SEL1L Is Required for Endoplasmic Reticulum-associated Degradation of Misfolded Luminal Proteins but not Transmembrane Proteins in Chicken DT40 Cell Line

Department of Biophysics, Graduate School of Science, Kyoto University, Sakyo-ku, Kyoto, Japan.
Cell Structure and Function (Impact Factor: 1.68). 08/2011; 36(2):187-95. DOI: 10.1247/csf.11018
Source: PubMed


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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    • "As spliced ERdj5 lacks 46 amino acids within a redox-inactive domain present in unspliced ERdj5 (i.e., the so-called Trxb1 domain; Hagiwara et al., 2011), this part is not likely to be responsible for engaging Sel1L. Identifying an ERdj5-Sel1L complex prompted us to examine whether Sel1L functions in CTA1 retrotranslocation, especially considering that ERAD substrate degradation can occur using a Sel1L- independent mechanism (Mueller et al., 2006; Bernasconi et al., 2010; Ninagawa et al., 2011; Stanley et al., 2011). CT coprecipitated with Sel1L, suggesting a physical interaction between them. "
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