Sec61p serves multiple roles in secretory precursor binding and translocation into the endoplasmic reticulum membrane

The evolutionarily conserved Sec61 protein complex mediates the translocati on of secretory proteins into the endoplasmic reticulum. To investigate the role of Sec61p, which is the main subunit of this complex, we generated re cessive, cold-sensitive alleles of sec61 that encode stably expressed prote ins with strong defects in translocation. The stage at which posttranslatio nal translocation was blocked was probed by chemical crosslinking of radiol abeled secretory precursors added to membranes isolated from wild-type and mutant strains. Two classes of sec61 mutants were distinguished. The first class of mutants was defective in preprotein docking onto a receptor site o f the translocon that included Sec61p itself. The second class of mutants a llowed docking of precursors onto the translocon but was defective in the A TP-dependent release of precursors from this site that in wild-type membran es leads to pore insertion and full translocation. Only mutants of the seco nd class were partially suppressed by overexpression of SEC63, which encode s a subunit of the Sec61 holoenzyme complex responsible for positioning Kar 2p (yeast BiP) at the translocation channel. These mutants thus define two early stages of translocation that require SEC61 function before precursor protein transfer across the endoplasmic reticulum membrane.