Figure 7. Sip1-GFP localized at the Golgi/endosome and Sip1 associated with the 4 subunits of the AP-1 complex.

(A) Binding assay involving Sip1 and the 4 subunits of the AP-1 complex. GST pull-down experiments performed with expressing chromosome-borne GST-Sip1 under the control of the nmt1 promoter; cells expressing GFP alone or Apm1-GFP, GFP-Apl2, Apl4-GFP or GFP-Aps1 were collected, and the lysates were incubated with purified full-length Sip1 fused GST protein. GST-tagged Sip1 was precipitated by glutathione beads, washed extensively, subjected to SDS-PAGE, and immunoblotted using anti-GFP or anti-GST antibodies. (B) The colocalization of Sip1-GFP or GFP-Sip1 with FM4-64 in wild-type cells. Wild-type (wt) cells, expressing chromosome-borne Sip1-GFP or expressing chromosome-borne GFP-Sip1 under the control of the nmt1 promoter, were examined by fluorescence microscopy under the repressed conditions. The cells were incubated with FM4-64 fluorescent dye for 5 min at 27°C to visualize the Golgi/endosomes. The fluorescence of the FM4-64 was examined under the fluorescence microscope. Arrowheads indicated the dot-like structures and Golgi/endosomes. Bar, 10 µm. (C) The partial colocalization of Sip1-GFP or GFP-Sip1 with Apm1-mCherry in wild-type cells. Wild-type cells expressing chromosome-borne Sip1-GFP or expressing chromosome-borne GFP-Sip1 under the control of the nmt1 promoter, were transformed with pREP1-Apm1-mCherry. The cells were cultured in EMM medium at 27°C and examined by fluorescence microscopy. Arrowheads indicated the colocalization of Sip1-GFP or GFP-Sip1 with Apm1-mCherry at Golgi/endosomes. Bar, 10 µm.