Figure 7

Cytosol to membrane translocation of FIP3 occurs concomitantly with dephosphorylation. Panel A Synchronized HeLa cells at prophase/metaphase (0 min) or telophase (70 min) were lysed in the presence of phosphatase inhibitors as described in methods. The lysates were then passed over a Phospho-Protein purification column (Qiagen). The column was washed to remove non-phosphorylated material, and phosphoproteins eluted. The amount of FIP3 and RCP were then analyzed by immunoblotting equal amounts of lysate or eluate, as shown. Data from a representative experiment is shown, repeated with similar results. Panel B. Synchronized HeLa cells were harvested immediately after release from nocodazole block. Half of the cell lysates were treated with alkaline phosphatase before fractionation of the lysates into cytosolic and membrane fractions. The levels of FIP3, TfR and RCP in all fractions were then analyzed by immunoblotting. Data from a representative experiment is shown, repeated three times. Panel C. Density gradient analysis of FIP3 association with HeLa membranes. Phosphatase-treated, recombinant FIP3 was incubated with either HeLa membranes or protein-free liposomes. These membranes (or vesicles) were then floated up through an Optiprep gradient. Fractions were collected and the distribution of proteins within the gradients was assayed by immunoblotting using the antibodies shown. The FIP3 sample incubated with HeLa membranes was blotted for Rab5, as a marker for endocytic membranes. The bottom and top of the Optiprep density gradient is marked. Data from a typical experiment, repeated with two different preparations of lipid vesicles and HeLa membranes is shown.