SEL1L is required for endoplasmic reticulum-associated degradation of misfolded luminal proteins but not transmembrane proteins in chicken DT40 cell line.
ABSTRACT Proteins misfolded in the endoplasmic reticulum (ER) are degraded in the cytosol by a ubiquitin-dependent proteasome system, a process collectively termed ER-associated degradation (ERAD). Unraveling the molecular mechanisms of mammalian ERAD progresses more slowly than that of yeast ERAD due to the laborious procedures required for gene targeting and the redundancy of components. Here, we utilized the chicken B lymphocyte-derived DT40 cell line, which exhibits an extremely high homologous recombination frequency, to analyze ERAD mechanisms in higher eukaryotes. We disrupted the SEL1L gene, which encodes the sole homologue of yeast Hrd3p in both chickens and mammals; Hrd3p is a binding partner of yeast Hrd1p, an E3 ubiquitin ligase. SEL1L-knockout cells grew only slightly more slowly than the wild-type cells. Pulse chase experiments revealed that chicken SEL1L was required for ERAD of misfolded luminal proteins such as glycosylated NHK and unglycosylated NHK-QQQ but dispensable for that of misfolded transmembrane proteins such as NHK(BACE) and CD3-δ, as in mammals. The defect of SEL1L-knockout cells in NHK degradation was restored by introduction of not only chicken SEL1L but also mouse and human SEL1L. Deletion analysis showed the importance of Sel1-like tetratricopeptide repeats but not the fibronectin II domain in the function of SEL1L. Thus, our reverse genetic approach using the chicken DT40 cell line will provide highly useful information regarding ERAD mechanisms in higher eukaryotes which express ERAD components redundantly.
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ABSTRACT: Folding-defective proteins must be cleared efficiently from the endoplasmic reticulum (ER) to prevent perturbation of the folding environment and to maintain cellular proteostasis. Misfolded proteins engage dislocation machineries (dislocons) built around E3 ubiquitin ligases that promote their transport across the ER membrane, their polyubiquitylation, and their proteasomal degradation. Here, we report on the intrinsic instability of the HRD1 dislocon and the constitutive, rapid turnover of the scaffold protein HERP. We show that HRD1 dislocon integrity relies on the presence of HRD1 clients that interrupt, in a dose-dependent manner, the UBC6e/RNF5/p97/proteasome-controlled relay that controls HERP turnover. We propose that ER-associated degradation (ERAD) deploys autoadaptive regulatory pathways, collectively defined as ERAD tuning, to rapidly adapt degradation activity to misfolded protein load and to preempt the unfolded protein response (UPR) activation.Molecular cell 11/2013; 56(6). · 14.46 Impact Factor
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ABSTRACT: Proteins misfolded in the endoplasmic reticulum are cleared by the ubiquitin-dependent proteasome system in the cytosol, a series of events collectively termed endoplasmic reticulum-associated degradation (ERAD). It was previously shown that SEL1L, a partner protein of the E3 ubiquitin ligase HRD1, is required for degradation of misfolded luminal proteins (ERAD-Ls substrates) but not misfolded transmembrane proteins (ERAD-Lm substrates) in both mammalian and chicken DT40 cells. Here, we analyzed ATF6, a type II transmembrane glycoprotein which serves as a sensor/transducer of the unfolded protein response, as a potential ERAD-Lm substrate in DT40 cells. Unexpectedly, degradation of endogenous ATF6 as well as exogenously expressed chicken and human ATF6 by the proteasome required SEL1L. Deletion analysis revealed that the luminal region of ATF6 is a determinant for SEL1L-dependent degradation. Chimeric analysis revealed that the luminal region of ATF6 conferred SEL1L dependency on type I transmembrane protein as well. In contrast, degradation of other known type I ERAD-Lm substrates (BACE457, TCR-α, CD3-δ and CD147) did not require SEL1L. Thus, ATF6 represents a novel type of ERAD-Lm substrate requiring SEL1L for degradation despite its transmembrane nature. In addition, endogenous ATF6 was markedly stabilized in wild-type cells treated with kifunensine, an inhibitor of α1,2-mannosidase in the ER, indicating that degradation of ATF6 required proper mannose trimming. Our further analyses revealed that the five ERAD-Lm substrates examined are classified into three subgroups based on their dependency on mannose trimming and SEL1L. Thus, ERAD-Lm substrates are degraded through much more diversified mechanisms in higher eukaryotes than previously thought.Journal of Biological Chemistry 09/2013; · 4.60 Impact Factor
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ABSTRACT: Glycoproteins misfolded in the endoplasmic reticulum (ER) are subjected to ER-associated glycoprotein degradation (gpERAD) in which Htm1-mediated mannose trimming from the oligosaccharide Man8GlcNAc2 to Man7GlcNAc2 is the rate-limiting step in yeast. In contrast, the roles of the three Htm1 homologues (EDEM1/2/3) in mammalian gpERAD have remained elusive, with a key controversy being whether EDEMs function as mannosidases or as lectins. We therefore conducted transcription activator-like effector nuclease-mediated gene knockout analysis in human cell line and found that all endogenous EDEMs possess mannosidase activity. Mannose trimming from Man8GlcNAc2 to Man7GlcNAc2 is performed mainly by EDEM3 and to a lesser extent by EDEM1. Most surprisingly, the upstream mannose trimming from Man9GlcNAc2 to Man8GlcNAc2 is conducted mainly by EDEM2, which was previously considered to lack enzymatic activity. Based on the presence of two rate-limiting steps in mammalian gpERAD, we propose that mammalian cells double check gpERAD substrates before destruction by evolving EDEM2, a novel-type Htm1 homologue that catalyzes the first mannose trimming step from Man9GlcNAc2.The Journal of Cell Biology 08/2014; 206(3):347-56. · 9.69 Impact Factor