ANALYSIS OF SUCCESSIVE ENDOCYTIC COMPARTMENTS ISOLATED FROM DICTYOSTELIUM-DISCOIDEUM BY MAGNETIC FRACTIONATION

A colloidal iron probe was fed to the amoeba Dictyostelium discoideum and chased for different intervals. Successive segments of the endocyt ic pathway were then isolated magnetically at high yield and purity. T here were approx. 500 endocytic vacuoles per cell; their diameters inc reased from approx. 0.1-0.2 mu m after 3 min of feeding to approx. 2 m u m after 15 min of feeding and 60 min of chase. The wave-like progres sion of ingested probes along the endocytic pathway suggested that the transfer of cargo involved a maturation mechanism rather than the shu ttling of cargo between stable compartments. The lifetime of primary p inosomes was calculated to be approx. 1 s. Multivesicular bodies were common in the 3 min fraction and abundant in 15 min lysosomes. alpha- and beta-adaptins of molecular masses of approx. 89 and 83 kDa were ri cher in the 3 min vesicles than in plasma membranes and later endocyti c vacuoles. Acid phosphatase, intrinsic vacuole acidity, the vacuolar proton pump protein and pump activity were present at all endocytic st ages but rose between the 3 min and 15 min vacuoles and declined there after. Bis(monoacylglycero)phosphate or BMP, a lipid characteristic of lysosomes, followed a similar time course; it contributed up to half of the total lipid in lysosomal vacuoles. We conclude that there is bo th continuity and differentiation along this endocytic pathway.