Sip1, an AP-1 Accessory Protein in Fission Yeast, Is Required for Localization of Rho3 GTPase
Figure 7
Sip1 links Rho3 to AP-1 complex.
(A) Binding assay involving Apm1 and Rho3 in wild-type, or sip1-i4 cells harboring the control vector or Sip1ΔN. GST pull-down experiments were performed using Apm1-GST expressed in wild-type (wt) and sip1-i4 mutant (sip1-i4) cells, which were transformed with the pDB248 multi-copy vector or the vector containing sip1ΔN expressed under the control of the nmt1 promoter. Cells that expressed GFP alone or GFP-Rho3 were harvested, and their lysates were incubated with purified full-length Apm1 fused to GST. Proteins bound to glutathione beads were analyzed by SDS-PAGE and visualized by autoradiography. Right panel: Quantitation of GFP-Rho3 beads protein levels by densitometry of the expressed bands against that of the lysate protein levels in wild-type cells, sip1-i4 cells or sip1-i4 cells with Sip1ΔN expression as shown in A. Data from at least three independent experiments are expressed as means ± standard deviations. (B) Subcellular localization of GFP-Rho3 in wild-type cells, sip1-i4 cells or sip1-i4 cells with Sip1ΔN expression. GFP-Rho3 expressed in wild-type (wt) and sip1-i4 mutant (sip1-i4) cells, which were transformed with the pDB248 multi-copy vector or the vector containing sip1ΔN expressed under the control of the nmt1 promoter. Cells were cultured in YPD medium at 27°C, following which they were incubated with FM4-64 dye for 5 min at 27°C to visualize the Golgi/endosomes. FM4-64 fluorescence was examined using a fluorescence microscope. Arrowheads indicate the dot-like structures of GFP-Rho3 and the Golgi/endosomes stained with FM4-64, double arrowheads indicate cytoplasmic accumulation of GFP-Rho3, and arrows indicate the concentrated fluorescence at the cell division site. Bar, 10 µm. (C) Percentage of cells in which Rho3 were localized at the cell division site in wild-type (wt) and sip1-i4 cells, which were transformed with the pDB248 multi-copy vector or the vector containing sip1ΔN expressed under the control of the nmt1 promoter. (D) Quantitative analysis for the number of Rho3 dots co-localizing with FM4-64/cells in wt and sip1-i4 cells, which were transformed with the pDB248 multi-copy vector or the vector containing sip1ΔN expressed under the control of the nmt1 promoter. Data are the means ± standard deviations of 3 independent experiments with 150 cells in B.