TRANSPORT OF AN EXTERNAL LYS-ASP-GLU-LEU (KDEL) PROTEIN FROM THE PLASMA-MEMBRANE TO THE ENDOPLASMIC-RETICULUM - STUDIES WITH CHOLERA-TOXIN IN VERO CELLS

The A2 chain of cholera toxin (CTX) contains a COOH-terminal Lys-Asp-G lu-Leu (KDEL) sequence. We have, therefore, analyzed by immunofluoresc ence and by subcellular fractionation in Vero cells whether CTX can be used to demonstrate a retrograde transport of KDEL proteins from the Golgi to the ER. Immunofluorescence studies reveal that after a pulse treatment with CTX, the CTX-A and B subunits (CTX-A and CTX-B) reach G olgi-like structures after 15-20 min (maximum after 30 min). Between 3 0 and 90 min, CTX-A (but not CTX-B) appear in the intermediate compart ment and in the ER, whereas the CTX-B are translocated to the lysosome s. Subcellular fractionation studies confirm these results: after CTX uptake for 15 min, CTX-A is associated only with endosomal and Golgi c ompartments. After 30 min, a small amount of CTX-A appears in the ER i n a trypsin-resistant form, and after 60 min, a significant amount app ears. CTX-A seems to be transported mainly in its oxidized form (CTX-A 1-S-S-CTX-A2) from the Golgi to the ER, where it becomes slowly reduce d to form free CTX Al and CTX-A2, as indicated by experiments in which cells were homogenized 30 and 90 min after the onset of CTX uptake in the presence of N-ethylmaleimide. Nocodazol applied after accumulatio n of CTX in the Golgi inhibits the appearance of CTX-A in the ER and d elays the increase of 3', 5'cAMP, indicating the participation of micr otubules in the retrograde Golgi-ER transport.