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
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.
Available from: Jeffrey Williams
- "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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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.
Available from: Maurizio Molinari
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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.
Available from: jbc.org
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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.
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