Figure 2.
Purified PI4KIIα and AP-3 interact in vitro. (A) AP-3 was purified from rat brain cytosol by immunoaffinity chromatography using a mAb (KF4) to the δ subunit of AP-3. The cytosol preincubated beads were washed extensively (see Material and Methods) and incubated with 125 nM purified GST-PI4KIIα (lanes 4–8 and 10) or 125 nM GST alone (lane 9). Bound GST-PI4KIIα was detected by SDS-PAGE and immunoblot using antibodies to the β3 subunit of AP-3 and GST. GST alone fails to bind to beads coated with AP-3 (lane 9). Control immunoprecipitations using beads without antibody (lanes 4 and 5), irrelevant antibody (SV2, lanes 6 and 7), or absence of cytosol (lane 8) display only background levels of bound GST-PI4KIIα, whereas beads coated with purified AP-3 display significant GST-PI4KIIα binding. GST and GST-PI4KIIα inputs (lanes 1 and 2) are 15% and cytosol input is 2.5%. (B) Purification, binding of AP-3 and GST-PI4KIIα, and detection of bound GST-PI4KIIα was performed as in A, except that the mAb used for AP-3 immunoaffinity purification was δ (anti-δ SA4 monoclonal). A peptide corresponding to the epitope recognized by the δ antibody was used to elute bound AP-3 (lanes 9 and 10). Inclusion of the peptide during AP-3 immunoaffinity purification out-competed the interaction between the antibody and AP-3 (lanes 9 and 10). As in A, GST-PI4KIIα failed to bind to uncoated beads (lane 4), beads coated with irrelevant antibody (lane 5, SV2), or to beads coated with δ antibody but lacking AP-3 (lane 6), and GST alone does not bind AP-3 (lane 7).