In eukaryotes approximately forty percent of all cellular proteins and many lipids are glycosylated in the lumen of the Golgi apparatus and endoplasmic reticulum (ER). The nucleotide substrates of these reactions must first be translocatedfrom the cytosol into the lumen of the above organelles via specific transporters which function as antiporters with the corresponding nucleoside monophosphate. This was discovered in our laboratoryand is known to occur in every eukaryote examined so far. Recent studies have shown that mutants in the cycle profoundly affect development of multicellular eukaryotes including C. elegans, and Drosophila. The first nucleotide sugar transporter disease in humans , Leukocyte adhesion deficiency syndrome II, a defect in the Golgi apparatus GDP-fucose transporter has recently been described. To understand the regulation of the above cycle we plan to pursue the following three major specific aims:1) To continue with our studies on the human GDP-fucose transporter. Specific questions being asked are whether the transporter proteins are partially active as a result of the mutations or whether they are inactive and other transporter(s) are using GDP-fucose as substrate. For this we will express these active transporters in bacteria and reconstitute the proteins into proteoliposomes. This system will also be used to determine the substrate binding site. (2 )To continue with our studies on the physiology of the three C. elegans' transportersfor UDP-Galactose/UDP-N-Acetylglucosamine discovered in our laboratory. We believe this nematodeto be a good model system to study these transporters in multicellular organisms, including humans. We plan to study the tissue and cellular location of the above transporters, the phenotypes of mutants in these genes and their glycoconjugates. This will allow a molecular understanding of the mutant phenotype. (3) Continue with our studies on the ire-1 dependent transcriptional upregulation of a novel UDP/GDPase, uda-1, of C. elegans. We hypothesize that this upregulation leads to increased supply of UDP-glucose into the ER/Golgi lumen, a novel regulatory mechanism in the nucleotide sugar transport/antiport cycle of mammals including humans. Together, the above studies should lead to a better understanding of the role of these transporters in multicellular eukaryotes and provide novel and important insights into the role of glycoconjugates during normal eukoryotic development and human disease.
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