Electromagnetic Purification of Endocytic Vacuoles and Acidosomes from Dictyostelium

Earlier studies have shown that, in Dictyostelium discoideum, ∼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 ∼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 ∼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 Mg2+-dependent manner. In addition, the procedure provides a rapid method of purifying acidosomes.