Retention and retrieval: both mechanisms cooperate to maintain calreticulin in the endoplasmic reticulum

Many soluble resident proteins of the endoplasmic reticulum share a COOH-terminal Lys-Asp-Glu-Leu (KDEL) sequence. Current opinion favours a model in which these proteins can escape from the endoplasmic reticulum (ER) by bulk flow and are recognized and sorted in the Golgi apparatus by binding to a specific KDEL-receptor, which returns them to the ER. Through biochemical, morphological and mutational analysis we have studied the mechanisms that determine the localization of calreticulin, a soluble 60 kDa KDEL-protein of the ER. Immunogold labelling established the ER localization of calreticulin in transfected and nontransfected COS cells. Although the ER cisternae in transfected cells were enormously dilated and heavily labelled by gold particles we found no significant label in any other compartment. In vivo pulse chase experiments with [35S]methionine followed by biochemical fractionation of calreticulin overexpressing COS cells (50- to 100-fold) revealed that only a minor part of labelled calreticulin leaves the ER. Retrieval from the Golgi was confirmed by a partial redistribution of the endogenous KDEL-receptor as shown by double immunofluorescence. These data suggest a KDEL-independent retention of calreticulin in the ER. Further supporting evidence has come from morphological in vivo studies using calreticulin-transfected and vesicular stomatitis virus (ts045)-infected COS cells. Stimulation of vesicular transport from the ER by releasing the temperature-dependent transport block for the viral G-protein resulted in a small but significant appearance of calreticulin in a post-ER compartment. In contrast a calreticulin mutant, which lacked the Ca(2+)-binding domain but included the KDEL sequence, could escape from the ER to a much higher extent. Secretion of the nonmutated calreticulin was very low (1-2% of total calreticulin in 3 hours) compared to the mutated form (18% in 3 hours). Deletion of the KDEL sequence led to an increase in secretion to 29% over a 3 hour period, which is much less than expected for a secretory protein. Taken together these results strongly support the hypothesis of two independently operating retention/retrieval mechanisms for calreticulin: one providing for direct retention in the ER with a very high capacity and having Ca(2+)-dependent properties; the other a KDEL-based retrieval system for escaped calreticulin present in the Golgi apparatus.