Kinetics of membrane internalization and recycling during pinocytosis in Dictyostelium discoideum

Abstract

Internalization and recycling of plasma membrane during pinocytosis in Dictyostelium discoideum was analyzed quantitatively. A labeling technique was used by which [3H]galactose could be enzymatically bound to and released from the plasma membrane. Label internalized with the plasma membrane was no longer accessible to enzymatic release and could therefore be distinguished quantitatively from label remaining on the cell surface. Internalization of labeled membrane components was measured as a function of pinocytotic uptake. Direct experimental evidence for membrane recycling was obtained by demonstrating that previously internalized label reappeared at the plasma membrane. The experimental data agree with a kinetic model requiring that a shuttle of membrane between two membrane compartments leads to the same surface concentration of label in both. The two compartments consist of the plasma membrane and of cytoplasmic vacuolar membranes; their relative membrane surface areas are 1 and 0.5, respectively. One surface area equivalent of the plasma membrane is internalized during a pinocytotic uptake amounting to 15% of the cell volume. At the observed rate of pinocytosis, this occurred once every 45 min. The average size of the primary pinosomes, as weighted according to their contribution to pinocytotic uptake, was calculated to be about 0.6 microns.