Internalization and recycling of plasma membrane glycoconjugates during pinocytosis in the macrophage cell line, P388D1. Kinetic evidence for compartmentation of internalized membranes.

An analysis was made of the pinocytosis-derived internalization and recycling of membrane in the macrophage cell line, P388D1. Plasma membrane glycoconjugates, reversibly labelled with [3H]galactose, were used as a membrane marker. Label internalized with the plasma membrane was no longer accessible to release by externally added beta-galactosidase and could therefore be distinguished quantitatively from label remaining on the cell surface. Direct experimental evidence for membrane recycling was obtained by demonstrating that previously internalized label reappeared at the cell surface. The composition of labelled membrane glycoconjugates, as analysed by SDS-polyacrylamide gel electrophoresis, remained unaltered before and after internalization. The label remained membrane-bound in an unmodified way during the entire period of 8 h investigated, corresponding to about twenty-four cycles of membrane flow. Membrane flow led to a steady-state distribution of label between the plasma membrane and intracellular membranes. The redistribution of label occurred with biphasic kinetics, which could be described as the sum of two exponential functions. This behavior is explained by presenting a model of membrane flow between the plasma membrane and two consecutive intracellular membrane compartments, which we assume to consist of pinosomal membranes and of pinosome-derived membrane of secondary lysosomes. The relative membrane surface areas turn out to be in the ratio of 100:12.5:7.3, respectively. At the observed rate of pinocytosis, the equivalent of the plasma membrane is internalized once every 21 min, in the form of primary pinosomes of the size 0.24 micrometer. The residence time of membranes in the pinosome compartment is about 3 min. The rate at which membranes enter the lysosomal compartment is 31 times lower than the rate of membrane internalization. We conclude that only 3% of the amount of membrane internalized at any one time subsequently enters the secondary lysosome compartment. After a residence time of 49 min this membrane fraction is finally recycled to the cell surface. The results are discussed in terms of mixing and sorting-out of pinosomal and lysosomal membranes.