Abstract
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.
Highlights
From the Department of Biochemistry, Biozentrum, University of Basel, Klingelbergstrasse 70, CH-4056 Basel, Switzerland and the $Department of Biochemistry, Biophysics, and Genetics, University of Colorado Health Sciences Center, Denver, Colorado 80262
Thisis in contrast to this study we have extended the use of sulfation for transport other cell types in which tyrosine sulfation was found sttoudies to proteins not naturally sulfated by introducing a tybe virtuallythe last modificationof the Golgi apparatus. rosine sulfation site by in vitro mutagenesis of the encoding
Our results suggest that tyrosine sulfation occurs befoDrNeA
Summary
Dept.of Biochemistry, Biozentrum, University of Basel, Klingelbergstrasse 70, CH-4056 Basel, Switzerland. Labeling with r5S]MethioninelCysteine"COS-7 cells expressing wild-type and mutant AlPiwere washed with phosphate-buffered saline (PBS), starvefdor 45 min in methionine-free medium (Selectamine kit, Life Technologies, Inc.), labeled for 30 min in 0.5 ml of methioninefree medium supplemented with 100 pCi/ml [35Slmethionine, washed with PBS, and chased for different times a t 37 "C in complete medium containing excess methionine. COS-7 cells expressing wild-type and mutant H1 were starved in medium lacking methionine and cysteine for 45 min, labeled with 100 pCi/ml [35Slmethionine and [35Slcysteine for 1 h, and chased in complete medium containing excess methionine and cysteine. Labeling with P5SISulfate-Cells were grown in 35-mm wells, washed with PBS, and incubated in sulfate-free medium (containing one-tenth of the normal concentration of methionine and cysteine, 350 mglliter NaHC03, buffered with 20 m~ Hepes, pH 7.2) for at least 1h a t 37 "C. The sheet waswetted with the same buffer and run at 750 V for approximately 45 min to separate tyrosine and sulfotyrosine. t3H1Tyrosine and sulf~-[~H]tyrosinwe ere quantified by liquid scintillation counting
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