Retrotranslocation of the chaperone calreticulin from the endoplasmic reticulum lumen to the cytosol

Abstract

Polypeptide folding and quality control in the endoplasmic reticulum (ER) are mediated by protein chaperones, including calreticulin (CRT). ER localization of CRT is specified by two types of targeting signals, an N-terminal hydrophobic signal sequence that directs insertion into the ER and a C-terminal KDEL sequence that is responsible for retention in the ER. CRT has been implicated in a number of cytoplasmic and nuclear processes, suggesting that there may be a pathway for generating cytosolic CRT. Here we show that CRT is fully inserted into the ER, undergoes processing by signal peptidase, and subsequently undergoes retrotranslocation to the cytoplasm. A transcription-based reporter assay revealed an important role for the C-terminal Ca(2+) binding domain in CRT retrotranslocation. Neither ubiquitylation nor proteasome activity was necessary for retrotranslocation, which indicates that the pathway is different from that used by unfolded proteins targeted for destruction. Forced expression of cytosolic CRT is sufficient to rescue a cell adhesion defect observed in mouse embryo fibroblasts from crt(-/-) mice. The ability of CRT to retrotranslocate from the ER lumen to the cytosol explains how CRT can change compartments and modulate cell adhesion, transcription, and translation.

FIG. 1.

Cytosolic fraction of CRT can be released from cells by treatment with digitonin. (A) The cells used throughout our studies were treated with 0.005% digitonin for 5 min. Antibody binding to CRT was quantitated using the Odyssey gel system, and the percent of cellular CRT released was calculated by dividing the amount of CRT in the released fraction by the amount of CRT present in intact cells. (B) Cell permeabilization assay to determine cytosolic and ER compartmentalization of endogenous proteins. Intact cells, digitonin-permeabilized cells (containing the ER fraction), and the released fractions were analyzed by immunoblotting. Permeabilization of the plasma membrane releases the cytosolic pool of CRT, which is not the case for chaperones restricted to the ER lumen (Grp94 and protein disulfide isomerase [PDI]). (C) The integrity of the ER membrane is preserved at low concentrations of digitonin. Cos7 cells were transfected with ER-targeted YFP. Cells were treated with digitonin (to permeabilize the plasma membrane) or Triton X-100 (to permeabilize all membranes) and probed with an antibody that detects YFP. At low concentrations of digitonin, the antibody cannot access YFP expressed within the ER.

FIG. 2.

CRT signal sequence directs efficient insertion into the ER. (A) CHO cells stably expressing YFP engineered with or without the N-terminal CRT signal sequence (YFP, targeted to ER; *YFP, targeted to cytoplasm) were used in the cell permeabilization assay. Except where indicated (*), the constructs used in this study contain an N-terminal signal sequence. CHO cells stably expressing mouse CRT either lacking or containing its signal sequence were assayed in the same manner, and detected using a CRT antibody that recognizes mouse but not hamster CRT. Note that the concentration of digitonin used was sufficient to permeabilize 90 to 95% of the cells (determined by trypan blue staining). The release efficiency of a protein will be influenced by protein-protein interactions and nuclear localization; thus, the fraction of CRT identified as cytosolic is probably an underestimate. (B) Signal sequence cleavage assay to establish that cytosolic CRT is processed through the ER. Diagram of constructs used to show N-terminal T7 epitope is removed by signal peptidase cleavage (blue arrow). (C) CHO cells stably expressing T7 epitope-tagged mouse CRT were assayed for signal sequence cleavage by immunofluorescence microscopy. T7 immunoreactivity is lost when CRT is synthesized as an ER protein but retained when CRT is synthesized as a cytosolic protein. (D) Cell permeabilization assay to show the T7 epitope was absent from cytosolic CRT that was synthesized as an ER protein (bkgd, background band).

FIG. 3.

CRT is fully inserted into the ER lumen prior to retrotranslocation. (A) Diagram of constructs used to show ER insertion. A minimal signal peptidase cleavage site from the CRT signal sequence (black box) was inserted between CRT and YFP. (B) Immunoblotting (anti-YFP) of the CRT-YFP fusions expressed in Cos7 cells. CRT-YFP targeted to the ER via the endogenous CRT signal sequence is cleaved at the minimal signal peptidase cleavage site, yielding the ∼25-kDa YFP. In the cell permeabilization assay, the cleaved YFP product is not released by digitonin. In contrast, the CRT-YFP fusion that is not targeted to the ER is not cleaved at the minimal signal peptidase cleavage site, but is released by digitonin in the cytosolic fraction.

FIG. 4.

Cytosolic CRT is protease resistant. Cells were treated with digitonin to release cytosolic proteins. The remaining permeabilized cells were put through two freeze-thaw cycles to permeabilize organelles. ER and cytosolic fractions were enriched for CRT by filtration over a MonoQ anion-exchange column. Peak fractions of each run were digested with trypsin (1:100 trypsin-protein) for 30 min at 30°C. Trypsin digests were then probed for CRT. As expected, CRT in the ER fraction was degraded by trypsin in the absence of Ca²⁺, whereas it was protease resistant in the presence of high Ca²⁺. Cytosolic CRT, however, was protease resistant in the presence and absence of Ca²⁺, suggesting that cytosolic CRT has a different structure. (B) The enriched cytosolic fractions do not contain trypsin inhibitors. Silver staining of the trypsin digests indicates that trypsin degrades cytosolic proteins in the presence and absence of Ca²⁺.

FIG. 5.

Transcription-based assay to measure retrotranslocation of CRT. (A) Design of the assay and constructs used for transfection. ERX is a fusion of the DNA-binding domain (DBD) from Gal4p and the activation domain (AD) from p53 and contains the N-terminal CRT signal sequence. Because ERX is retained in the ER, it generates only a background level of luciferase activity. Protein disulfide isomerase (PDI) is an ER lumenal protein similar in size and isoelectric point to CRT. (C) CRT promotes retrotranslocation of ERX, measured by the level of luciferase activity (CRT-ERX). (D) CRT domains involved in retrotranslocation. Deletion of the N domain of CRT resulted in a high level of retrotranslocation, whereas deletion of the C domain resulted in no retrotranslocation. (E) The C domain is sufficient for retrotranslocation when fused to ERX or protein disulfide isomerase. Luciferase assays were normalized to cotransfected Renilla luciferase, performed in triplicate, and are plotted with the standard deviation. The levels of ERX reporters were analyzed by immunoblotting on a single gel, but only the region of the gel containing the ERX fusion is shown.

FIG. 6.

Retrotranslocation of CRT is not linked to protein degradation. (A) Assay for ubiquitylation of CRT. HA epitope-tagged CRT was transfected into Cos7 cells in the absence (lanes 1 to 6) and presence (lanes 7 to 12) of T7 epitope-tagged ubiquitin (T7-Ub), in either the absence or presence of the proteasome inhibitor MG132 (40 μM). CRT-HA was immunoprecipitated and blotted using the HA (lanes 1 to 12) and T7 (lanes 13 to 24) antibodies. Ubiquitylated forms of CRT were not detected after MG132 treatment, and CRT was not immunoprecipitated by the HA antibody, indicating that CRT is not a substrate for the proteasome. The ladder of endogenous proteins detected with the T7 antibody demonstrates MG132 inhibition of the proteasome (lane 22). (B) Proteasome activity is not required for retrotranslocation of CRT. NIH 3T3 cells were transfected with *ERX, CRT-ERX, and ERX. After overnight treatment with MG132, retrotranslocation of *ERX and CRT-ERX was not affected, whereas ERX activity increased twofold.

FIG. 7.

CRT functions outside of the ER. (A) crt^−/− MEFs displayed a reduced level of motility in a transwell migration assay compared to crt^+/+ MEFs. (B) CRT expression enhances cell binding to extracellular matrix. crt^+/+ and crt^−/− MEFs were assayed for binding to collagen type IV. Only crt^+/+ MEFs responded to collagen IV. (C) Cytoplasmic CRT promotes cell adhesion. Transiently transfected CRT targeted directly to the cytoplasm (*CRT) stimulated crt^−/− cell binding to collagen type IV. Expression levels of transiently transfected CRT (lane 1) and *CRT (lanes 2 to 4) in crt^−/− cells are shown.

FIG. 8.

Targets and functions of CRT within and outside of the ER lumen. See text for discussion. GR, AR, RAR, and VDR, glucocorticoid, androgen, retinoic acid, and vitamin D receptors, respectively.