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.
Mesh Terms:
Amino Acid Sequence, Binding Sites, Conserved Sequence, Gene Deletion, Genes, Fungal, Lysine, Methylation, Molecular Sequence Data, Peptide Elongation Factor 1, Protein Methyltransferases, Protein Processing, Post-Translational, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins, Sequence Homology, Amino Acid
J. Biol. Chem.
Date: Aug. 06, 2010
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