Kv. Nolta et al., ANALYSIS OF SUCCESSIVE ENDOCYTIC COMPARTMENTS ISOLATED FROM DICTYOSTELIUM-DISCOIDEUM BY MAGNETIC FRACTIONATION, Biochimica et biophysica acta. Molecular cell research, 1224(2), 1994, pp. 237-246
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.