Little is known about the mechanism of sorting and packaging of secretory proteins that transit from the Golgi complex to the cell surface. Although some proteins use clathrin to traverse the endosome en route to the plasma membrane, others do not, and, until now, the general view has been that the direct path out of the trans-Golgi network (TGN) may involve tubular carriers formed without the intervention of coat proteins. To examine this limb of the secretory pathway, we have examined two membrane proteins: a biosynthetic enzyme, chitin synthase III (Chs3p), and a mating cell fusion protein, Fus1p, which travel from the TGN/endosome membrane to the plasma membrane in yeast.
Left: Chitin rings stained with calcofluor
Right: Chs3-GFP viewed by fluorescence microscopy (note the presence of Chs3p at both the bud neck and within intracellular compartments)
In cells with mutations that prevent Chs3p from reaching the cell surface, chitin rings and bud neck staining are not present.
Genetic and biochemical analyses have now shown that Chs3p and Fus1p are conveyed from the TGN by a novel coat protein complex we call exomer. Exomer consists of five subunits: Chs5p, Chs6p, and nonstoichiometric levels of three paralogs of Chs6p. Exomer is a large, ~1-MDa complex that binds membranes in the presence of a GTP-activated form of Arf1p, a small GTPase implicated in other coat protein assembly events. Unlike other coats, however, exomer is responsible for the traffic of a nonessential set of surface proteins. We are exploring the sorting of plasma membrane proteins into mature secretory vesicles by immunoisolating vesicles from cells blocked late in the secretory pathway.
Analysis of Vesicle Budding from the ER