Evidence that the hamster tunicamycin resistance gene encodes UDP-GlcNAc:dolichol phosphate N-acetylglucosamine-1-phosphate transferase.
A cDNA clone isolated from Chinese hamster ovary cells conferred elevated GlcNAc-1-P-transferase (GPT) activity and resistance to tunicamycin in transfected cells (Zhu, X., and Lehrman, M. A. (1990) J. Biol. Chem. 265, 14250-14255). It had been assumed that this cDNA, termed TRG for tunicamycin resistance gene, encoded GPT enzyme. However, other functions were not ruled out. Thus, by one of several mechanisms, the TRG protein could have instead functioned by activation of the transfected host's endogenous GPT enzyme. To analyze the biochemical function of the TRG protein, hamster TRG cDNA was stably expressed at high levels in Chinese hamster ovary cells. In addition, several antipeptide polyclonal antibodies directed against the predicted TRG protein were obtained. With these tools in hand, experiments were performed to test the hypothesis that the TRG encodes GPT enzyme, as well as to rule out other possible functions for the TRG protein. These experiments included examination of the effects of solubilization of membranes on TRG-dependent GPT activity, the apparent binding of tunicamycin to the TRG protein, and the immunoadsorption of GPT activity with TRG protein-specific antibodies. From these results, we conclude that the hamster TRG most likely encodes GPT enzyme.
Zhu
X
X
Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas 75235.
Zeng
Y
Y
Lehrman
M A
MA
D000818:Animals; D015153:Blotting, Western; D016466:CHO Cells; D006224:Cricetinae; D004247:DNA / Q000235:genetics; D004351:Drug Resistance / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D005455:Fluorescent Antibody Technique; D009844:Oligosaccharides / Q000378:metabolism; D010770:Phosphotransferases / Q000235:genetics* / Q000378:metabolism; D010835:Phytohemagglutinins / Q000633:toxicity; D017026:Swainsonine / Q000494:pharmacology; D014162:Transfection; D017855:Transferases (Other Substituted Phosphate Groups); D014415:Tunicamycin / Q000494:pharmacology*
D009844:Oligosaccharides; D010835:Phytohemagglutinins; D014415:Tunicamycin; D004247:DNA; D010770:Phosphotransferases; D017855:Transferases (Other Substituted Phosphate Groups); C030012:UDP-N-acetylglucosamine-lysosomal-enzyme-N-acetylglucosaminephosphotransferase; D017026:Swainsonine