Cloning, sequence, and expression of a cDNA encoding hamster UDP-GlcNAc:dolichol phosphate N-acetylglucosamine-1-phosphate transferase.
UDP-GlcNAc:dolichol phosphate N-acetylglucosamine-1-phosphate transferase (GPT) catalyzes the initial reaction required for synthesis of dolichol-P-P-oligosaccharides. We report here on the sequence and expression of a full-length cDNA clone encoding hamster GPT. The cDNA predicts a protein of 408 amino acid residues including 10 hydrophobic segments. Several portions of the hamster GPT sequence constituting one-third of the protein have 60% or greater identity with yeast GPT, and one-half of the conserved sequence falls within the hydrophobic segments. In addition, hamster GPT has two copies of a putative dolichol recognition sequence recently identified in three yeast enzymes that interact with dolichol. The protein lacks KDEL or DEKKMP-type carboxyl-terminal ER sorting sequences. When expressed in COS-1 cells, the cDNA causes a 5-7-fold increase of GPT activity in membrane fractions. The activity was completely inhibitable by tunicamycin, and the primary product was shown to be GlcNAc-pyrophosphoryldolichol. This cDNA represents the first enzyme of the dolichol-oligosaccharide biosynthetic pathway to be cloned from a vertebrate source and demonstrates structural homology between the enzymes of the yeast and mammalian pathways.
Zhu
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Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas 75235.
Lehrman
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D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D006224:Cricetinae; D004247:DNA / Q000235:genetics*; D015723:Gene Library; D008961:Models, Structural; D008969:Molecular Sequence Data; D010770:Phosphotransferases / Q000235:genetics* / Q000378:metabolism; D011487:Protein Conformation; D012333:RNA, Messenger / Q000235:genetics; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D012689:Sequence Homology, Nucleic Acid; D014162:Transfection; D017855:Transferases (Other Substituted Phosphate Groups)
D012333:RNA, Messenger; D004247:DNA; D010770:Phosphotransferases; D017855:Transferases (Other Substituted Phosphate Groups); C023222:UDPacetylglucosamine-dolichyl-phosphate acetylglucosamine-1-phosphate transferase