Sip1, an AP-1 Accessory Protein in Fission Yeast, Is Required for Localization of Rho3 GTPase
Figure 3
Functional and physical interactions between Rho3 and Sip1.
(A) Rho3 suppresses sip1-i4 mutant cells (sip1-i4) in a GTP- and effector domain-dependent manner. The sip1-i4 cells were transformed with the pDB248 multi-copy vector or the vector containing rho3+, rho3GV, rho3TN, and rho3EV expressed from its endogenous promoter. These cells were streaked onto YES plates and then incubated at 27°C for 4 d or at 36°C for 3 d, respectively. (B) Binding assay for Sip1 and Rho3. GST pull-down experiments were performed using chromosome-borne GST-Sip1 expressed under the control of the nmt1 promoter. Cells expressing GFP alone, or GFP-Rho3, GFP-Rho3GV, GFP-Rho3TN, or GFP-Rho3EV were harvested and their lysates were incubated with the purified full-length Sip1 fused GST protein. GST-tagged Sip1 was precipitated with glutathione beads, washed extensively, subjected to SDS-PAGE, immunoblotted using anti-GFP or anti-GST antibodies and visualized by autoradiography. Lower panel: Quantitation of GFP-tagged various mutant forms of Rho3 beads protein levels by densitometry of the expressed bands against that of the lysate protein levels as shown in B. Data from at least three independent experiments are expressed as means ± standard deviations.