We are studying the exchange of membrane between the surface and the internal vacuolar system that occurs during endocytosis. As one aspect of this study, we are examining the distribution of membrane peptides with the aid of monoclonal antibodies that we have raised to Acanthamoeba membranes. One monoclonal, MC1, is specific to a carbohydrate epitope that occurs on a number of membrane peptides. A second monoclonal, MC3, reacts with peptide(s) that run as a small cluster of bands on SDS gels. Freeze-fracture replicas of cells labeled simultaneously with both antibodies and a different size colloidal gold probe for each antibody, showed that the antigens are uniformly distributed and intermingled in the plane of the membrane. Fluorescent labeling showed that both epitopes are present in all internal vacuole membranes as well. We worked out procedures for immunolabeling cells embedded in plastic in order to quantitatively assess antigen distribution in internal membranes relative to the plasma membrane. Preliminary measurements of label distribution in electron micrographs indicate that MC1 antigens are more concentrated in the vacuolar membrane and MC3 antigens are more concentrated in the plasma membrane. We are investigating whether differential uptake or return of antigens during the membrane recycling that accompanies endocytosis can account for this distribution.
