ERp57 functions as a subunit of specific complexes formed with the ER lectins calreticulin and calnexin

Abstract

ERp57 is a lumenal protein of the endoplasmic reticulum (ER) and a member of the protein disulfide isomerase (PDI) family. In contrast to archetypal PDI, ERp57 interacts specifically with newly synthesized glycoproteins. In this study we demonstrate that ERp57 forms discrete complexes with the ER lectins, calnexin and calreticulin. Specific ERp57/calreticulin complexes exist in canine pancreatic microsomes, as demonstrated by SDS-PAGE after cross-linking, and by native electrophoresis in the absence of cross-linking. After in vitro translation and import into microsomes, radiolabeled ERp57 can be cross-linked to endogenous calreticulin and calnexin while radiolabeled PDI cannot. Likewise, radiolabeled calreticulin is cross-linked to endogenous ERp57 but not PDI. Similar results were obtained in Lec23 cells, which lack the glucosidase I necessary to produce glycoprotein substrates capable of binding to calnexin and calreticulin. This observation indicates that ERp57 interacts with both of the ER lectins in the absence of their glycoprotein substrate. This result was confirmed by a specific interaction between in vitro synthesized calreticulin and ERp57 prepared in solution in the absence of other ER components. We conclude that ERp57 forms complexes with both calnexin and calreticulin and propose that it is these complexes that can specifically modulate glycoprotein folding within the ER lumen.

Figure 1

The association of endogenous ER proteins. Canine pancreatic microsomes were treated with either DMSO (solvent control, lanes 1, 3, 5, and 7) or 1 mM BMH (lanes 2, 4, 6, and 8). The samples were then separated on an 8% SDS polyacrylamide gel and transferred to nitrocellulose. Antisera to calreticulin (CRT, lanes 1 and 2), ERp57 (lanes 3 and 4), calnexin (CNX, lanes 5 and 6), and PDI (lanes 7 and 8) were used for immunodetection.

Figure 2

Native gel analysis of ER protein complexes. The lumenal contents of canine pancreatic microsomes were separated by blue native gel electrophoresis and then transferred to PVDF. The proteins were detected by immunoblotting with antisera raised against calreticulin (CRT, lanes 1–3), ERp57 (lanes 4–6), and PDI (lanes 7–9). The samples were untreated (lanes 1, 4, and 7); heated to 95°C before electrophoresis (lanes 2, 5 and 8), or treated with 1 mM BMH before isolation of the lumenal contents (lanes 3, 6, and 9).

Figure 3

Interactions of in vitro synthesized calreticulin, ERp57, and PDI with the endogenous ER proteins of microsomes. Calreticulin (CRT, lanes 1–6), ERp57 (lanes 7–12), and PDI (lanes 13–18) RNAs were translated in a rabbit reticulocyte lysate system in the presence of microsomes. After termination of translation, the microsomal fraction was isolated and, where indicated, the samples were treated with the cross-linking reagent BMH. The reaction was quenched, and the samples denatured with 1% SDS before immunoprecipitation with the antisera indicated: CRT, anticalreticulin; NRS, control nonrelated serum; CNX, anti-calnexin; ERp57, anti-ERp57; PDI, anti-PDI. The samples were analyzed on an 8% SDS-polyacrylamide gel. The identity of imported polypeptides with cleaved signal sequences (indicated by arrows) was confirmed by protease protection and comparison with unprocessed polypeptides bearing signal sequences (our unpublished observations).

Figure 4

Interactions of in vitro synthesized calreticulin, ERp57, and PDI with the endogenous ER proteins of semipermeabilized cells. Parental CHO cells (panel A) and glucosidase I-deficient Lec23 CHO cells (panel B) were selectively permeabilized with digitonin. Calreticulin (CRT, lanes 1–6), ERp57 (lanes 7–12), and PDI (lanes 13–18) RNAs were translated in a rabbit reticulocyte lysate system in the presence of the semipermeabilized mammalian cells. After termination of translation, the cells were washed and, where indicated, the samples were treated with the cross-linking reagent BMH. The samples were quenched and denatured with 1% SDS before immunoprecipitation with the antisera indicated: CRT, anti-calreticulin; NRS, control nonrelated serum; CNX, anti-calnexin; ERp57, anti-ERp57; PDI, anti-PDI. The samples were analyzed on an 8% SDS-polyacrylamide gel.

Figure 5

Interactions between in vitro translated calreticulin, ERp57, and PDI in solution. The products of a translation reaction carried out in the presence of [³⁵S]methionine were mixed with the products of a translation reaction carried out with unlabeled methionine, and the resulting protein complexes were cross-linked with BMH. ³⁵S-labeled calreticulin (panel A), ERp57 (panel B), and PDI (panel C) were mixed with unlabeled calreticulin (CRT, lanes 1–4), ERp57 (lanes 5–8), and PDI (lanes 9–12). After cross-linking and SDS denaturation, immunoprecipitation was carried out with the antisera indicated (see Figure 3 for key). The samples were analyzed on an 8% SDS-polyacrylamide gel. The identity of full-length precursor proteins (i.e., with signal sequences; indicated by arrowheads) was experimentally confirmed (our unpublished observations).

Figure 6

A proposed model of glycoprotein folding. Calreticulin (and calnexin, its integral membrane protein homologue; not shown) acts as a lectin by binding to monoglucosylated oligosaccharide chains present on newly synthesized glycoproteins. By virtue of its association with calreticulin, ERp57 is brought into contact with the glycoprotein. ERp57 modulates the folding of the glycoprotein, illustrated here as disulfide bond formation, but possibly by other mechanisms. The glycoprotein is released from calreticulin upon glucose trimming by glucosidase II. If the folding has been successful, the native glycoprotein is free to continue along the secretory pathway. If the glycoprotein is incompletely folded, the folding sensor UDP-glucose:glycoprotein glucosyl transferase (UGGT) adds back a single glucose residue, and the glycoprotein undergoes another round of lectin binding.