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 underwent retrograde transport to the ER, as evidenced by the transfer of N-linked carbohydrate to their acceptor site. The KDEL-tagged glycopeptides (approximately 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.
Miesenböck
G
G
Cellular Biochemistry and Biophysics Program, Memorial Sloan-Kettering Cancer Center, New York 10021, USA.
Rothman
J E
JE
D000595:Amino Acid Sequence; D000818:Animals; D001692:Biological Transport; D016466:CHO Cells; D006224:Cricetinae; D004705:Endocytosis; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D006023:Glycoproteins; D006031:Glycosylation; D006056:Golgi Apparatus / Q000378:metabolism*; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism*; D008565:Membrane Proteins; D008954:Models, Biological; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000378:metabolism; D010455:Peptides / Q000138:chemical synthesis / Q000378:metabolism*; D021382:Protein Sorting Signals; D016271:Proto-Oncogene Proteins c-myc / Q000235:genetics; D018000:Receptors, Peptide / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000378:metabolism
D006023:Glycoproteins; C064459:KDEL receptor; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D009842:Oligopeptides; D010455:Peptides; D021382:Protein Sorting Signals; D016271:Proto-Oncogene Proteins c-myc; D018000:Receptors, Peptide; D011993:Recombinant Fusion Proteins; C073762:lysyl-aspartyl-glutamyl-leucine