CABP1, A CALCIUM-BINDING PROTEIN OF THE THIOREDOXIN FAMILY, IS A RESIDENT KDEL PROTEIN OF THE ER AND NOT OF THE INTERMEDIATE COMPARTMENT

A cDNA encoding rat CaBP1 has been isolated and sequenced. The deduced polypeptide chain consists of 440 amino acids including two internal thioredoxin-like domains and a C-terminal KDEL retention/retrieval sig nal. Regarding the high degree of identity to the hamster protein P5, CaBP1 is considered to be the homologous rat protein. Previous work ha s suggested that CaBP1 is a resident luminal protein of the intermedia te compartment (Schweizer, A., Peter, F., Nguyen Van, P., Soling, H. D . and Hauri, H. P. (1993) fur. J. Cell Biol. 60, 366-370). Our conclus ion that CaBP1 is a resident protein of the endoplasmic reticulum and not of the intermediate compartment is based on three different approa ches: subcellular fractionation, indirect immunofluorescence and overe xpression of CaBP1. Subcellular fractionation of Vero cells in a veloc ity controlled step gradient led to copurification of CaBP1-containing vesicles and several marker proteins for the ER including calreticuli n and alpha-SSRP. The intermediate compartment, as defined by a monocl onal antibody against the marker protein p53 (ERGIC-53), could be sepa rated from these ER markers. Double immunofluorescence analysed by las er scanning microscopy showed no significant colocalization between Ca BP1 and p53, but between CaBP1 and calreticulin. In additional experim ents, Vero cells were infected with VSV tsO45. At 15 degrees C the VSV -G protein accumulated in punctuate structures representing the interm ediate compartment, while CaBP1 maintained its original reticular loca lization. Even after high-level overexpression in COS cells, CaBP1 was not detected in the intermediate compartment, but was efficiently ret ained in the ER as judged by light microscopy.