FIG. 7.

The NXT1- and nucleoporin-binding domains of TAP are required for export activity. (A) Plasmids expressing the indicated portions of TAP (Fig. 5) fused to RevM10 were transfected into 293T cells and tested for activity as described in the legend to Fig. 1. A plasmid expressing NXT1 was cotransfected as indicated. Plasmids expressing Rev or RevM10 were also tested as controls. The values shown are averages of duplicate SEAP-normalized p24 levels. (B) Lysates of 293T cells transfected with plasmids expressing C-terminal deletion mutant RevM10-TAP were subjected to IP-Western blot analysis as described in the legend to Fig. 4A. The values on the right are molecular weights in thousands. (C) FLAG-RevM10-TAP and NXT1 binding in vitro. Full-length RevM10-Tap (61-619) and the indicated RevM10-TAP deletion mutant proteins were assayed for in vitro NXT1-binding activity. ³⁵S-labeled FLAG-RevM10-TAP fusion proteins and NXT1 protein were synthesized in vitro in rabbit reticulocyte lysates. FLAG-RevM10-TAP lysates were mixed with NXT1 lysates and allowed to bind. FLAG-RevM10-TAP proteins were immunoprecipitated with M2 anti-FLAG monoclonal antibody or a control irrelevant antibody, resolved by SDS-PAGE, and visualized by autoradiography. The bands corresponding to the proteins are indicated.