The Essential Functions of Molecular Chaperones and Folding Enzymes in Maintaining Endoplasmic Reticulum Homeostasis

Abstract

It has been estimated that up to one-third of the proteins encoded by the human genome enter the endoplasmic reticulum (ER) as extended polypeptide chains where they undergo covalent modifications, fold into their native structures, and assemble into oligomeric protein complexes. The fidelity of these processes is critical to support organellar, cellular, and organismal health, and is perhaps best underscored by the growing number of disease-causing mutations that reduce the fidelity of protein biogenesis in the ER. To meet demands encountered by the diverse protein clientele that mature in the ER, this organelle is populated with a cadre of molecular chaperones that prevent protein aggregation, facilitate protein disulfide isomerization, and lower the activation energy barrier of cis-trans prolyl isomerization. Components of the lectin (glycan-binding) chaperone system also reside within the ER and play numerous roles during protein biogenesis. In addition, the ER houses multiple homologs of select chaperones that can recognize and act upon diverse peptide signatures. Moreover, redundancy helps ensure that folding-compromised substrates are unable to overwhelm essential ER-resident chaperones and enzymes. In contrast, the ER in higher eukaryotic cells possesses a single member of the Hsp70, Hsp90, and Hsp110 chaperone families, even though several homologs of these molecules reside in the cytoplasm. In this review, we discuss specific functions of the many factors that maintain ER quality control, highlight some of their interactions, and describe the vulnerabilities that arise from the absence of multiple members of some chaperone families.

Keywords: chaperones; co-chaperones; protein folding; proteostasis; unfolded protein response (UPR).

Figure 1.. The ER is a dedicated compartment for the synthesis and maturation of one-third of the proteins encoded in the human genome, which are subject to quality control mechanisms executed by a variety of chaperones and folding enzymes.

Nascent polypeptide chains enter the ER co-translationally where they will be engaged by either the (a) lectin or (b) Hsp70 (BiP) chaperone system. In both cases, co-chaperones participate in their maturation. If successful (c), the secretory pathway client is freed from the chaperones and exits the ER for the Golgi in COPII vesicles. However, if the nascent protein fails to fold (d), it is targeted for ERAD and retrotranslocated to the cytosol for proteasomal degradation.

Figure 2.. The ER lumenal Hsp70, BiP, acts as a hub to manage ER proteostasis.

Established BiP (Kar2 in yeast) partners are shown by shared dashed black lines, and a genetic/functional interaction with calnexin (Cne1 in yeast) is shown by grey dashed lines. The interactions were established: between BiP and SEC63 (Sec63 in yeast) [246, 247]; between BiP and ERdj3 (Scj1-Jem1 in yeast) in yeast and mammals [33, 96]; between BiP and GRP170 (Lhs1 in yeast) [65, 68]; between BiP and SIL1 (Sil1 in yeast) [65, 66]; and between BiP and a specific PDI, p5/PDIA6, in mammalian cells [36]. Isolated multimeric complexes have been described that contain BiP, GRP94, ERp72, GRP170, and ERdj3 [204], depicted by a black star, [207] indicated with a red number sign, and [206] depicted by a yellow dollar sign, respectively. The complexes identified by Meunier et al. and Fujimori et al. also contained SDF2-L1, and Meunier et al. additionally isolated UGGT, cyclophilin B, and CaBP1 (also known as p5—see above—or PDIA6) as components of the multimeric BiP complex.

Figure 3.. BiP is an essential chaperone in yeast, early in mouse development, and in cultured mammalian cells.

a. Disruption of Kar2 in yeast reveals it to be essential for survival [218]. b. Attempts to produce a mouse that was null for BiP/GRP78 using constitutive Cre-mediated gene excision yielded no null embryos after embryonic day 3.5, as shown in the cartoon depiction, revealing an early requirement for this chaperone [222]. c. Regulated expression of tissue-specific Cre recombinase to disrupt BiP revealed its essential nature in multiple tested tissues [220, 221]. d. The AB5 subtilase cytotoxin kills mammalian cells by cleaving BiP between the NBD and SBDs, thereby disrupting its function [223].

Figure 4.. GRP94 loss is tolerated in some but not all tissues.

a. Yeast lack an ER orthologue of this Hsp90 family member, and thus maturation of secretory pathway clients synthesized in yeast is GRP94-independent. b. Constitutive disruption of GRP94 using Cre-mediated excision reveals GRP94 is required for mouse development [225]. c. Tissue-specific excision of GRP94 demonstrates its requirement for hematopoiesis differentiation, production of Treg immune cells, normal hepatic function, and pancreatic β islet cell development [–250]. d. Embryonic stem cells obtained from the constitutive GRP94 knock-out mouse can be induced to differentiate into adipocytes, hepatocytes, and neurons, but not myocytes [225]. Loss of GRP94 has been reported in a number of cell lines including a pre-B cell line. See text for additional details.

Figure 5.. GRP170 is an essential gene in all mammalian tissues tested, but its loss is tolerated in yeast.

a. Lhs1/GRP170 is not essential in S. cerevisiae, but disruption of the other ER NEF, Sil1 results in a synthetic lethality phenotype [251]. b. Attempts to create ORP150/GRP170 null mice were performed by crossing heterozygous GRP170^+/− mice. No viable homozygous GRP170^−/− mice were recovered in over 100 matings indicting that deletion of this gene results in embryonic lethality [226]. c. A line of mice was engineered with inducible Cre-mediated, nephron-specific disruption of GRP170 [228]. After induction of the Cre recombinase, these mice exhibited hallmarks of acute kidney injury (AKI), which was accompanied by UPR activation and apoptosis.This demonstrates that GRP170 is critical for kidney function. d. Mouse embryonic fibroblasts were obtained from homozygous GRP170^fl/fl mice and transduced with an inducible Cre construct. [229]. Upon Cre expression, as GRP170 levels fell, the UPR was activated, the steady-state binding of BiP to a client increased, degradation of a misfolded BiP substrate was reduced, and BiP accumulated in NP40-insoluble fractions. This disruption of BiP functions and ER homeostasis culminated in apoptosis revealing GRP170 to be essential in MEFs.