The aim of the proposed research is to investigate membrane protein sorting during endocytosis and organelle biogenesis in Xenopus oocytes, using both endogenous membrane markers and exogenous markers introduced by microinjection of mRNA. Accumulation of yolk proteins in these cells is accomplished by the receptor-mediated endocytosis of a precursor, vitellogenin, which undergoes condensation and is then stored for prolonged periods of time in an abundant and very dense organelle, the yolk platelet (YP). Transport to this final storage site occurs via a less dense YP compartment which has lysosomal enzyme activity. The ability to isolate membrane fractions from both the beginning (plasma membrane = PM) and the end (YPs) of this unusual endocytic pathway makes it a particularly good system to study protein sorting during endocytosis. To this end, the protein and glycoprotein composition of oocyte PM and YP membranes will be analyzed and compared to determine their degree of similarity. Immunological probes for endogenous oocyte membrane proteins will be developed by raising monoclonal and ployclonal antibodies against specific populations of glycoprotein isolated by lectin affinity chromatography. These antibodies will be used to investigate intracellular site(s) of PM protein sorting during endocytosis, using immunocytochemical localization and subcellular fractionation to detect antigens. Cell surface and intravesicular iodination will be used to selectively label membrane proteins of the PM and various endocytic compartments, using the increasing density resulting from yolk protein condensation to distinguish early and late organelles of the endocytic pathway. The levels of expression and intracellular behavior of exogenous Fc and asialoglycoprotein receptors and lysosomal membrane glycoproteins introduced by microinjection of mRNA will also be examined, to assess the usefullness of the oocyte microinjection system for studies of membrane sorting. Pulse-chase experiments will be used to analyze the routes taken by endogenous PM and YP proteins and exogenous lysosomal membrane proteins to their target organelles. These studies will contribute to our knowledge of the sorting of membrane proteins during endocytosis and targeting to specific intracellular locations, knowledge which is fundamental to an understanding of both normal and abnormal cell physiology.
| Wall, D A; Holguin, R (1992) Expression of the chicken hepatic glycoprotein receptor in Xenopus oocytes: conservation of ligand and receptor targeting signals. J Cell Biochem 48:248-61 |
