Analysis of successive endocytic compartments isolated from Dictyostelium discoideum by magnetic fractionation

Abstract

A colloidal iron probe was fed to the amoeba Dictyostelium discoideum and chased for different intervals. Successive segments of the endocytic pathway were then isolated magnetically at high yield and purity. There were approx. 500 endocytic vacuoles per cell; their diameters increased from approx. 0.1-0.2 microns after 3 min of feeding to approx. 2 microns after 15 min of feeding and 60 min of chase. The wave-like progression of ingested probes along the endocytic pathway suggested that the transfer of cargo involved a maturation mechanism rather than the shuttling of cargo between stable compartments. The lifetime of primary pinosomes 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 richer in the 3 min vesicles than in plasma membranes and later endocytic vacuoles. Acid phosphatase, intrinsic vacuole acidity, the vacuolar proton pump protein and pump activity were present at all endocytic stages but rose between the 3 min and 15 min vacuoles and declined thereafter. Bis(monoacyglycero)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 both continuity and differentiation along this endocytic pathway.