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Abstract
BackgroundThe 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.ResultsWe 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 sec61∆L7 than in wildtype cells. Although Sec61∆L7 channels were unstable in detergent, co-translational protein integration into the ER membrane, proteasome binding to Sec61∆L7 channels, and formation of hetero-heptameric Sec complexes were not affected.ConclusionsWe 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.
Highlights
The Sec61 channel mediates protein translocation across the endoplasmic reticulum (ER) membrane during secretory protein biogenesis, and likely during export of misfolded proteins for ER-associated degradation (ERAD)
Transformants were selected on minimal media without leucine, and the wildtype SEC61 plasmid was counterselected on plates containing 5′- fluoroorotic acid (5′-FOA)
Upon sequencing we found that in the mutant amino acids 305–371 of Sec61p had been replaced with two amino acids, arginine and glutamate, only, which is equivalent to deletion of the entire L7 and the luminal ends of transmembrane domains (TMDs) 7 and 8 (Figure 1C)
Summary
The Sec channel mediates protein translocation across the endoplasmic reticulum (ER) membrane during secretory protein biogenesis, and likely during export of misfolded proteins for ER-associated degradation (ERAD). 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]. In yeast proteins are imported co-translationally into the ER through a proteinaceous channel formed by the Sec complex [3,4]. This heterotrimeric complex consists of the channel-forming Sec protein, and two small proteins, Sss1p and Sbh1p, which stabilize the channel and mediate interactions with other protein complexes [3,5,6].
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