Abstract
This chapter describes an in vitro assay that reconstitutes vesicular transport between the endoplasmic reticulum (ER) and Golgi complex in the yeast Saccharornyces cerevisiae. In this assay, the processing of a precursor of the secreted pheromone α- factor is followed. Prepro- α– factor is synthesized as a 19- k Da precursor molecule that contains four tandem repeats of the mature protein. The in vitro transport assay is composed of four elements—namely, a donor compartment, acceptor membranes, soluble transport factors, and an ATPregenerating system. The donor compartment is provided by permeabilized yeast cells (PYCs) that have radiolabeled pro-a-factor translocated into the lumen of the ER. The studies show that when vesicles containing the 26- k Da species are incubated with acceptor membranes, cytosol, and an ATP-regenerating system, pro-a-factor is processed to a 28-kDa species before it is converted to the high molecular weight reaction product. We have proposed that 28- k Da pro- α -factor is formed in an early subcompartment of the Golgi complex prior to its conversion to the high molecular weight species.
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