Figure 7. Functional interaction of Rho3 with the clathrin adaptor-protein complex.

(A) Rho3 suppressed the temperature-sensitive growth of deletion mutants of individual adaptin subunit of the AP-1 complex. The Δapl2, Δapl4, and Δaps1 cells were transformed with either the pDB248 multicopy vector or with the rho3⁺-containing vector. The cells were then streaked onto plates containing YPD, and then incubated at 27°C for 4 d or at 35°C for 3 d, respectively. (B) Rho3 suppressed the immunosuppressant sensitivity of deletion mutants of individual adaptin subunit of the AP-1 complex. Cells as indicated in (A) were spotted onto YPD or YPD plus 0.5 µg/mL FK506 and incubated at 27°C for 4 d. (C) Binding assay involving Rho3 and the 4 subunits of the AP-1 complex. GST pull-down experiments performed with GST alone or Apm1-GST, GST-Apl2, Apl4-GST or GST-Aps1; cells expressing GFP-Rho3 were collected, and the lysates were incubated with purified full-length Apm1 fused GST, GST-Apl2, Apl4-GST or GST-Aps1 or the control GST protein. GST and GST-tagged adaptin subunits were precipitated by glutathione beads, washed extensively, subjected to SDS-PAGE, and immunoblotted using anti-GFP or anti-GST antibodies.