tailieunhanh - Báo cáo khoa học: Comparative importance in vivo of conserved glutamate residues in the EX7E motif retaining glycosyltransferase Gpi3p, the UDP-GlcNAc-binding subunit of the first enzyme in glycosylphosphatidylinositol assembly

Saccharomyces cerevisiaeGpi3p is the UDP-GlcNAc-bind-ing andpresumed catalytic subunit of the enzyme that forms GlcNAc-phosphatidylinositol in glycosylphosphatidylinosi-tol biosynthesis. It is anessential proteinwithanEX7 Emotif that is conserved in four families of retaining glycosyl-transferases. All Gpi3ps contain a cysteine residue four residues C-terminal to EX7E. To test their importance for Gpi3p functionin vivo,Glu289 and297 in theEX7 Emotif of S. cerevisiaeGpi3p, as well as Cys301, were altered by site-specificmutagenesis, and themutant proteins tested for their ability to complement nonviable GPI3-deleted haploids | Eur. J. Biochem. 270 4507-4514 2003 FEBS 2003 doi Comparative importance in vivo of conserved glutamate residues in the EX7E motif retaining glycosyltransferase Gpi3p the UDP-GlcNAc-binding subunit of the first enzyme in glycosylphosphatidylinositol assembly Zlatka Kostova1 Benjamin C. Yan1 Saulius Vainauskas2 Roberta Schwartz2 Anant K. Menon2 and Peter Orlean1 1 Department of Biochemistry University of Illinois at Urbana-Champaign Urbana IL USA 2Department of Biochemistry University of Wisconsin-Madison Madison WI USA Saccharomyces cerevisiae Gpi3p is the UDP-GlcNAc-bind-ing and presumed catalytic subunit of the enzyme that forms GlcNAc-phosphatidylinositol in glycosylphosphatidylinositol biosynthesis. It is an essential protein with an EX7E motif that is conserved in four families of retaining glycosyltransferases. All Gpi3ps contain a cysteine residue four residues C-terminal to EX7E. To test their importance for Gpi3p function in vivo Glu289 and 297 in the EX7E motif of S. cerevisiae Gpi3p as well as Cys301 were altered by sitespecific mutagenesis and the mutant proteins tested for their ability to complement nonviable GPI3-deleted haploids. Gpi3p-C301A supported growth but membranes from C301A-expressing cells had low in vitro N-acetylglucosami-nylphosphatidylinositol GlcNAc-PI synthetic activity. Haploids harboring Gpi3p-E289A proved viable although slow growing but Gpi3-E297A did not support growth. The E289D and E297D mutants both supported growth at 25 C but whereas the E289D strain grew at 37 C the E297D mutant did not. Membranes from E289D mutants had severely reduced in vitro GlcNAc-PIsynthetic activity and E297D membranes had none. The mutation of the first Gluinthe EX7E motif of Schizosaccharomycespombe Gpi3p Glu277 to Asp complemented the lethal null mutation in gpi3 and supported growth at 37 C but the E285D mutant was nonviable. Our results suggest that the second Glu residue of the EX7E motif in Gpi3p is of greater