Chapter 8 Reconstitution of Transport from the ER to the Golgi Complex in Yeast Using Microsomes and Permeabilized Yeast Cells

Abstract

We have developed a highly efficient in vitro-transport assay that couples translocation across the ER membrane and transport to the Golgi complex using the secreted pheromone alpha-factor as a marker protein. Radiolabeled prepro-alpha-factor of high specific radioactivity is obtained by in vitro-translating this protein in a yeast lysate. Prepro-alpha-factor synthesized in vitro is then translocated directly into microsomes or the ER of permeabilized yeast cells. Conversion of the 26-kDa ER form of pro-alpha-factor to the high molecular weight Golgi form is dependent on the presence of ATP and soluble and membrane-bound factors. Differential centrifugation and fractionation on a sucrose gradient have shown that the ER and Golgi forms of alpha-factor are enriched in separate compartments after the transport reaction. These and other findings (see Ruohola et al., 1988, for a more complete discussion) indicate that conversion to the high molecular weight form of alpha-factor is the result of authentic intercompartmental transport. Permeabilized mammalian cells have been used to reconstitute transport from the ER to the Golgi complex. In these systems (Becker et al., 1987; Simons and Virta, 1987), a viral membrane glycoprotein protein (vesicular stomatitis virus G protein) is used as the marker protein. This protein is radiolabeled with [35S]methionine during virus infection, either before or after the cells are permeabilized. Radiolabeled G protein, residing in the ER, is then transported to the Golgi complex in the presence of an ATP-regenerating system. In the mammalian system the donor and acceptor compartments are retained within the permeabilized cells (Simons and Virta, 1987); however, on occasion the addition of an exogenous acceptor compartment is required (Beckers et al., 1987). The assay we developed (Ruohola et al., 1988) differs from the mammalian assay (Beckers et al., 1987) in that we introduce radiolabeled marker protein into the ER in vitro during translocation rather than during virus infection. In addition, in our assay the acceptor Golgi compartment is always provided exogenously to the permeabilized cells. Therefore, if acceptor membranes are present in the PYC, they are not utilized. Because the permeabilized cells and the S3 fraction are prepared differently, the conditions used to prepare the cells may lead to inactivation or loss of the acceptor compartment. The in vitro assay will enable us to purify components involved in transporting proteins from the lumen of the ER to the Golgi complex. Antibody prepared to purified components can be used to clone the genes that code for these proteins.(ABSTRACT TRUNCATED AT 400 WORDS)