The proposed project will use Xenopus oocytes as a model system to investigate the mechanisms of postendocytotic compartmentation in cells. Among the many advantages offered by these cells is the ability to easily isolate the intermediate vesicles involved in the compartmentation of a specific ligand. This is of particular interest because these intermediate vesicles have been shown to modulate the regulatory effects of endocytotically incorporated proteins in other cell types. The first part of this study will define the number of intermediate compartments in these cells by analyzing membrane protein components using one dimensional SDS gel electrophoresis, two dimensional gel analysis and silver staining. The next two phases of the project will explore the mechanisms of compartmentation of proteins incorporated by either receptor-mediated or fluid-phase pinocytosis. This will entail sucrose gradient fractionation studies of the distribution of incorporated substances under both normal and perturbed conditions. This portion of the project seeks to determine whether compartment fusion is a random or directed event and to ascertain what factors are involved in normal compartmentation. The final aspect of the project will investigate the cellular modulation of compartmentation. This will be done by determining to what extent different cellular parameters contribute to changes in the net rate of ligand uptake observed when oocytes are exposed to hormones or are in different stages of development. Parameter analysis will be accomplished using a computer-assisted steady-state model of endocytosis. Coordinate control of ligand uptake and subsequent intracellular compartmentation will be assessed by examining the kinetics of ligand transfer using SDS gel electrophoresis and sucrose gradient fractionation. At the conclusion of these studies it is anticipated that many of the mechanisms involved in postendocytotic compartmentation will have been elucidated.
| Opresko, L K; Karpf, R A (1987) Specific proteolysis regulates fusion between endocytic compartments in Xenopus oocytes. Cell 51:557-68 |
| Opresko, L K; Wiley, H S (1987) Receptor-mediated endocytosis in Xenopus oocytes. I. Characterization of the vitellogenin receptor system. J Biol Chem 262:4109-15 |
| Opresko, L K; Wiley, H S (1987) Receptor-mediated endocytosis in Xenopus oocytes. II. Evidence for two novel mechanisms of hormonal regulation. J Biol Chem 262:4116-23 |
