Strong evidence now implicates an involvement of oncogenic human ras proteins in human carcinogenesis. Unexpectedly, the three human ras proteins represent only a small branch of larger superfamily of ras- related proteins. It is the general aim of the proposal to use high resolution electron microscopy (EM) involving immunogold and cryo- immunogold technologies to understand the role of the ras-related rab and ARF small GTP-binding protein in regulation of membrane trafficking of protein between the ER and the cis Golgi compartment.
The specific aims of this proposal are: (1) To characterize through application of immunogold technologies the morphological role of the rab and ARF families of ras-related GTP-binding proteins in vesicle formation and fusion. Specifically, the effect of trans dominant mutations and chimera (raba/rabb, rab/ras and ARFa/ARFb) will be used to define differences in the role of membrane localization, GTP-binding and GTP-hydrolysis in membrane trafficking of vesicular stomatitis virus glycoprotein (VSV-G) from the ER to the cis Golgi compartment in vivo using transient expression systems. (2) To use mutant recombinant proteins, selected biochemical reagents (i.e., GTPgammaS) an peptide reagents encoding functional domains of the rab and ARF proteins to inhibit transport in vitro using permeabilized cells in order to characterize the morphological consequences of inhibition using immunogold technologies. (3) To morphologically characterize novel ER to Golgi intermediates which accumulate in the absence of GTP hydrolysis and in the presence of mutant rab and ARF proteins in order to characterize their structure and their relationship to the molecular events involved in vesicle fission and fusion. By understanding the principles of localization, GTP binding and GTP hydrolysis for rab, ras and ARF proteins we will gain functional insight into the biochemical properties that account for their differences in the regulation of membrane trafficking, signal transduction and cell proliferation. These will in turn add to our understanding of potentially feasibility of drugs to specifically inhibit ras function during oncogenesis.
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