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 signal. Regarding the high degree of identity to the hamster protein P5, CaBP1 is considered to be the homologous rat protein. Previous work has suggested that CaBP1 is a resident luminal protein of the intermediate compartment (Schweizer, A., Peter, F., Nguyen Van, P., Söling, H.D. and Hauri, H.P. (1993) Eur. J. Cell Biol. 60, 366-370). Our conclusion that CaBP1 is a resident protein of the endoplasmic reticulum and not of the intermediate compartment is based on three different approaches: subcellular fractionation, indirect immunofluorescence and overexpression of CaBP1. Subcellular fractionation of Vero cells in a velocity controlled step gradient led to copurification of CaBP1-containing vesicles and several marker proteins for the ER including calreticulin and alpha-SSRP. The intermediate compartment, as defined by a monoclonal antibody against the marker protein p53 (ERGIC-53), could be separated from these ER markers. Double immunofluorescence analysed by laser scanning microscopy showed no significant colocalization between CaBP1 and p53, but between CaBP1 and calreticulin. In addition experiments, Vero cells were infected with VSV tsO45. At 15 degrees C the VSV-G protein accumulated in punctuate structures representing the intermediate compartment, while CaBP1 maintained its original reticular localization. Even after high-level overexpression in COS cells, CaBP1 was not detected in the intermediate compartment, but was efficiently retained in the ER as judged by light microscopy.
Füllekrug
J
J
Abt. Klinische Biochemie, University of Göttingen, FRG.
Sönnichsen
B
B
Wünsch
U
U
Arseven
K
K
Nguyen Van
P
P
Söling
H D
HD
Mieskes
G
G
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002135:Calcium-Binding Proteins / Q000032:analysis* / Q000737:chemistry / Q000235:genetics; D002460:Cell Line; D002522:Chlorocebus aethiops; D018076:DNA, Complementary / Q000235:genetics; D004721:Endoplasmic Reticulum / Q000737:chemistry*; D007535:Isomerases / Q000032:analysis* / Q000737:chemistry / Q000235:genetics; D008969:Molecular Sequence Data; D051381:Rats; D018353:Rhabdoviridae Infections / Q000378:metabolism; D013280:Stomatitis / Q000378:metabolism; D019749:Sulfur-Sulfur Bond Isomerases; D013879:Thioredoxins; D014709:Vero Cells; D014721:Vesicular stomatitis Indiana virus / Q000502:physiology; D014764:Viral Proteins / Q000378:metabolism
D002135:Calcium-Binding Proteins; D018076:DNA, Complementary; D014764:Viral Proteins; D013879:Thioredoxins; D007535:Isomerases; C085577:Ca2+-binding protein-1; D019749:Sulfur-Sulfur Bond Isomerases