With past NIH support we have developed techniques for (a) the selective radioiodination of endocytic membrane proteins, (b) the characterization of endocytic compartments by analytical centrifugation, and (c) the assay of pinocytic content exocytosis. These techniques have produced evidence for (a) rapid shuttling of endocytic membrane proteins to the cell surface, (b) the progressive processing of endocytic components of the vacuolar apparatus at both a pre-lysosomal and lysosomal stage, and (c) extensive exocytosis of endocytic contents from pinosomes, the major reversible endocytic compartment in fibroblasts. We wish, as described in this amended renewal proposal, to apply these techniques in concert to compare and contrast in molecular terms the processing of the membrane versus the contents of pinosomes and to determine the points of overlap between endocytic pathway(s) and plasma membrane biosynthetic pathway(s). The intracellular processing of these vesicles is more complex than envisioned even a few years ago and is little understood. The processing of pinocytic vesicles will be studied with respect to both content and membrane. The G protein of vesicular stomatitis virus (SVS) infected cells will be used as the model plasma membrane protein. Much of the methodology of contemporary cell biology will be employed. The research proposed is part of a long-term effort to relate, in molecular terms, pinocytic processes to: (a) regional specialization of the cell surface, (b) the turnover of cell surface proteins, (c) the maintenance of organelle integrity, and (d) the transport of newly synthesized proteins to the cell surface. The understanding of pinocytic processes should be important to strategies to target drugs, enzymes, and DNA to cells. This should be of therapeutic value. Knowledge of how the cell surface turns over should be important in being able to modulate cell interaction and growth which in turn should be fundamental to the progress of medicine.
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