title: 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.
authors.affiliation: Department of Biophysics, Graduate School of Science, Kyoto University, Sakyo-ku, Kyoto, Japan.
authors.forename: Satoshi
authors.initials: S
authors.lastname: Ninagawa
authors.forename: Tetsuya
authors.initials: T
authors.lastname: Okada
authors.forename: Shunichi
authors.initials: S
authors.lastname: Takeda
authors.forename: Kazutoshi
authors.initials: K
authors.lastname: Mori
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D002645:Chickens; D004721:Endoplasmic Reticulum / Q000201:enzymology / Q000378:metabolism*; D055786:Gene Knockout Techniques; D006031:Glycosylation; D006801:Humans; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment; D044767:Ubiquitin-Protein Ligases / Q000737:chemistry / Q000378:metabolism; D000515:alpha 1-Antitrypsin / Q000235:genetics / Q000378:metabolism
chemical_list: D008562:Membrane Glycoproteins; D011506:Proteins; C117384:SEL1L protein, human; D000515:alpha 1-Antitrypsin; D044767:Ubiquitin-Protein Ligases; D046988:Proteasome Endopeptidase Complex