Colin J. Stirling

Signal Sequence Recognition in Posttranslational Protein Transport across the Yeast ER Membrane

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.

DETERMINATION OF THE TRANSMEMBRANE TOPOLOGY OF YEAST SEC61P, AN ESSENTIAL COMPONENT OF THE ENDOPLASMIC RETICULUM TRANSLOCATION COMPLEX

Sec61p is a highly conserved integral membrane protein that plays a role in the formation of a protein-conducting channel required for the translocation of polypeptides into, and across, the membrane of the endoplasmic reticulum. As a major step toward elucidating the structure of the endoplasmic reticulum translocation apparatus, we have determined the transmembrane topology of Sec61p using a combination of C-terminal reporter-domain fusions and the in situ digestion of specifically inserted factor Xa protease cleavage sites. Our data indicate the presence of 10 transmembrane domains, including several with surprisingly limited hydrophobicity. Furthermore, we provide evidence for complex intramolecular interactions in which these weakly hydrophobic domains require C-terminal sequences for their correct topogenesis. The incorporation of sequences with limited hydrophobicity into the bilayer may play a vital role in the formation of an aqueous membrane channel required for the translocation of hydrophilic polypeptide chains.

LHS1 and SIL1 provide a lumenal function that is essential for protein translocation into the endoplasmic reticulum

Lhs1p is an Hsp70‐related chaperone localized in the endoplasmic reticulum (ER) lumen. Δlhs1 mutant cells are viable but are constitutively induced for the unfolded protein response (UPR). Here, we demonstrate a severe growth defect in Δire1Δlhs1 double mutant cells in which the UPR can no longer be induced. In addition, we have identified a UPR‐ regulated gene, SIL1, whose overexpression is sufficient to suppress the Δire1Δlhs1 growth defect. SIL1 encodes an ER‐localized protein that interacts directly with the ATPase domain of Kar2p (BiP), suggesting some role in modulating the activity of this vital chaperone. SIL1 is a non‐essential gene but the Δlhs1Δsil1 double mutation is lethal and correlates with a complete block of protein translocation into the ER. We conclude that the IRE1‐dependent induction of SIL1 is a vital adaptation in Δlhs1 cells, and that the activities associated with the Lhs1 and Sil1 proteins constitute an essential function required for protein translocation into the ER. The Sil1 protein appears widespread amongst eukaryotes, with homologues in Yarrowia lipolytica (Sls1p), Drosophila and mammals.

Sec63p and Kar2p are required for the translocation of SRP-dependent precursors into the yeast endoplasmic reticulum in vivo

The translocation of secretory polypeptides into the endoplasmic reticulum (ER) occurs at the translocon, a pore‐forming structure that orchestrates the transport and maturation of polypeptides at the ER membrane. In yeast, targeting of secretory precursors to the translocon can occur by two distinct pathways that are distinguished by their dependence upon the signal recognition particle (SRP). The SRP‐dependent pathway requires SRP and its membrane‐bound receptor, whereas the SRP‐independent pathway requires a separate receptor complex consisting of Sec62p, Sec63p, Sec71p, Sec72p plus lumenal Kar2p/BiP. Here we demonstrate that Sec63p and Kar2p are also required for the SRP‐dependent targeting pathway in vivo. Furthermore, we demonstrate multiple roles for Sec63p, at least one of which is exclusive to the SRP‐independent pathway.

N-Terminal Acetylation Inhibits Protein Targeting to the Endoplasmic Reticulum

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.

Intracellular catalysis of disulfide bond formation by the human sulfhydryl oxidase, QSOX1.

The discovery that the flavoprotein oxidase, Erv2p, provides oxidizing potential for disulfide bond formation in yeast, has led to investigations into the roles of the mammalian homologues of this protein. Mammalian homologues of Erv2p include QSOX (sulfhydryl oxidases) from human lung fibroblasts, guinea-pig endometrial cells and rat seminal vesicles. In the present study we show that, when expressed in mammalian cells, the longer version of human QSOX1 protein (hQSOX1a) is a transmembrane protein localized primarily to the Golgi apparatus. We also present the first evidence showing that hQSOX1a can act in vivo as an oxidase. Overexpression of hQSOX1a suppresses the lethality of a complete deletion of ERO1 (endoplasmic reticulum oxidase 1) in yeast and restores disulfide bond formation, as assayed by the folding of the secretory protein carboxypeptidase Y.

Sec61p is required for ERAD-L: genetic dissection of the translocation and ERAD-L functions of Sec61p using novel derivatives of CPY*

Misfolded proteins in the endoplasmic reticulum (ER) are exported to the cytosol for degradation by the proteasome in a process known as ER-associated degradation (ERAD). CPY* is a well characterized ERAD substrate whose degradation is dependent upon the Hrd1 complex. However, although the functions of some of the components of this complex are known, the nature of the protein dislocation channel remains obscure. Sec61p has been suggested as an obvious candidate because of its role as a protein-conducting channel through which polypeptides are initially translocated into the ER. However, it has not yet been possible to functionally dissect any role for Sec61p in dislocation from its essential function in translocation. By changing the translocation properties of a series of novel ERAD substrates, we are able to separate these two events and find that functional Sec61p is essential for the ERAD-L pathway.

The yeast CDC9 gene encodes both a nuclear and a mitochondrial form of DNA ligase I

BACKGROUND The yeast CDC9 gene encodes a DNA ligase I activity required during nuclear DNA replication to ligate the Okazaki fragments formed when the lagging DNA strand is synthesised. The only other DNA ligase predicted from the yeast genome sequence, DNL4/LIG4, is specifically involved in a non-homologous DNA end-joining reaction. What then is the source of the DNA ligase activity required for replication of the yeast mitochondrial genome? RESULTS We report that CDC9 encodes two distinct polypeptides expressed from consecutive in-frame AUG codons. Translational initiation at these two sites gives rise to polypeptides differing by a 23 residue amino-terminal extension, which corresponds to a functional mitochondrial pre-sequence sufficient to direct import into yeast mitochondria. Initiation at the first AUG codon results in a 755 amino-acid polypeptide that is imported into mitochondria, whereupon the pre-sequence is proteolytically removed to yield the mature mitochondrial form of Cdc9p. Initiation at the second AUG codon produces a 732 amino-acid polypeptide, which is localised to the nucleus. Cells expressing only the nuclear isoform were found to be specifically defective in the maintenance of the mitochondrial genome. CONCLUSIONS CDC9 encodes two distinct forms of DNA ligase I. The first is targeted to the mitochondrion and is required for propagation and maintenance of mitochondrial DNA, the second localises to the nucleus and is sufficient for the essential cell-division function associated with this gene.

Ssh1p determines the translocation and dislocation capacities of the yeast endoplasmic reticulum.

Sec61p is required both for protein translocation and dislocation across the membrane of the endoplasmic reticulum (ER). However, the cellular role of the Sec61p homolog Ssh1p has not been clearly defined. We show that deltassh1 mutant cells have strong defects in both SRP-dependent and -independent translocation. Moreover, these cells were also found to be induced for the unfolded protein response and to be defective in dislocation of a misfolded ER protein. In addition, deltassh1 mutant cells rapidly became respiratory deficient. The other defects discussed above were suppressed in the respiratory-deficient state or under conditions where the rate of polypeptide translation was artificially reduced. These data identify Ssh1p as a component of a second, functionally distinct translocon in the yeast ER membrane.

A novel subfamily of Hsp70s in the endoplasmic reticulum.

The endoplasmic reticulum contains a number of proteins involved in the processing of secretory polypeptides. These include BiP, which is an Hsp70-family member highly conserved throughout evolution. BiP is known to be intimately involved in several aspects of protein biogenesis, but our understanding of these events has been complicated by the recent description of a novel Hsp70-related protein in yeast, Lhauthorp, whose functions overlap with those of BiP. Current indications are that this protein is distributed widely among eukaryotes and that it represents a distinct subfamily of the Hsp70 class of molecular chaperones.