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

Department of Molecular and Cell Biology, Harvard University, Cambridge, Massachusetts, United States
Cell (Impact Factor: 32.24). 10/1998; 94(6):795-807. DOI: 10.1016/S0092-8674(00)81738-9
Source: PubMed


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.

    • "In addition to co-translational translocation, post-translational translocation occurs in S. cerevisiae in SRP-independent manner (Plath et al., 1998). Translation is carried out in cytosol before the initiation of translocation. "
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    ABSTRACT: Saccharomyces cerevisiae is widely used as a producer of heterologous proteins of medical and industrial interest. Numerous efforts have been made to overcome bottlenecks in protein expression and secretion. However, the effect of engineering protein translocation to heterologous protein secretion has not been studied extensively in S. cerevisiae. In this work, we confirmed that heterologous protein expression in S. cerevisiae induces the unfolded protein response (UPR). To enhance protein folding capacity, the endoplasmic reticulum (ER) chaperone protein BiP and the disulfide isomerase Pdi1p were each over-expressed, and the secretion of three heterologous proteins, β-glucosidase, endoglucanase and α-amylase, was improved. The impact of engineering key translocation components was also studied. The over-expression of co-translational translocation components Srp14p and Srp54p enhanced the secretion of three heterologous proteins (β-glucosidase, endoglucanase and α-amylase), but over-expressing the cytosolic chaperone Ssa1p (involved in post-translational translocation) enhanced the secretion of only β-glucosidase. By engineering both co-translational translocation and protein folding, we obtained strains with β-glucosidase, endoglucanase and α-amylase activities increased by 72%, 60% and 103% compared to the controls. Our results show that protein translocation may be a limiting factor for heterologous protein production. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.
    No preview · Article · Apr 2015 · Biotechnology and Bioengineering
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    • "The core of the translocon channel appears to be mainly formed by a-and g-subunits (Osborne, Rapoport, & van den Berg, 2005). Cross-linking experiments have established that signal sequences of secretory proteins (and signal anchors of membrane proteins) are in contact with Sec61a during translocation (Pilon, Romisch, Quach, & Schekman, 1998; Plath, Mothes, Wilkinson, Stirling, & Rapoport, 1998). This contact determines the orientation of signal sequence in the ER membrane. "
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    ABSTRACT: Insulin is an essential hormone for maintaining metabolic homeostasis in the body. To make fully bioactive insulin, pancreatic beta cells initiate synthesis of the insulin precursor, preproinsulin, at the cytosolic side of the endoplasmic reticulum (ER), whereupon it undergoes co- and post-translational translocation across the ER membrane. Preproinsulin is cleaved by signal peptidase to form proinsulin that folds on the luminal side of the ER, forming three evolutionarily conserved disulfide bonds. Properly folded proinsulin forms dimers and exits from the ER, trafficking through Golgi complex into immature secretory granules wherein C-peptide is endoproteolytically excised, allowing fully bioactive two-chain insulin to ultimately be stored in mature granules for insulin secretion. Although insulin biosynthesis has been intensely studied in recent decades, the earliest events, including proinsulin entry and exit from the ER, have been relatively understudied. However, over the past 5 years, more than 20 new insulin gene mutations have been reported to cause a new syndrome termed Mutant INS-gene-induced Diabetes of Youth (MIDY). Although these mutants have not been completely characterized, most of them affect proinsulin entry and exit from the ER. Here, we summarize our current knowledge about the early events of insulin biosynthesis and review recent advances in understanding how defects in these events may lead to pancreatic beta cell failure.
    Full-text · Article · Feb 2014 · Vitamins & Hormones
    • "Simultaneously, the diameter of the pore ring seems to widen and the cytoplasmic vestibule increases (Egea and Stroud, 2010). The signal anchor sequence moves through the cytoplasmic vestibule and is intercalated by helices 2b and 7 of the lateral gate (Fig. 10.6), as indicated by both structural (Becker et al., 2009; Egea and Stroud, 2010) as well as cross-linking studies (Plath et al., 1998). Blocking the opening of the lateral gate by disulfide bridges inhibits protein transport (du Plessis et al., 2009). "
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    ABSTRACT: Membrane proteins execute a plethora of essential functions in bacterial cells and there- fore bacteria utilize efficient strategies to ensure that these proteins are properly targeted and inserted into the membrane. Most bacterial inner membrane proteins are recognized early during their synthesis, i.e. co-translationally by the bacterial signal recognition particle (SRP), which delivers the ribosome-nascent chain (RNC) via its interaction with the mem- brane-bound SRP receptor to the SecYEG translocon, a highly dynamic and evolutionarily conserved protein conducting channel. Membrane protein insertion via SecYEG is coupled to on going polypeptide chain elongation at the ribosome and the emerging transmembrane helices exit the SecYEG channel laterally into the lipid phase. Lateral release and folding of transmembrane helices is most likely facilitated by YidC, which transiently associates with the SecYEG translocon. YidC has also been shown to facilitate insertion of inner mem- brane proteins independently of the SecYEG translocon. The targeting of outer membrane proteins to SecYEG occurs predominantly post-translationally by the SecA/SecB pathway and thus follows the same route as periplasmic proteins. In this chapter, we summarize the current knowledge on membrane protein targeting and transport/integration by either SecYEG or YidC.
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