Previous studies from our laboratory have provided in vivo and in vitro evidence for a nucleotide sugar transporter/antiport cycle in the Golgi apparatus of higher and lower eukaryotes. We have hypothesized that this is a novel regulatory step in post-translational modifications which occur in the lumen of the Golgi apparatus and affect most membrane and secreted proteins in the cell. We plan to further understand how this cycle is regulated under physiologic and pathologic conditions by pursuing the following directions: (1) To clone the Golgi transporter of GDP-fucose and determine whether mutations in the protein per se, regulatory regions of the gene or extragenic ones, are the cause for the hypofucosylation and specific decrease in Golgi GDP-fucose transport in patients with leukocyte adhesion deficiency syndrome II, the first disease identified as a possible consequence of a defective Golgi nucleotide sugar transporter. (2) In collaboration with Dr. Alok Mitra we will determine the substrate recognition sites and 3D structure of the murine Golgi transporter for CMP-sialic acid and the canine and K. lactis transporters for UDP-GIcNAc. The crystal structures will be determined by 2D crystallization, electron cryomicroscopy and image analysis. Crosslinking studies with nucleotide sugar analogs will aid in the identification of amino acids implicated in substrate binding. (3) As a continuation to our recent studies with SQV-7 of C. elegans, we will attempt to determine the function of some of the other 8 putative nucleotide sugar transporters in the genome of this nematode. (4) We will study regulation of glycosylation through nucleotide sugar transporters by using self-complementary chimeric oligonucleotides to generate cell lines that lack or have altered levels of specific nucleotide sugar transporters. (5) We will determine the role of putative Golgi GDPases/UDPases of C. elegans by searching for orthologues which correct the S. cerevisiae GDPase (gda1).
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