Misfolded proteins in the endoplasmic reticulum (ER) are destroyed by a pathway termed ER-associated protein degradation (ERAD). Glycans are often removed from glycosylated ERAD substrates in the cytosol before substrate degradation, which maintains the efficiency of the proteasome. Png1, a deglycosylating enzyme, has long been suspected, but not proven, to be crucial in this process. We demonstrate that the efficient degradation of glycosylated ricin A chain requires the Png1–Rad23 complex, suggesting that this complex couples protein deglycosylation and degradation. Rad23 is a ubiquitin (Ub) binding protein involved in the transfer of ubiquitylated substrates to the proteasome. How Rad23 achieves its substrate specificity is unknown. We show that Rad23 binds various regulators of proteolysis to facilitate the degradation of distinct substrates. We propose that the substrate specificity of Rad23 and other Ub binding proteins is determined by their interactions with various cofactors involved in specific degradation pathways.

1-Nitropyrene (1-NP) is one of the most abundant nitrated polycyclic aromatic hydrocarbons (NPAHs) in diesel exhaust particulate matter (DEP) and is a main contributor of direct-acting mutagenicity in DEP. Therefore, the metabolites of 1-NP are expected to be a biomarker for assessment of exposure to DEP. In this study, a highly specific and sensitive analytical method using liquid chromatography with tandem mass spectrometry (LC-MS/MS) was developed to determine urinary 1-NP metabolites. After enzymatic hydrolysis of the conjugated metabolites, the analytes were selectively extracted from the urine matrix with blue rayon. The eluate from the rayon was further purified on an acidic alumina cartridge. Hydroxy-N-acetyl-1-aminopyrenes (6-and 8-OHNAAP) and hydroxy-1-nitropyrenes (3-, 6-, and 8-OHNP) in human urine were identified by their retention times and MS/MS spectra and quantified by using deuterated internal standards. 1-NP metabolites were quantified in urine from all healthy, nonoccupationally exposed subjects. 6-OHNAAP, 8-OHNAAP, 6-OHNP, and 8-OHNP (means of 117, 109, 203, and 137 pmol/mol creatinine, respectively) were the most abundant isomers in human urine. This report is the first to demonstrate the presence of OHNAAPs and OHNPs in human urine, in agreement with previous in vivo and in vitro studies that predicted that these metabolites should be excreted into human urine. This method for determining urinary 1-NP metabolites should be useful for the surveillance of exposure to NPAHs and DEP and will facilitate the study of cancer risk associated with these exposures.

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 ␣-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.In eukaryotes, the folding and assembly of nascent polypeptide chains in the endoplasmic reticulum (ER) 3 are closely monitored by an ER quality control system, which ensures that only correctly folded/assembled proteins are delivered to their respective destinations (1). Terminally misfolded proteins are destroyed by a process called ER-associated degradation (ERAD) (2, 3). ERAD involves the extraction of proteins from the ER to the cytosol followed by proteasome-mediated degradation. Depending on the position of the misfolded region, the recognition step can occur on the luminal side (ERAD-L), the cytosolic side (ERAD-C), or in the ER membrane itself (ERAD-M) (4). Several key components of the ER quality control and ERAD systems have been identified to date, but the mechanisms by which the substrates are selectively recognized remain elusive.The cytoplasmic peptide:N-glycanase (PNGase; Png1 in yeast) is a deglycosylating enzyme involved in the ERAD process (5). Many ERAD substrates are presumed to be deglycosylated by PNGase during the degradation process (6, 7), and the involvement of PNGase activity in the antigen presentation process has been suggested in mammalian cells (8). However, most of the tested ERAD substrates considered susceptible for deglycosylation were efficiently degraded, even when PNGase activity was inhibited (9), making it difficult to assess the biological significance of this enzyme in the ERAD process. Nevertheless, our finding that the ricin A-chain nontoxic mutant (RTA), an ERAD-L substrate, is degraded in a Png1-dependent fashion in Saccharomyces cerevisiae (10) prompted us to further characterize the mechanisms involved in the PNGase-dependent ERAD pathway.To gai...

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