Proteomic analysis of a developmentally regulated secretory vesicle

Secretion of spore coat proteins from the prespore secretory vesicles (PSVs ) in Dictyostelium discoideum is a signal mediated event that underlies ter minal cell differentiation, and represents an important case of development ally regulated secretion. In order to study the biochemical mechanisms that govern the regulated fusion of the PSVs with the plasma membrane and the s ubsequent secretion of their cargo, we purified this organelle from prespor e cells. Analysis of protein extracts of highly purified PSVs indicated tha t, in addition to the cargo of structural spore coat proteins, many more pr oteins are associated with the PSVs. Their identification is paramount to t he understanding of the mechanism of regulated secretion in this system. In this study we have taken the first comprehensive proteomic approach to the analysis of an entire, previously uncharacterized, organelle, with the goa l of identifying the major proteins associated with the PSVs. We show that in addition to the structural spore coat proteins, the PSVs contain the enz ymes needed for proper spore coat assembly (thioredoxin 2 and 3), regulator y proteins which we predict receive and transduce the developmental signal for secretion (rab7 GTPase, PI-3 kinase, NDP kinase and the calcium binding proteins calfumirin-1 and calreticulin) as well as proteins that interact with the cytoskeleton to mediate movement of the PSVs to the plasma membran e (actin binding proteins coactosin and profilin 1). In addition, the resul ts suggest that proteins can play multiple roles in the cell, and that prot ein function can be dictated in part by subcellular localization. The ident ification of the PSV proteins is allowing us to develop testable hypotheses about the roles of these proteins within the functional context of develop mentally regulated secretion.