Both potential dolichol recognition sequences of hamster GlcNAc-1- phosphate transferase are necessary for normal enzyme function

A potential dolichol recognition sequence (PDRS) with the 11-residue consensus F-I/V-X-F/Y-X-X-I-P-F-X-F/Y can be found in each of five microsomal enzymes that interact with dolichol or a dolichol derivative. However, no direct evidence demonstrating a role for the PDRSs has been reported. Hamster UDP-GlcNAc:dolichol phosphate N-acetylglucosamine-1-phosphate transferase (GPT) differs from the other enzymes by having two PDRSs. Stable CHO-K1 transfectants were created that expressed elevated amounts of normal GPT, GPT with a scramble mutation at the first PDRS (nearest the amino terminus), or GPT with a triple alanine-replacement mutation at the second PDRS. The mutant enzymes had no detectable catalytic activity in vivo, but were fully capable of conferring cellular resistance to the GPT inhibitor tunicamycin. In vitro studies with membrane preparations confirmed that the mutant enzymes were catalytically inactive and also showed that their recovery in microsomes was diminished compared with normal enzyme. These data demonstrate that each PDRS of hamster GPT is necessary for normal enzyme function. The implications of these data for possible roles of the PDRSs are discussed.