BiP Acts as a Molecular Ratchet during Posttranslational Transport of Prepro-α Factor across the ER Membrane

Abstract

We have addressed the mechanism by which proteins are posttranslationally transported across the membrane of the yeast endoplasmic reticulum (ER). We demonstrate that BiP (Kar2p), a member of the Hsp70 family resident in the ER lumen, acts as a molecular ratchet during translocation of the secretory protein prepro-alpha factor through the channel formed by the Sec complex. Multiple BiP molecules associate with each translocation substrate following interaction with the J domain of the Sec63p component of the Sec complex. Bound BiP minimizes passive backward movements of the substrate through the channel, and BiP's subsequent dissociation results in a free polypeptide in the ER lumen. Antibodies against the substrate can replace BiP, indicating that a Brownian ratchet is sufficient to achieve translocation.

The Role of p58IPK in Protecting the Stressed Endoplasmic Reticulum

“…This process involves yet unidentified lumenal factor(s), possibly including BiP, whose titration during acute ER stress leads to reversible inhibition of the initiation of translocation (Kang et al, 2006). The idea that the availability of lumenal proteins could influence translocation is consistent with previous observations demonstrating that lumenal chaperones can either facilitate forward translocation or prevent backward slipping of translocating nascent chains (Nicchitta and Blobel, 1993;Brodsky et al, 1995;Matlack et al, 1999;Tyedmers et al, 2003). Thus, the initial commitment to ER translocation may be linked in a substrate-selective manner to chaperone availability in the lumen, providing a means to match substrate flux with maturation capacity.…”

Firefly luciferase mutants as sensors of proteome stress

“…Hsp70 members are highly multifunctional proteins that have been shown to play a key role in proteome maintenance, such as in de novo protein folding (co-or post-translational), protein translocation across membranes (Lyman and Schekman, 1997;Matlack et al, 1999;Young et al, 2003), refolding of stress damaged proteins (Ben-Zvi et al, 2004;Schroder et al, 1993;Sharma et al, 2010), in preventing protein aggregation (Auluck et al, 2002;Broadley and Hartl, 2009;Klucken et al, 2004;Sakahira et al, 2002;Warrick et al, 1999), disaggregation (Ben-Zvi and Goloubinoff, 2001;Diamant et al, 2000;Liberek et al, 2008;Shorter, 2011) and degradation of irreparable misfolded proteins (Bercovich et al, 1997;Fisher et al, 1997;Urushitani et al, 2004). These essential and diverse cellular functions of Hsp70 are attributed to its physical interaction with various co-chaperones such as Hsp40, NEFs and with proteins such as HIP, HOP and CHIP.…”

Emerging features of ER resident J-proteins in plants

“…24,25 Sec63p, a transmembrane protein with a luminal J-domain and a cytosolic Sec63/Brl domain, is required for assembly of functional ER translocons. 26 The J-domain of Sec63p, and not the Sec63/Brl domain, was recently shown to be required for ERAD of soluble proteins in yeast.…”