THE CAPACITY TO RETRIEVE ESCAPED ER PROTEINS EXTENDS TO THE TRANS-MOST CISTERNA OF THE GOLGI STACK

To explore how far into the Golgi stack the capacity to retrieve KDEL proteins extends, we have introduced an exogenous probe (the peptide YHPNSTCSEKDEL) into the TGN of living cells. For this purpose, a CHO cell line expressing a c-myc-tagged version of the transmembrane protein TGN38-which cycles between the TGN and the cell surface-was generated. The cells internalized peptides that were disulfide bonded to anti-myc antibodies and accumulated the peptide-antibody complexes in the TGN. Peptides released from these complexes under-went retrograde transport to the ER, as evidenced by the transfer of N-linked carbohydrate to their acceptor site. The KDEL-tagged glycopeptides (similar to 10% of the endocytosed load) behaved like endogenous ER residents: they stayed intracellular, and their oligosaccharide side chains remained sensitive to endoglycosidase H. An option thus exists to extract ER residents even at the most distant pole of the Golgi stack, suggesting that sorting of resident from exported ER proteins may occur in a multistage process akin to fractional distillation.