Topology and functional domains of Sec63p, an endoplasmic reticulum membrane protein required for secretory protein translocation.

Abstract

). Antibody directed against a recombinant form of the protein detects a 73-kDa polypeptide which, by immunofluorescence microscopy, is localized to the nuclear envelope-ER network. Cell fractionation and protease protection experiments confirm the prediction that Sec63p is an integral membrane protein. A series of SEC63-SUC2 fusion genes was created to assess the topology of Sec63p within the ER membrane. The largest hybrid proteins are unglycosylated, suggesting that the carboxyl terminus of Sec63p faces the cytosol. Invertase fusion to a loop in Sec63p that is flanked by two putative transmembrane domains produces an extensively glycosylated hybrid protein. This loop, which is homologous to the amino terminus of the Escherichia coli heat shock protein, DnaJ, is likely to face the ER lumen. By analogy to the interaction of the DnaJ and Hsp70-like DnaK proteins in E. coli, the DnaJ loop of Sec63p may recruit luminal Hsp7O (BiP/GRP78/Kar2p) to the translocation apparatus. Mutations in two highly conserved positions of the DnaJ loop and short deletions of the carboxyl terminus inactivate Sec63p activity. Sec63p associates with several other proteins, including Sec6lp, a 31.5-kDa glycoprotein, and a 23-kDa protein, and together with these proteins may constitute part of the polypeptide translocation apparatus. A nonfunctional DnaJ domain mutant allele does not interfere with the formation of the Sec63p/Sec6lp/gp3l.5/p23 complex.We have previously described a set of temperature-sensitive lethal mutants of Saccharomyces cerevisiae that fail to localize a signal peptide-bearing cytosolic enzyme chimera to the lumen of the endoplasmic reticulum (ER) (14, 38). Characterization of these mutants showed that the products of four genes, SEC61, SEC62, SEC63, and SEC65, are required for translocation of secretory precursor proteins across the ER membrane (14, 38, 45a). Additional alleles of SEC63 that inhibit protein import into the nucleus have also been described (39). In addition to the SEC genes described above, several other genes are required for the proper targeting and insertion of presecretory proteins into the ER. Hsp7O homologs appear to be required in the cytosol and ER lumen for translocation to occur. Cytosolic Hsp7O, encoded by the SSA genes, is required for efficient translocation of prepro-a-factor into the ER both in vivo and in vitro (8,12). An ER luminal Hsp7O homolog, BiP (the KAR2 gene product), has been identified, and some alleles of this gene block translocation of presecretory proteins (33,37,50 pletely purified replication system, the DnaJ and DnaK proteins are required to mediate the disassembly of the A 0-some complex, which activates the helicase activity of DnaB protein to initiate X DNA replication (2, 27, 52). The homology of Sec63p to DnaJ and the requirement for Hsp7O homologs for efficient translocation suggest that one role of Sec63p may be to interact with an Hsp7O homolog to promote protein translocation.To gain a better understanding of Sec63p and its function, we have analyzed the int...

A squalene synthase protein degradation method for improved sesquiterpene production in Saccharomyces cerevisiae

“…Consistent with this, the GFP flouorescence in this region overlapped with red fluorescence from RFP-tagged Sec63p (Fig. 3b) , a protein known to localise to the ER (Feldheim et al, 1992). In addition to the ER, GFP signal was also observed in optically bright regions connecting to the ER ( Fig.…”

“…Fluorescent microscopy showed that GFP was located in the perinuclear/peripheral organelles, a region recognised as the ER (Feldheim et al, 1992). Consistent with this, the GFP flouorescence in this region overlapped with red fluorescence from RFP-tagged Sec63p (Fig.…”

Mdj1p, a novel chaperone of the DnaJ family, is involved in mitochondrial biogenesis and protein folding

“…The expectation that additional proteins are involved has been nourished by the observation that in E. coli the Hsp70 homolog, DnaK, acts in concert with two other heat shock proteins, DnaJ and GrpE (reviewed by Georgopoulos, 1992). Furthermore, in S. cerevisiae, the Hsp70 homologs Ssalp and Kar2p are thought to interact with the DnaJ homologs Ydjl p and Sec83p, respectively (CapIan et al, 1992;Sadler et al, 1989;Feldheim et al, 1992).…”

The Role of p58IPK in Protecting the Stressed Endoplasmic Reticulum

“…These results point to a new functional assignment for p58 IPK in serving as a BiP cochaperone to optimize protein folding homeostasis in the ER. Because p58 IPK represents one among several (at least five others) ER localized Jdomain proteins that interface with BiP (Feldheim et al, 1992;Brightman et al, 1995;Shen et al, 2002;Hosoda et al, 2003;Shen and Hendershot, 2005), its absence would result in an ER lumen that is only modestly compromised in overall folding capacity. The functional consequences of this slightly lower protein maturation capacity would be obscured under all but the most taxing conditions and can explain each of the following phenotypic observations in cells and animals lacking p58 IPK .…”