Abstract
Protein translocation across the endoplasmic reticulum membrane occurs at the Sec61 translocon. This has two essential subunits, the channel-forming multispanning membrane protein Sec61p/Sec61α and the tail-anchored Sss1p/Sec61γ, which has been proposed to “clamp” the channel. We have analyzed the function of Sss1p using a series of domain mutants and found that both the cytosolic and transmembrane clamp domains of Sss1p are essential for protein translocation. Our data reveal that the cytosolic domain is required for Sec61p interaction but that the transmembrane clamp domain is required to complete activation of the translocon after precursor targeting to Sec61p.
Highlights
The translocon is a highly conserved protein complex that forms a channel for the translocation of proteins across the endoplasmic reticulum (ER)2 membrane
In addition we show that the TM domain is required for Sss1p membrane association but that the cytosolic domain is essential for interaction with Sec61p and with Sec61p TM9
Our results demonstrate that Sss1p is required after signal sequence interaction with the translocon to complete channel activation and that the clamp domain of Sss1p is a key regulator of translocon function
Summary
Growth of Yeast Cells—Yeast strains (Table 1) were grown at 30 °C in YP medium (2% peptone, 1% yeast extract) containing 2% glucose and 0.02% adenine (YPAD) or in minimal medium (0.67% yeast nitrogen base) with 2% glucose or galactose with appropriate supplements for selective growth. Cells were grown in minimal galactose medium to an A600 nm 0.2, harvested, and resuspended in YPAD at the same density. The resulting plasmid (pJKB2) was used for site-directed mutagenesis (QuikChange, Stratagene) to create the G57L mutation (primers SSS1-G1, G1a: see supplementary Table S1 for primer sequences). The G62L mutation (primers SSS1-G2, G2a) was introduced into the G57L plasmid, and this G57L/G62L double mutation was used in turn to add the G65L mutation (primers SSS1-G3, G3a) and generate the G57L/G62L/G65L triple. MATa/MAT␣, ade2-1/ade, ura3-1/ura, his3-11,-15/his3-11,-15, leu2-3,-112/leu2-3,-112, trp1-1/trp, CAN1-100/CAN1-100 MAT␣, ⌬sec61::HIS3, ade, ura, his3-11,-15, leu2-3,-112, trp, CAN1-100, ͓pBW7͔ MATa/MAT␣, SSS1/⌬sss1::KanMX4, ade2-1/ade, ura3-1/ura, his3-11,-15/his3-11,-15, leu2-3,-112/leu, trp1-1/trp, CAN1-100/CAN1-100 MAT␣, ⌬sss1::KanMX4, ade, ura, his3-11,-15, leu2-3,-112, trp, CAN1-100, ͓FKp53͔ MATa, ⌬sss1::KanMX4, ade, ura, his3-11,-15, leu2-3,-112, trp, CAN1-100, ͓FKp53͔ MATa, ⌬sss1::KanMX4, ade, ura, his3-11,-15, leu2-3,-112, trp, CAN1-100, ͓pCM214͔ MATa, sss1⌬12::KanMX6, ade, ura, his3-11,-15, leu2-3,-112, trp, CAN1-100, ͓pCM214͔ MAT␣, ⌬sss1::KanMX4, ⌬sec61::HIS3, ade, ura, his3-11,-15, leu2-3,-112, trp, CAN1-100, ͓pCM203͔ MAT␣, ⌬sss1::KanMX4, ⌬sec61::HIS3, ade, ura, his3-11,-15, leu2-3,-112, trp, CAN1-100,
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