The overall aim of this research is to understand the molecular mechanisms by which substrates gain access to the glycosyltransferases during terminal glycosylation of secreted glycoproteins in the Golgi apparatus. Studies from several laboratories, including this one, have demonstrated that specific carrier proteins are involved in transporting nucleotide sugars into the Golgi lumen and nucleotide products of the transferases out of the lumen. The specific goals of the project are to: (1) identify the proteins responsible for UDP-galactose and UMP transport across Golgi membranes; (2) isolate and characterize the transport proteins; and (3) determine how the transporters are oriented in the membrane, and how they are distributed relative to galactosyltransferases. Perhaps the most striking thing about living cells is the very high degree of order and organization which is required for life processes to occur. The cell is compartmentalized into specific regions which carry out specific functions, and this compartmentalization allows for a high degree of regulation of those functions with minimal interference from other cellular functions. Some of these "compartments" consist of membrane- enclosed intracellular organelles, within which specific biochemical reactions occur. In the case of post-translational processing of "secretory" proteins (including membrane proteins and lysosomal proteins, the biosyntheses of which also utilize the "secretory pathway"), late post-translational modification by the addition of terminal sugars (and other substituents) takes place within the lumen of the Golgi apparatus, a complex collection of membrane-bound biosynthetic compartments to which the nascent proteins are transported. The sugar transferase reactions require the participation of the membrane-bound transferase enzymes, the nascent glycoprotein, and the nucleotide-linked "activated" sugar substrates. The activated nucleotide sugars are not biosynthesized within the Golgi lumen, but elsewhere in the cell (usually in the cytoplasm). These compounds do not readily cross biological membranes. Similarly, the resulting reaction product of the transferase reaction, free nucleotide, also does not cross membranes readily. Yet, it is critical for the nucleotide sugars to enter the lumen, and also critical for the reaction products (which would inhibit the reaction if allowed to acumulate) to exit from the site of the reaction. By focusing on the transport and compartmentalized utilization of a particular nucleotide sugar, UDP-galactose, his research will increase our understanding of how this delicate, yet critical, compartmentalization works in the living cell.
