The Brl domain in Sec63p is required for assembly of functional endoplasmic reticulum translocons.

Protein translocation into the endoplasmic reticulum occurs at pore-forming structures known as translocons. In yeast, two different targeting pathways converge at a translocation pore formed by the Sec61 complex. The signal recognition particle-dependent pathway targets nascent precursors co-translationally, whereas the Sec62p-dependent pathway targets polypeptides post-translationally. In addition to the Sec61 complex, both pathways also require Sec63p, an integral membrane protein of the Hsp40 family, and Kar2p, a soluble Hsp70 located in the ER lumen. Using a series of mutant alleles, we demonstrate that a conserved Brl (Brr2-like) domain in the COOH-terminal cytosolic region of Sec63p is essential for function both in vivo and in vitro. We further demonstrate that this domain is required for assembly of two oligomeric complexes of 350 and 380 kDa, respectively. The larger of these corresponds to the heptameric "SEC complex" required for post-translational translocation. However, the 350-kDa complex represents a newly defined hexameric SEC' complex comprising Sec61p, Sss1p, Sbh1p, Sec63p, Sec71p, and Sec72p. Our data indicate that the SEC' complex is required for co-translational protein translocation across the yeast ER membrane.

An in vitro assay using overexpressed yeast SRP demonstrates that cotranslational translocation is dependent upon the J-domain of Sec63p.

The signal recognition particle (SRP) is required for co-translational targeting of polypeptides to the endoplasmic reticulum (ER). Once at the membrane, the precursor interacts with a complex proteinaceous machinery that mediates its translocation across the bilayer. Genetic studies in yeast have identified a number of genes whose products are involved in this complex process. These mutants offer a potentially valuable resource with which to analyze the biochemical role played by each component in the pathway. However, such analyses have been hampered by the failure to reconstitute an efficient in vitro assay for SRP-dependent translocation. We report the construction of two multicopy vectors that allow overexpression of all seven gene products required to make SRP in the yeast Saccharomyces cerevisiae. The overexpressed subunits assemble into intact and functional SRP particles, and we further demonstrate that in vitro reconstitution of co-translational translocation is greatly enhanced using cytosol from the overexpression strain. We use this assay to demonstrate that Sec63p is required for co-translational translocation in vitro and specifically identify the "J-domain" of Sec63p as crucial for this pathway.

Identification of novel protein-protein interactions at the cytosolic surface of the Sec63 complex in the yeast ER membrane.

Precursors of secretory proteins are targeted to the membrane of the endoplasmic reticulum by specific protein complexes that recognize their signal sequence. All eukaryotic cells investigated so far have been found to possess the signal recognition particle (SRP) that targets the majority of precursors to the translocation machinery. In Saccharomyces cerevisiae a number of proteins are translocated independently of SRP. These precursors rely on a different signal sequence-binding complex, which includes Sec62p, Sec63p, Sec71p and Sec72p. Identifying interactions between individual components of this tetrameric protein complex is important in the understanding of its function. We demonstrate a specific interaction between the only two essential proteins in this complex, Sec62p and Sec63p. Second, we show evidence of homodimerization of Sec72p molecules and further identify the YLR301w gene product as a novel in vivo interacting partner of Sec72p. Finally, we determine the authentic N-terminus of Sec62p and describe interacting subdomains of both Sec62p and Sec63p.