Abstract
This chapter describes the morphologic, biochemical assays used to study the role of Rab8, and Rab11a in the exit from the trans-Golgi network (TGN) of cargo proteins destined for the plasma membrane. The functional analysis of Rab proteins is facilitated by the possibility of producing mutant forms that behave as dominant negative inhibitors of membrane transport. Mutants are generated by site-directed mutagenesis of conserved domains critical for nucleotide binding or hydrolysis. A recombinant vaccinia virus expression system is used to establish a biochemical assay useful for studying the function of Rab proteins in exocytic transport. This system was utilized on account of several important advantages. First, the high transfection efficiencies obtained with this system makes it particularly well suited for the development of biochemical assays that rely on having a uniform population of cells. Second, this system enables the coexpression of multiple proteins with relative ease. Thus, multiple Rab proteins could be overexpressed in combination together with relevant transport markers.
Published Version
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