We have analyzed how the signal sequence of prepro-alpha-factor is recognized during the first step of posttranslational protein transport into the yeast endoplasmic reticulum. Cross-linking studies indicate that the signal sequence interacts in a Kar2p- and ATP-independent reaction with Sec61p, the multispanning membrane component of the protein-conducting channel, by intercalation into transmembrane domains 2 and 7. While bound to Sec61p, the signal sequence forms a helix that is contacted on one side by Sec62p and Sec71p. The binding site is located at the interface of the protein channel and the lipid bilayer. Signal sequence recognition in cotranslational translocation in mammals appears to occur similarly. These results suggest a general mechanism by which the signal sequence could open the channel for polypeptide transport.

Yeast mutants defective in the translocation of soluble secretory proteins into the lumen of the endoplasmic reticulum (sec6l, sec62, sec63) are not impaired in the assembly and glycosylation of the type II membrane protein dipeptidylaminopeptidase B (DPAPB) or of a chimeric membrane protein consisting of the multiple membrane-spanning domain of yeast hydroxymethylglutaryl CoA reductase (HMG1) fused to yeast histidinol dehydrogenase (HIS4C). This chimera is assembled in wild-type or mutant cells such that the His4c protein is oriented to the ER lumen and thus is not available for conversion of cytosolic histidinol to histidine. Cells harboring the chimera have been used to select new translocation defective sec mutants. Temperature-sensitive lethal mutations defining two complementation groups have been isolated: a new allele of sec6l and a single isolate of a new gene sec65. The new isolates are defective in the assembly of DPAPB, as well as the secretory protein a-factor precursor. Thus, the chimeric membrane protein allows the selection of more restrictive sec mutations rather than defining genes that are required only for membrane protein assembly. The SEC61 gene was cloned, sequenced, and used to raise polyclonal antiserum that detected the Sec6l protein. The gene encodes a 53-kDa protein with five to eight potential membrane-spanning domains, and Sec6lp antiserum detects an integral protein localized to the endoplasmic reticulum membrane. Sec6lp appears to play a crucial role in the insertion of secretory and membrane polypeptides into the endoplasmic reticulum.

Hsp70s are a ubiquitous family of molecular chaperones involved in many cellular processes. Two Hsp70s, Lhs1p and Kar2p, are required for protein biogenesis in the yeast endoplasmic reticulum. Here, we found that Lhs1p and Kar2p specifically interacted to couple, and coordinately regulate, their respective activities. Lhs1p stimulated Kar2p by providing a specific nucleotide exchange activity, whereas Kar2p reciprocally activated the Lhs1p adenosine triphosphatase (ATPase). The two ATPase activities are coupled, and their coordinated regulation is essential for normal function in vivo.

Amino-terminal acetylation is probably the most common protein modification in eukaryotes with as many as 50%–80% of proteins reportedly altered in this way. Here we report a systematic analysis of the predicted N-terminal processing of cytosolic proteins versus those destined to be sorted to the secretory pathway. While cytosolic proteins were profoundly biased in favour of processing, we found an equal and opposite bias against such modification for secretory proteins. Mutations in secretory signal sequences that led to their acetylation resulted in mis-sorting to the cytosol in a manner that was dependent upon the N-terminal processing machinery. Hence N-terminal acetylation represents an early determining step in the cellular sorting of nascent polypeptides that appears to be conserved across a wide range of species.

The yeast genome sequencing project predicts an open reading frame (YKL073) that would encode a novel member of the Hsp70 family of molecular chaperones. We report that this 881 codon reading frame represents a functional gene expressing a 113–119 kDa glycoprotein localized within the lumen of the endoplasmic reticulum (ER). We therefore propose to designate this gene LHS1 (Lumenal Hsp Seventy). Our studies indicate that LHS1 is regulated by the unfolded protein response pathway, as evidenced by its transcriptional induction in cells treated with tunicamycin, and in various mutants defective in precursor processing (sec11–7, sec53–6 and sec59–1). LHS1 is not essential for viability, but an Lhs1 null mutant strain exhibits a coordinated induction of genes regulated by the unfolded protein response indicating a role for Lhs1p in protein folding in the ER. Furthermore, the null mutation is synthetically lethal in combination with (delta)ire1, thus activation of the unfolded protein response pathway is essential for cells to tolerate loss of Lhs1p. Synthetically lethality is also seen with mutations in KAR2, strongly suggesting that Kar2p and Lhs1p have overlapping functions. The Lhs1 null mutant exhibits a severe constitutive defect in the translocation of several secretory preproteins. We therefore propose that Lhs1p is a molecular chaperone of the ER lumen involved in both polypeptide translocation and subsequent protein folding.