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: 1.65). 08/2011; 36(2):187-95.
Source: PubMed

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.

  • [Show abstract] [Hide abstract]
    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; · 14.61 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Cholera toxin (CT) traffics from the host cell surface to the endoplasmic reticulum (ER) where the toxin's catalytic CTA1 subunit retro-translocates to the cytosol to induce toxicity. In the ER, CT is captured by the E3 ubiquitin ligase Hrd1 via an undefined mechanism to prepare for retro-translocation. Using loss- and gain-of function approaches, we demonstrate that the ER-resident factor ERdj5 promotes CTA1 retro-translocation, in part, via its J domain. This Hsp70 cochaperone regulates binding between CTA and the ER Hsp70 BiP, a chaperone previously implicated in toxin retro-translocation. Importantly, ERdj5 interacts with the Hrd1 adapter Sel1L directly through Sel1L's N-terminal lumenal domain, thereby linking ERdj5 to the Hrd1 complex. Sel1L itself also binds CTA and facilitates toxin retro-translocation. By contrast, EDEM1 and OS-9, two established Sel1L binding partners, do not play significant roles in CTA1 retro-translocation. Our results thus identify two ER factors that promote ER-to-cytosol transport of CTA1. They also indicate ERdj5, by binding to Sel1L, triggers BiP-toxin interaction proximal to the Hrd1 complex. We postulate this scenario enables the Hrd1-associated retro-translocation machinery to capture the toxin efficiently once the toxin is released from BiP.
    Molecular biology of the cell 01/2013; · 5.98 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The endoplasmic reticulum-associated degradation (ERAD) machinery selects native and misfolded polypeptides for dislocation across the ER membrane and proteasomal degradation. Regulated degradation of native proteins is an important aspect of cell physiology. For example, it contributes to the control of lipid biosynthesis, calcium homeostasis and ERAD capacity by setting the turnover rate of crucial regulators of these pathways. In contrast, degradation of native proteins has pathologic relevance when caused by viral or bacterial infections, or when it occurs as a consequence of dysregulated ERAD activity. The efficient disposal of misfolded proteins prevents toxic depositions and persistent sequestration of molecular chaperones that could induce cellular stress and perturb maintenance of cellular proteostasis. In the first section of this review, we survey the available literature on mechanisms of selection of native and non-native proteins for degradation from the ER and on how pathogens hijack them. In the second section, we highlight the mechanisms of ERAD activity adaptation to changes in the ER environment with a particular emphasis on the post-translational regulatory mechanisms collectively defined as ERAD tuning.
    Traffic 03/2013; · 4.65 Impact Factor