The long-term goals of this research proposal are the following: (a) To understand the cellular mechanisms which regulate the glycosylation of proteins and lipids in the rough endoplasmic reticulum and Golgi apparatus and, (b) to establish the role of protein and lipid glycosylation in organelle topography and compartmentation during secretion and membrane biogenesis. We propose to pursue the following specific aims towards achieving these goals: (1) To continue with our studies on the mechanisms of glycosylation in the Golgi apparatus of mammalian cells. We will use biochemical and molecular biological approaches to purify the Golgi CMPNeuAc and UDP-Galactose transporters and to clone the respective genes. Using our recently described proteoliposome reconstitution system, we will use affinity and conventional chromatography to purify the transporters. We will also rescue plasmids, containing cDNAs of the Golgi UDP-Galactose and CMPNeuAc transporters, from transfectants of mutant Chinese hamster ovary cells, which are deficient in the above transport activities. Antibodies against the transporters will be made from the proteins or based on the cDNAs and used in immunoelectronmicroscopy studies to determine whether the transporters are polarized within the Golgi apparatus. In conjunction with proteases, the antibodies will also be used to study the arrangement of the transporter proteins in the Golgi membrane. The cDNAs will be used to study the structure of the transporter genes and how the expression of these proteins is regulated. (2) To continue with our studies on the subcellular organization and topography of glycosylation in the Golgi apparatus of yeast. We will transform, with a genomic library made from wild-type DNA, a mutant of K. lacis. recently characterized by us to be deficient in UDP-GlcNAc transport into Golgi-like vesicles in order to isolate and characterize the Golgi UDP-GlcNAc transporter gene. We will clone and disrupt the gene of the Golgi membrane GDPase, a lumenal marker enzyme from S. cerevisiae recently purified in our laboratory and hypothesized to be necessary for Golgi mannosylation. Antibodies against the GDPase and the K. lactis UDP-GlcNAc transporters (obtained via their DNA sequence) will be used to study, (a) by immunoelectronmicroscopy, their location within the cell, and (b) via sensitivity towards proteases to establish the proteins' topography in the Golgi membrane. (3) To continue with our studies on the topography of glycosylation in the rough endoplasmic reticulum. Using membrane impermeable probes, we will attempt to demonstrate, directly, translocation of dolichol-oligosaccharide derivatives from the cytosolic side of the membrane into the lumen. Reconstitution studies with endoplasmic reticulum (ER) membrane proteins and liposomes will be attempted to demonstrate the occurrence of a dolichol-oligosaccharide translocator protein in the ER membme.
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