Figure 6.

Resistance of AP-1 to Tris extraction in two-stage binding assays. Golgi membranes were primed in a first-stage incubation with either ARF·GTP or ARF·GTPγS. After recovery of the membranes, either cytosol or affinity-purified AP-1 was mixed with the primed membranes on ice as indicated, followed by a further incubation on ice for the second-stage binding step. Membranes were pelleted and then extracted with 1 M Tris, and the pellets (TP) and supernatants (TS) were then separated. Unprimed Golgi membranes were used as a control to determine background (Bkg) binding of AP-1. Membranes from two-stage reactions not extracted with Tris were used to show total (Tot) AP-1 recruitment. Notice that on membranes primed with ARF·GTP and reincubated with either cytosol or pure AP-1, the bound AP-1 is fully Tris sensitive, whereas in the presence of ARF·GTPγS, the bound AP-1 is resistant to Tris extraction.