The long term broad objective of this proposal is to uncover molecular mechanisms mediating cellular protein transport and sorting through the secretory pathway. Recent studies are revealing a central role for the cytoskeleton in the function and organization of the secretory pathway. Cytoskeletal regulation in the secretory pathway has been linked to cell transformation. Thus, this work will provide insight into cancer etiology and the invasive behavior of cancer cells, A signaling pathway has been characterized that connects proteins involved in COPI transport vesicle assembly at the Golgi with proteins that regulate the assembly of the cytoskeleton. This pathway depends on a cargo-protein-sensitive binding interaction between the coat protein, coatomer, and the GTP-binding protein Cdc42. Activation of this pathway leads to the recruitment of a specific actin-binding protein, mAbp1 to the Golgi. Both upstream (Cdc42) and downstream (mAbp1) components of this signaling pathway are involved in protein transport at the Golgi apparatus. Preliminary results link the coatomer/Cdc42-dependent signaling pathway to the recruitment of the molecular motor dynein to the membrane. It is hypothesized that a role for vesicle-coat dependent signaling to the cytoskeleton is to provide precise temporal regulation between the processes involved in transport vesicle assembly and cytoskeleton-mediated vesicle scission and translocation, As a first step toward proving this hypothesis, the following aims will be carried out:
Aim 1) To reconstitute coatomerdependent cytoskeletal regulation on liposomes, Aim 2) To define the domains of mAbpl that are important for its recruitment to the Golgi.
Aim 3) to determine how dynein recruitment responds to coatomer-Cdc42 signaling at the Golgi.
Aim 4) to determine which p24-family vesicle cargo proteins influence coatomer/Cdc42 signaling.
These aims will define the role of the coatomer/Cdc42 complex in cytoskeletal regulation, define the role of coatomer-dependent actin signaling in dynein recruitment and identify novel proteins that function in cytoskeletal regulation at the Golgi.
