The long-term goal of my research is to elucidate the mechanisms that mediate cargo selection and export from the endoplasmic reticulum (ER). The cytosolic COPII coat complex is the basic sorting machine that mediates ER export. Selective low affinity interactions of COPII components with ER export signals on cargo) proteins support cargo selection and incorporation into COPII vesicles. We hypothesized that these low affinity interactions are transiently transformed into high avidity multivalent protein-lipid and protein-protein interactions at budding sites to enable ER export. The Sar1p GTPase may control this """"""""membrane transformation"""""""", through effector activation and membrane binding. Membrane remodeling may be essential to support the dynamic and localized nature of coat assembly and disassembly. Dynamic lipid re-modeling may also be required to enable localized membrane curvature and vesicle fission. In preliminary results, we demonstrate that local membrane re-modeling, through activation of phospholipase D and PI-kinases, may contribute to coat assembly and ER export. The involved enzymatic activities will be characterized in biochemical assays, and the temporal re-modeling of ER membranes, will be analyzed using morphological in vitro assays with fluorescent lipid-binding peptide reporters. In preliminary results, we also demonstrate that Sar1p interactions with re-modeled bilayers support vesicle fission. We will characterize the molecular basis for Sar1p and COPII Sec23/24 complex interactions with lipid bilayers. The role of coat-lipid interactions in vesicle formation, cargo selection and vesicle fission, will be studied using combined biochemical morphological and biophysical vitro and in vivo assays. The results may provide a general model to describe the mechanism and dynamics of membranes deformation, cargo selection and vesicle fission in vesicular traffic. ? ?
