Glycosyltransferases have an inverted membrane topology that consists of short amino-terminal cytoplasmic tail, a hydrophobic anchor domain and the carboxyl-terminal portion of the protein that carries the catalytic domain. In the present studies we have identified a transmembrane region of the enzyme by transfection of Cos-7 cells with the amino-terminal deletions of beta-14galactosyltransferase cDNA. By exchanging the sequence segment of this enzyme with the corresponding region of other glycosyltransferases and using catalytic domain of 1-4galactosyltransferase as a reporter protein we have identified a transmembrane domain of alpha-1-3galactosyltransferase and of alpha-2-- 6sialyltransferase. These studies have shown that the amino-terminal signal anchor domain, the transmembrane region of glycosyltransferases is essential for the stability and targeting of these enzymes to the Golgi apparatus in mammalian cells. For the functional analysis of the carboxyl-terminal catalytic domain of beta-1-4galactosyltransferase the deletions and site directed mutants of the cDNAs were constructed in the pGEX vectors and expressed in E. coli. Either the full length or the amino-terminal deleted bovine galactosyltransferase were expressed as fusion proteins, connected to the COOH-terminus of glutathione S- transferase (GST), and purified on glutathione-affinity columns, cleaved by site-specific protease from GST, and each analyzed for GT and lactose synthetase (LS) enzymatic activities. Full length bovine GT (402 aa), and the proteins with deletions in the amino-terminal end up to residue 130 (GT 130) were active in GT and LS assays. Both GT and LS activities were lost when residues 130-142 were deleted. Within this deletion is the Cys 134 which when modified by site-specific mutagenesis to serine (GTcl34s) or alanine (GTcl34a) gives about 1% of the activity of GT 130. These results show that the Cys 134 which has been shown to be in a disulphide bridge with Cys 234 is necessary to impart the enzymatic function to the protein.
