Abstract
Import of secretory proteins into the Endoplasmic Reticulum (ER) is an established function of the Sec61 channel. The contribution of the Sec61 channel to export of misfolded proteins from the ER for degradation by proteasomes is still controversial, but the proteasome 19S regulatory particle (RP) is necessary and sufficient for extraction of specific misfolded proteins from the ER, and binds directly to the Sec61 channel. In this work we have identified an import-competent sec61 mutant, S353C, carrying a point mutation in ER-lumenal loop 7 which reduces affinity of the cytoplasmic face of the Sec61 channel for the 19S RP. This indicates that the interaction between the 19S RP and the Sec61 channel is dependent on conformational changes in Sec61p hinging on loop 7. The sec61-S353C mutant had no measurable ER import defects and did not cause ER stress in intact cells, but reduced ER-export of a 19S RP-dependent misfolded protein when proteasomes were limiting in a cell-free assay. Our data suggest that the interaction between the 19S RP and the Sec61 channel is essential for the export of specific substrates from the ER to the cytosol for proteasomal degradation.
Highlights
Proteins destined for secretion enter the secretory pathway by translocation through the Sec61 channel in the membrane of the endoplasmic reticulum (ER) [1]
To analyze whether our new sec61 mutants were defective in biosynthetic protein import into the ER, we transformed the strains with reporter plasmids encoding a fusion of the carboxypeptidase Y (CPY) signal peptide to the ura3–52 RPT1FH::YIplac21 (URA3) gene, or a fusion of the Pho8p signal anchor to the URA3 gene
In the yeast strains used the chromosomal copy of the URA3 gene was deleted, the cells were only able to grow in the absence of uracil if the fusion protein remained in the cytosol due to a protein import defect into the ER (Fig. 1C, top) [29]
Summary
Proteins destined for secretion enter the secretory pathway by translocation through the Sec channel in the membrane of the endoplasmic reticulum (ER) [1]. Part of the controversy stems from the fact that most sec mutants initially characterized for ERAD had defects in transport across the ER membrane in both directions, which made it difficult to differentiate direct effects on ERAD-related export from indirect effects caused by altered import of ER-resident proteins required for ERAD [3, 4, 5]. The Sec channel consists of three proteins, Sec61p, Sbh1p, and Sss1p in yeast, equivalent to Sec α, β, γ in mammals [6]. This channel on its own mediates cotranslational protein import into the ER, during which the ribosome binds to Sec61p and Sbh1p [7, 8]. A mutation in PLOS ONE | DOI:10.1371/journal.pone.0117260 February 6, 2015
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