The overall goal of the work proposed here is to further understanding of protein traffic through the secretory pathway of the eucaryotic cell. This process is crucial for the generation and maintenance of cellular compartment, and hence for viability. GTC (for Golgi Transport Complex) is a novel hetero-oligomeric protein complex that has recently been identified and purified based on its ability to facilitate transport through the Golgi in vitro. A cDNA fore one of the subunits was cloned, and used to generate recombinant protein for antibody production. Use of the antibody in subcellular fractionation and indirect immunofluorescence studies showed that GTC is localized to the Golgi.
The specific aims of the work proposed here are designed to elucidate the role of GTC in the secretory pathway. To do this, a high yield GTC purification will be developed employing immunoblotting, rather than GTC activity, as a detection method. Peptide sequence data will be obtained from the subunits in order to clone cDNAs for each of the polypeptides. Recombinant GTC-subunits will be used to generate polyclonal and monoclonal anti-GTC antibodies for structural and functional analyses. The structural studies will entail a rigorous determination of GTC subunit stoichiometry, morphological examination by deep-etch electron microscopy, localization of the subunits within the complex, an attempt to generate functional subcomplexes, and nearest neighbor analyses by chemical cross-linking and perhaps two-hybrid studies. The intracellular localization of GTC will be analyzed in detail using subcellular fractionation, and confocal and immuno-gold microscopy. Potential residence within the Golgi-matrix or on COPI-coated vesicles will be examined biochemically GTC function will be analyzed in vivo, in cells over-expressing or under-expressing GTC subunits to determine at what stage of the early secretory pathway GTC functions. These results will be corroborated through use of semi-intact cell systems that monitor several early transport steps. In an effort to determine whether GTC affects vesicle budding, or membrane docking/fusion the potential GTC requirement in two in vitro assays will be examined; one reconstitutes only vesicle budding, and the other measures only membrane docking/fusion. Lastly, since GTC is tightly associated with the Golgi membrane, experiments to identify and purify a putative GTC receptor will be undertaken. These studies will provide the first insights into the role of this novel protein complex in the process of intracellular protein traffic.
| Oka, Toshihiko; Ungar, Daniel; Hughson, Frederick M et al. (2004) The COG and COPI complexes interact to control the abundance of GEARs, a subset of Golgi integral membrane proteins. Mol Biol Cell 15:2423-35 |
| Ungar, Daniel; Oka, Toshihiko; Brittle, Elizabeth E et al. (2002) Characterization of a mammalian Golgi-localized protein complex, COG, that is required for normal Golgi morphology and function. J Cell Biol 157:405-15 |
