The long-term goal of the proposed research is to understand the molecular mechanisms by which nucleotide sugars are transported across intracellular membranes and how such transport systems regulate glycoconjugate biosynthesis in mammalian cells. The proposed research will focus on determining how uridine nucleotide sugars are transported across the Golgi apparatus membrane. UDP- galactose transport across the Golgi membrane will be characterized using Golgi vesicles isolated from rat liver. This characterization will include determination of the kinetic constants and driving force for UDP-galactose transport. The UDP- N-acetylglucosamine and UDP-galactose transporters will be solubilized and reconstituted into liposomes, as a first step toward the ultimate purification of these transporter proteins. A cDNA encoding for the UDP-galactose transporter will be isolated utilizing an expression cloning strategy to clone by complementation analysis using a chinese hamster ovary cell mutant which is defective in UDP-galactose transport. %%% Glycoconjugates, including glycoproteins and glycolipids, are critical to the life of all cells. They are synthesized by the stepwise addition of covalently linked simple sugars to nascent structures (proteins or lipids). These reactions generally proceed by the transfer of the sugar moiety from a biosynthetic activated precursor, or nucleotide-sugar, to the nascent glycoconjugate. In eukaryotes, these glycosyltransfer reactions usually take place within the lumens of intracellular membrane-bounded compartments (e.g. endoplasmic reticulum and Golgi apparatus). Most nucleotide sugars (e.g., UDP-galactose, UDP-N-acetylglucosamine) are synthesized in the cytosol, and because of their ionic charge, cannot passively diffuse across a biological membrane. Therefore, specific transporters exist in the intracellular membranes which effect the translocation of the nucleotide sugar precursors from the cytosol into the lumen. This project focuses on the transport of UDP-galactose across the Golgi apparatus membrane, and in particular on the biochemical description of the membrane transporter. The work will lead to the eventual understanding of how this class of transport protein operates, and also will further our understanding of the functions and organization of the Golgi apparatus, a subcellular organelle which plays a central role in protein sorting and secretion, and in the biosynthesis of glycoproteins and glycolipids. The results of these studies may have practical application in engineering cells to produce various specific glycoconjugates on a commercial scale.
