RETENTION AND RETRIEVAL - BOTH MECHANISMS COOPERATE TO MAINTAIN CALRETICULIN IN THE ENDOPLASMIC-RETICULUM

Many soluble resident proteins of the endoplasmic reticulum share a CO OH-terminal Lys-Asp-Glu-Leu (KDEL) sequence. Current opinion favours a model in which these proteins can escape from the endoplasmic reticul um (ER) by bulk flow and are recognized and sorted in the Golgi appara tus 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 calreticuli n, a soluble 60 kDa KDEL-protein of the ER. Immunogold labelling estab lished the ER localization of calreticulin in transfected and nontrans fected 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 experi ments with [S-35]methionine followed by biochemical fractionation of c alreticulin overexpressing COS cells (50- to 100-fold) revealed that o nly a minor part of labelled calreticulin leaves the ER. Retrieval fro m the Golgi was confirmed by a partial redistribution of the endogenou s KDEL-receptor as shown by double immunofluorescence. These data sugg est a KDEL-independent retention of calreticulin in the ER. Further su pporting evidence has come from morphological in vivo studies using ca lreticulin-transfected and vesicular stomatitis virus (ts045)-infected COS cells. Stimulation of vesicular transport from the ER by releasin g the temperature-dependent transport block for the viral G-protein re sulted in a small but significant appearance of calreticulin in a post -ER compartment. In contrast a calreticulin mutant, which lacked the C a2+-binding domain but included the KDEL sequence, could escape from t he ER to a much higher extent. Secretion of the nonmutated calreticuli n 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 a n increase in secretion to 29% over a 3 hour period, which is much les s than expected for a secretory protein. Taken together these results strongly support the hypothesis of two independently operating retenti on/retrieval mechanisms for calreticulin: one providing for direct ret ention in the ER with a very high capacity and having Ca2+-dependent p roperties; the other a KDEL-based retrieval system for escaped calreti culin present in the Golgi apparatus.