Tagging secretory and membrane proteins with a tyrosine sulfation site. Tyrosine sulfation precedes galactosylation and sialylation in COS-7 cells.
Sulfation of proteins on tyrosines is a late Golgi modification that can be used to label proteins with [35S]sulfate for the analysis of post-Golgi transport. To extend the use of this modification to proteins not naturally sulfated, we fused a tyrosine sulfation site, the carboxyl-terminal nonapeptide of cholecystokinin precursor, to the carboxyl terminus of two normally unsulfated proteins: alpha 1-proteinase inhibitor, a secretory protein, and subunit H1 of the asialoglycoprotein receptor; a type II membrane protein. The tagged proteins were efficiently sulfated in transfected COS-7 and Madin-Darby canine kidney cells. Specifically in COS-7 cells, the proteins were sulfated before they were galactosylated and sialylated and were converted to the mature forms with a half-time of approximately 2-3 min. This is in contrast to other cell types in which tyrosine sulfation was found to be virtually the last modification of the Golgi apparatus. Our results suggest that tyrosine sulfation occurs before the trans-Golgi in transfected COS-7 cells.
Leitinger
B
B
Department of Biochemistry, University of Basel, Switzerland.
Brown
J L
JL
Spiess
M
M
D000595:Amino Acid Sequence; D000818:Animals; D037263:Asialoglycoprotein Receptor; D001483:Base Sequence; D002241:Carbohydrates / Q000032:analysis; D002460:Cell Line; D002766:Cholecystokinin / Q000096:biosynthesis*; D004285:Dogs; D006056:Golgi Apparatus / Q000378:metabolism; D007668:Kidney; D007700:Kinetics; D008565:Membrane Proteins / Q000096:biosynthesis; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D011499:Protein Processing, Post-Translational; D051381:Rats; D011956:Receptors, Cell Surface / Q000096:biosynthesis*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D013431:Sulfates / Q000378:metabolism*; D013997:Time Factors; D014162:Transfection; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000032:analysis / Q000378:metabolism; D000515:alpha 1-Antitrypsin / Q000096:biosynthesis*
D037263:Asialoglycoprotein Receptor; D002241:Carbohydrates; D008565:Membrane Proteins; D009838:Oligodeoxyribonucleotides; D011956:Receptors, Cell Surface; D011993:Recombinant Fusion Proteins; D013431:Sulfates; D000515:alpha 1-Antitrypsin; C005662:tyrosine O-sulfate; D014443:Tyrosine; D002766:Cholecystokinin