Localization of GRP78 to mitochondria under the unfolded protein response

Abstract

The ubiquitously expressed molecular chaperone GRP78 (78 kDa glucose-regulated protein) generally localizes to the ER (endoplasmic reticulum). GRP78 is specifically induced in cells under the UPR (unfolded protein response), which can be elicited by treatments with calcium ionophore A23187 and sarcoplasmic/endoplasmic reticulum Ca2+-ATPase inhibitor TG (thapsigargin). By using confocal microscopy, we have demonstrated that GRP78 was concentrated in the perinuclear region and co-localized with the ER marker proteins, calnexin and PDI (protein disulphide-isomerase), in cells under normal growth conditions. However, treatments with A23187 and TG led to diminish its ER targeting, resulting in redirection into a cytoplasmic vesicular pattern, and overlapping with the mitochondrial marker MitoTracker. Cellular fractionation and protease digestion of isolated mitochondria from ER-stressed cells suggested that a significant portion of GRP78 is localized to the mitochondria and is protease-resistant. Localizations of GRP78 in ER and mitochondria were confirmed by using immunoelectron microscopy. In ER-stressed cells, GRP78 mainly localized within the mitochondria and decorated the mitochondrial membrane compartment. Submitochondrial fractionation studies indicated further that the mitochondria-resided GRP78 is mainly located in the intermembrane space, inner membrane and matrix, but is not associated with the outer membrane. Furthermore, radioactive labelling followed by subcellular fractionation showed that a significant portion of the newly synthesized GRP78 is localized to the mitochondria in cells under UPR. Taken together, our results indicate that, at least under certain circumstances, the ER-resided chaperone GRP78 can be retargeted to mitochondria and thereby may be involved in correlating UPR signalling between these two organelles.

Molecular cloning and expression analysis of glucose-regulated protein 78 (GRP78) gene in silkworm Bombyx mori

“…GRP78, the ER member of the HSP70 family, is regulated by heat and/or other stresses (Shiu et al 1977;Sun et al 2006;Quinones et al 2008). In this paper, we verified that thermal stress and starvation can induce the up-regulation of Bmgrp78 expression in silkworms, especially in head tissues.…”

The Escherichia coli Subtilase Cytotoxin A Subunit Specifically Cleaves Cell-surface GRP78 Protein and Abolishes COOH-terminal-dependent Signaling

“…Previous studies using transmembrane prediction programs have suggested that GRP78 has four potential transmembrane domains at the cell surface: I (amino acids (aa) [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17], II (aa 29 -45), III (aa 222-242), and IV (aa 414 -431) (34). However, the studies performed here demonstrate that at least residues 416 -417 are exposed on the cell surface, as this is the sole SubA cleavage site, and we observe cell-surface cleavage.…”

“…Normal cellular function is, therefore, reestablished by reducing intermediate protein aggregates, increasing protein folding, regulating Ca 2ϩ , and repressing translation (1). In addition to its role as a molecular chaperone in the ER, GRP78 is found in the cytoplasm, nucleus, and mitochondria, and it exists in secreted and plasma membrane-associated forms (2)(3)(4)(5)(6). At the plasma membrane, GRP78 acts as a signaling receptor for activated ␣ 2 -macroglobulin (␣ 2 M*) (7,8).…”

microRNAs: Innovative Targets for Cerebral Ischemia and Stroke

“…GRP75 directly interacts with both VDAC and IP3R, playing a central role in scaffolding this ER-mitochondrial complex [87]. Translocation of GRP78 from ER to mitochondria observed following ER stress [88] or ischemia-like stress [86] may also play a key role in the ER-mitochondria crosstalk during cerebral ischemia [86]. Chaperone complexes at both the ER and mitochondrion orchestrate the regulation of Ca 2+ signaling between these two organelles and control bioenergetics, cell survival, and cell death decisions.…”