Photoaffinity labeling and characterization of the cloned purine-cytosine transport system in Saccharomyces cerevisiae.

Abstract

8-Azido[2-3H]adenine was used as a photoaffinity label for the purine-cytosine transport system. After irradiation in the presence of the photoaffinity label, the cells were converted into protoplasts, their plasma membranes were purified, and the membrane proteins were extracted and separated by NaDodSO4/PAGE [sodium dodecyl sulfate/polyacrylamide gel electrophoresis]. The radioactivity was specifically incorporated into a protein with a MW of 120,000. Photoaffinity labeling of this protein could be blocked by irradiation in the presence of natural substrates for the transport system. The MW as determined by NaDodSO4/PAGE was twice the value calculated from mRNA analysis of the cloned gene. Incubation of exponentially growing cells with tunicamycin, an antibiotic that inhibits glycosylation of proteins, resulted in a 40% decrease in the overall initial uptake rate, which correlates with the reduction of the labeled MW 120,000 protein. Treatment of the extracted labeled plasma membrane proteins with glycosidic enzymes resulted in disappearance of the MW 120,000 peak and the appearance of new peaks at MW 60,000 and 73,000. The purine-cytosine transport protein is a glycoprotein.