ER residential chaperone GRP78 unconventionally relocalizes to the cell surface via endosomal transport

Abstract

Despite new advances on the functions of ER chaperones at the cell surface, the translocation mechanisms whereby these chaperones can escape from the ER to the cell surface are just emerging. Previously we reported that in many cancer types, upon ER stress, IRE1α binds to and triggers SRC activation resulting in KDEL receptor dispersion from the Golgi and suppression of retrograde transport. In this study, using a combination of molecular, biochemical, and imaging approaches, we discovered that in colon and lung cancer, upon ER stress, ER chaperones, such as GRP78 bypass the Golgi and unconventionally traffic to the cell surface via endosomal transport mediated by Rab GTPases (Rab4, 11 and 15). Such unconventional transport is driven by membrane fusion between ER-derived vesicles and endosomes requiring the v-SNARE BET1 and t-SNARE Syntaxin 13. Furthermore, GRP78 loading into ER-derived vesicles requires the co-chaperone DNAJC3 that is regulated by ER-stress induced PERK-AKT-mTOR signaling.

Keywords: Endoplasmic reticulum stress; Endosome; GRP78; Unconventional trafficking.

Fig. 1

csGRP78 expression is independent of Golgi integrity and SRC in colon cancer. HCT116 cells were A treated with BFA with or without Tg or B transfected with dominant negative (DN) Arf1-HA or HA-STX5 or empty vector with or without Tg treatment. The indicated proteins from whole cell lysate (WCL) and cell surface (CS) were analyzed by Western blot with GAPDH serving as loading control for WCL and EphB4 as positive control for BFA treatment. C As in A, except four additional colon cancer cell lines (RKO, LS180, HT-29, and SW480) were analyzed. MSI and MSS denote microsatellite instability and stability, respectively. D As in B, except that the indicated cells were transfected with shRNA-expressing plasmid against SRC or empty vector and selected by puromycin

Fig. 2

Unconventional relocalization of ER chaperones requires Rab4 and Rab11. A Summary of the contributions of Rabs 4, 5, 11, and 15 to trafficking of proteins to the cell surface. B HCT116 cells were transfected with the dominant negative (DN) form of the indicated Rabs or empty vector, with or without Tg treatment. The indicated proteins from the WCL and CS were analyzed by Western blot, and normalized against their respective loading controls (n  =  3). C As in B, except that the cells were transfected with HA-Rab15 DN or empty vector. The relative csGRP78 level under each condition was quantified, normalized against their respective loading controls, and graphed below (n  =  3)

Fig. 3

GRP78 localizes with markers of the early and recycling endosomes following ER stress. A HCT116 cells were treated with Tg for 18 h and subjected to immunofluorescent (IF) staining for GRP78 (green) or EEA1 (red). Co-staining of both proteins (yellow) is shown in the merged image. Pearson R was calculated per cell (n  =  10). Scale bar, 10 µm. B HCT116 cells were treated with Tg for 18 h and subjected to IF staining for GRP78 (green) or TfnR (red). Co-staining of both proteins (yellow) is shown in the merged image. Pearson R was calculated per cell (n = 10). Scale bar, 10 µm. C Summary of early and recycling endosome involvement in the ER stress-induced chaperone relocalization

Fig. 4

STX13 mediates GRP78 relocalization onto the cell surface. A HCT116 cells were transfected with DN Myc-STX13 or empty vector with or without Tg treatment. The indicated proteins from the whole cell lysate (WCL) and cell surface (CS) were analyzed by Western blot, and normalized against their respective loading controls (n  =  3). B Same as A, except that the ER stressors DTT, tunicamycin (Tu) and 2-deoxyglucose (2DG) were used. C HCT116 cells were transfected with Myc-STX13 with or without Tg treatment. Organelles containing Myc-STX13 were immunoprecipitated (IP) using an anti-Myc antibody. Target proteins were analyzed by Western Blot. D HCT116 cells were treated with Tg for 18 h and subjected to proximity ligation assay (PLA) for GRP78 and STX13. Positive stain was indicated as yellow puncta. The total number of yellow puncta was normalized against the number of cells in the micrograph and graphed (n  =  3, from three independent experiments). Scale bar, 10 µm. E Summary of STX13 involvement in the ER stress-induced chaperone relocalization

Fig. 5

The ER v-SNARE BET1 mediates GRP78 localization into the endosome. A Comparison of STX5, GS27, BET1, and Sec22b transcript levels in HCT116 cell from the GENT database (n  =  5). B HCT116 cells were transfected with DN BET1 or empty vector with or without Tg treatment. The indicated proteins from the whole cell lysate (WCL) and cell surface (CS) were analyzed by Western blot. Conditions were labeled 1 to 4 (1—Control; 2—HA-BET1 DN; 3—Tg; 4—Tg + HA-BET1 DN) and relative csGRP78 level (normalized to their respective loading controls) under each condition was quantified and graphed (n  =  3). C Same as B, but with DN Sec22b. D Cells were transfected with Myc-STX13 and HA-BET1 with or without Tg treatment. Organelles containing HA-BET1 were subjected to IP using an anti-HA antibody. The indicated proteins from the IP and WCL were analyzed by Western Blot. E same as D except the cells were transfected with HA-Sec22b and Myc-STX13. F HCT116 cells were treated with Tg for 18 h and subjected to proximity ligation assay (PLA) for STX13 and BET1. Positive stain was indicated as yellow puncta. The total number of yellow puncta was normalized against the number of cells in the micrograph and graphed (n  =  3, from three independent experiments). Scale bar, 10 µm. G Summary of BET1 and STX13 involvement in the ER stress-induced chaperone relocalization

Fig. 6

The co-chaperone DNAJC3 mediates GRP78 localization into BET1 vesicles. A Flag GRP78 wild-type (WT), G227D, T453D, or R197H was transfected into HCT116 cells. The indicated proteins from the whole cell lysate (WCL) and cell surface (CS) were analyzed by Western blot. B HCT116 cells were transfected with siRNA targeting DNAJC3 or siControl with or without Tg treatment. The indicated proteins from the WCL and CS were analyzed by Western blot. C Flag GRP78 (WT), G227D, T453D, R197H mutant or empty vector (pcDNA3) was transfected into HCT116 cells with HA-BET1. Organelles containing HA-BET1 were subjected to IP using an anti-HA antibody. The indicated proteins from the IP and WCL were analyzed by Western Blot. D HCT116 cells were transfected HA-BET1 with siControl or siRNA targeting DNAJC3 with or without Tg treatment. Organelles containing HA-BET1 were subjected to IP using an anti-HA antibody. The indicated proteins from the IP and WCL were analyzed by Western Blot. E HCT116 cells were treated with siRNA targeting DNAJC3 and then treated with Tg for 18 h and subjected to proximity ligation assay (PLA) for GRP78 and BET1. Positive stain was indicated as yellow puncta. The total number of yellow puncta was normalized against the number of cells in the micrograph and graphed (n  =  3, from three independent experiments). Scale bar, 10 µm. F Summary of DNAJC3 and BET1 involvement in the ER stress-induced chaperone relocalization

Fig. 7

PERK regulates ER stress-induced unconventional transport of GRP78. A HCT116 cells were transfected with siRNA against ATF6, plasmid expressing shRNA against IRE1α, or siRNA against PERK, with or without Tg treatment. siControl and empty vector served as negative controls. The indicated proteins from the WCL and CS were analyzed by Western blot. B Same as A, except cells were treated with GSK2606414 or BFA with or without Tg. C Same as A except cells were transfected with empty vector or HA-PERK with or without Tg treatment. Conditions were labeled 1 to 4 (1—Control; 2—HA-PERK; 3—Tg; 4—Tg + HA-PERK) and relative csGRP78 level (normalized against their respective loading controls) under each condition was quantified and graphed. (n  =  3). D Summary of PERK involvement in the ER stress-induced chaperone relocalization

Fig. 8

PERK-AKT-mTOR axis drives DNAJC3 expression. A HCT116 cells were transfected with siRNA against PERK, or siControl with or without Tg treatment. The indicated proteins from the whole cell lysate (WCL) and cell surface (CS) were analyzed by Western blot. B Same as A, except cells were treated with GSK2606414 with or without Tg. C HCT116 cells were treated with Tg for the indicated times, and the indicated proteins from the WCL and CS were analyzed by WB. D HCT116 cells were transfected with siRNA against PERK or siControl with or without Tg treatment. The indicated proteins from the WCL were analyzed by Western blot and normalized against their respective loading controls (n  =  3). DNAJC3 levels under each treatment condition was quantitated and graphed below. E Same as D, except cells were either nontreated or treated with Tg in combination with GSK2606414, MK-2206, or rapamacyin (Rapa) as indicated on top (n  =  3). F HCT116 cells were treated with MK-2206 or Rapa alone or in combination with Tg. The indicated proteins from the WCL and CS were analyzed by Western blot. (G) Summary of PERK involvement in the ER stress-induced chaperone relocalization

Fig. 9

Summary of the mechanism of ER chaperone translocation via endosomal transport to the cell surface in response to ER stress in colon cancer