Identification of an Htm1 (EDEM)-dependent, Mns1-independent Endoplasmic Reticulum-associated Degradation (ERAD) pathway in Saccharomyces cerevisiae: application of a novel assay for glycoprotein ERAD.
Endoplasmic reticulum (ER)-associated degradation (ERAD) is a quality control system for newly synthesized proteins in the ER; nonfunctional proteins, which fail to form their correct folding state, are then degraded. The cytoplasmic peptide:N-glycanase is a deglycosylating enzyme that is involved in the ERAD and releases N-glycans from misfolded glycoproteins/glycopeptides. We have previously identified a mutant plant toxin protein, RTA (ricin A-chain nontoxic mutant), as the first in vivo Png1 (the cytoplasmic peptide:N-glycanase in Saccharomyces cerevisiae)-dependent ERAD substrate. Here, we report a new genetic device to assay the Png1-dependent ERAD pathway using the new model protein designated RTL (RTA-transmembrane-Leu2). Our extensive studies using different yeast mutants identified various factors involved in RTL degradation. The degradation of RTA/RTL was independent of functional Sec61 but was dependent on Der1. Interestingly, ER-mannosidase Mns1 was not involved in RTA degradation, but it was dependent on Htm1 (ERAD-related alpha-mannosidase in yeast) and Yos9 (a putative degradation lectin), indicating that mannose trimming by Mns1 is not essential for efficient ERAD of RTA/RTL. The newly established RTL assay will allow us to gain further insight into the mechanisms involved in the Png1-dependent ERAD-L pathway.
Hosomi
Akira
A
Glycometabolome Team, RIKEN Advanced Science Institute, Wako, Saitama 351-0198, Japan.
Tanabe
Kaori
K
Hirayama
Hiroto
H
Kim
Ikjin
I
Rao
Hai
H
Suzuki
Tadashi
T
D001483:Base Sequence; D001671:Biochemistry / Q000379:methods; D003513:Cycloheximide / Q000737:chemistry; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D006023:Glycoproteins / Q000737:chemistry*; D006031:Glycosylation; D008361:Mannosidases / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000378:metabolism; D008969:Molecular Sequence Data; D010957:Plasmids / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex / Q000737:chemistry; D011487:Protein Conformation; D000069816:SEC Translocation Channels; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*; D025801:Ubiquitin / Q000737:chemistry; D043323:alpha-Mannosidase / Q000737:chemistry* / Q000378:metabolism*
C098515:DER1 protein, S cerevisiae; D006023:Glycoproteins; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D000069816:SEC Translocation Channels; C499383:SEC61 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D025801:Ubiquitin; D003513:Cycloheximide; C434527:MNL1 protein, S cerevisiae; D008361:Mannosidases; D043323:alpha-Mannosidase; D046988:Proteasome Endopeptidase Complex