomethylated RBsp