ERAD and protein import defects in a sec61 mutant lacking ER-lumenal loop 7
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RESEARCH ARTICLE
Open Access
ERAD and protein import defects in a sec61 mutant lacking ER-lumenal loop 7 Thomas Tretter1, Fábio P Pereira1, Ozlem Ulucan2, Volkhard Helms2, Susanne Allan3, Kai-Uwe Kalies3 and Karin Römisch1*
Abstract Background: The Sec61 channel mediates protein translocation across the endoplasmic reticulum (ER) membrane during secretory protein biogenesis, and likely also during export of misfolded proteins for ER-associated degradation (ERAD). The mechanisms of channel opening for the different modes of translocation are not understood so far, but the position of the large ER-lumenal loop 7 of Sec61p suggests a decisive role. Results: We show here that the Y345H mutation in L7 which causes diabetes in the mouse displays no ER import defects in yeast, but a delay in misfolded protein export. A complete deletion of L7 in Sec61p resulted in viable, cold- and tunicamycin-hypersensitive yeast cells with strong defects in posttranslational protein import of soluble proteins into the ER, and in ERAD of soluble substrates. Membrane protein ERAD was only moderately slower in sec61ΔL7 than in wildtype cells. Although Sec61ΔL7 channels were unstable in detergent, co-translational protein integration into the ER membrane, proteasome binding to Sec61ΔL7 channels, and formation of hetero-heptameric Sec complexes were not affected. Conclusions: We conclude that L7 of Sec61p is required for initiation of posttranslational soluble protein import into and misfolded soluble protein export from the ER, suggesting a key role for L7 in transverse gating of the Sec61 channel. Keywords: Protein translocation, Endoplasmic Reticulum, Sec61 channel, ERAD
Background Protein secretion starts with protein translocation into the endoplasmic reticulum (ER) where secretory proteins mature into a functional three-dimensional conformation before they are packaged into ER-to-Golgi transport vesicles [1]. Proteins that fail to fold in the ER are not allowed to enter these vesicles, and are initially retained in the ER [1]. Most are subsequently exported to the cytosol and degraded by proteasomes, a process called ER-associated degradation (ERAD) [2]. In yeast proteins are imported co-translationally into the ER through a proteinaceous channel formed by the Sec61 complex [3,4]. This heterotrimeric complex consists of the channel-forming Sec61 protein, and two small proteins, Sss1p and Sbh1p, which stabilize the channel and mediate interactions with other protein complexes [3,5,6]. During posttranslational import * Correspondence: [email protected] 1 Department of Microbiology, Faculty of Natural Sciences and Technology VIII, Saarland University, Campus A1.5, 66123 Saarbrücken, Germany Full list of author information is available at the end of the article
into the yeast ER the Sec61 channel collaborates with the heterotetrameric Sec63 complex (Sec63p, Sec62p, Sec71p, Sec72p) forming the heptameric Sec complex [3,4]. In yeast transmembrane proteins follow the co-translational pathway, whereas soluble proteins are impo
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