An Interaction Map of Endoplasmic Reticulum Chaperones and Foldases

Abstract

Chaperones and foldases in the endoplasmic reticulum (ER) ensure correct protein folding. Extensive protein-protein interaction maps have defined the organization and function of many cellular complexes, but ER complexes are under-represented. Consequently, chaperone and foldase networks in the ER are largely uncharacterized. Using complementary ER-specific methods, we have mapped interactions between ER-lumenal chaperones and foldases and describe their organization in multiprotein complexes. We identify new functional chaperone modules, including interactions between protein-disulfide isomerases and peptidylprolyl cis-trans-isomerases. We have examined in detail a novel ERp72-cyclophilin B complex that enhances the rate of folding of immunoglobulin G. Deletion analysis and NMR reveal a conserved surface of cyclophilin B that interacts with polyacidic stretches of ERp72 and GRp94. Mutagenesis within this highly charged surface region abrogates interactions with its chaperone partners and reveals a new mechanism of ER protein-protein interaction. This ability of cyclophilin B to interact with different partners using the same molecular surface suggests that ER-chaperone/foldase partnerships may switch depending on the needs of different substrates, illustrating the flexibility of multichap-

Protein Folding in the Endoplasmic Reticulum

“…It may compete with PPIases for this position, as CypB was found to share this binding site on calnexin and calreticulin (Kozlov et al 2010). For other oxidoreductases, chaperone activity has not been found (yet) but it is clear that the ER-resident folding assistants work in large (mostly transient) complexes rather than alone (Meunier et al 2002;Kleizen and Braakman 2004;Jansen et al 2012). …”

“…This suggests an abundant role for proline isomerization during disulfide bond formation and isomerization and vice versa. Inspiring then are the findings that individual PPIases and PDIs were found to interact (Jansen et al 2012) and that cyclophilin B (CypB) associates with the tip of the finger domain of calnexin and calreticulin, sharing its binding site with ERp57 0 s b 0 domain (Kozlov et al 2010). Whether these are stable interactions or whether the lectin chaperones bring an alternating enzyme to the folding protein remains to be seen.…”

“…The few proteins that have been subjected to PPIase inhibition during their folding were affected by both CsA and FK506, suggesting action from both PPIase families on the same protein. These studies do not allow distinction between both enzyme families acting on the same substrate molecule versus one acting on one folding protein and the other on another, but the cyclophilins and FKBPs have not been found in the same resident ER protein complexes (Meunier et al 2002;Kleizen and Braakman 2004;Jansen et al 2012), so perhaps they work through different interactions in different chaperone complexes on different substrates or at different times during substrate folding. Studies on family members have shown that purified FKBPs lose their high sequence specificity and turn into effective broad PPIases when attached to or collaborating with a chaperone (Knappe et al 2007;Jakob and Schmid 2009).…”

Redox controls UPR to control redox

“…PDIA6-mediated control of IRE1 and PERK signaling might be governed by low affinity interactions between the sensor and the enzyme, which could be one reason why PDIA6 is so abundant. Moreover, although PDIA5 also binds to BiP in detectable amounts (Jansen et al, 2012), the non-covalent complex between PDIA6 and BiP appears to be particularly prominent, as shown by several laboratories (Eletto et al, 2014;Jessop et al, 2009;Meunier et al, 2002). Thus, just like the UPR sensors themselves, PDIA6 might be sequestered (or recruited to the vicinity of BiP targets for the catalysis of oxidative folding) under basal conditions through interacting with BiP (Fig.…”

The Contribution of Systematic Approaches to Characterizing the Proteins and Functions of the Endoplasmic Reticulum

“…This methodology was used, for example, to predict PPIs in the glycosylation complex (Yan et al 2005), but never systematically used to predict all ER PPIs. Another approach is the split ire1 (Urech et al 2003), used to create an interaction map of ER chaperones and foldases (Jansen et al 2012). However, the widest map that was recently created is based on a genome-wide dihydro folate reductase (DHFR) protein fragment complementation assay (Tarassov et al 2008).…”