Electromagnetic purification of endocytic vacuoles and acidosomes from Dictyostelium.
Earlier studies have shown that, in Dictyostelium discoideum, approximately 90% of the vacuolar proton pump (V-H(+)-ATPase) activity is present in a buoyant membrane fraction called "acidosomes." In the presence of Mg2+, acidosomes and endocytic vacuoles copurified on equilibrium sucrose gradients, suggesting their reversible association. The association depended on Mg2+ and cytosolic proteins (H. Padh et al., 1991, J. Biol. Chem. 266, 5514-5520, 12123-12126). To further characterize the putative association of acidosomes and endocytic vacuoles, cells were fed dextran-coated superparamagnetic iron colloid plus FITC-dextran to load and label their endocytic vacuoles. The endocytic vesicles were then purified approximately 20-fold at > 60% yield by their retention on a column of fine steel wire in an electromagnetic field in the absence of Mg2+. The fraction retained on a magnet column contained only about 5% of total cellular V-H(+)-ATPase and traces of other organelle markers. In the presence of 1.5 mM Mg2+, however, the retention of V-H(+)-ATPase as well as FITC-dextran was approximately 60% with only traces of contaminant markers. When such preparations were washed with buffer lacking Mg2+ while still in the magnetic field, the endocytic marker (FITC-dextran) remained on the column while V-H(+)-ATPase was eluted selectively. The elute was shown by negative-stain electron microscopy to contain purified acidosomes (saccular membranes studded with V-H(+)-ATPase). The parent material, recovered from the column in the presence of Mg2+, was rich in endocytic vacuoles bearing colloidal iron. In an electron microscope, the endocytic vacuoles were often seen associated with pump-studded acidosomes. The results independently support and extend earlier observation that acidosomes and endocytic vacuoles physically associate in a Mg(2+)-dependent manner. In addition, the procedure provides a rapid method of purifying acidosomes.
Padh
H
H
Center for Biotechnology, Northwestern University, Evanston, Illinois 60208.
D000143:Acids; D000818:Animals; D002458:Cell Fractionation / Q000379:methods*; D004023:Dictyostelium / Q000201:enzymology / Q000648:ultrastructure; D004574:Electromagnetic Fields; D004705:Endocytosis; D008274:Magnesium / Q000494:pharmacology; D006180:Proton-Translocating ATPases / Q000302:isolation & purification / Q000378:metabolism; D014617:Vacuoles / Q000187:drug effects / Q000201:enzymology / Q000648:ultrastructure
D000143:Acids; D006180:Proton-Translocating ATPases; D008274:Magnesium