Figure 3.

PI4Kβ/rab11 association is regulated by GTP. (A) For quantitative yeast two-hybrid assay, S. cerevisiae Y190 cells were transformed with rab11Q70L, rab11S25N, or rab11wt in the bait vector pGBT9 and with PI4Kβ in pACT2. Yeast cells were grown on selective medium (-Trp, -Leu, -His), and β-galactosidase activity was determined as described in MATERIAL AND METHODS and expressed as Miller units. (B) COS-1 cells were transfected with 5 μg of cDNA encoding Myc-tagged PI4Kβ. After 2 d, cells were lysed and detergent lysates (1 ml), and 20 μl of this lysate was analyzed by Western blotting (Lysate). GST pull-down assays were performed using 5 μg of GST, GST-rab4, GST-rab11, GST-rab11Q70L, or GST-rab11S25N (pull down). Proteins bound to GSH beads were analyzed by Western blotting by using anti-Myc (WB, pull down). As controls, Myc-PI4Kβ present in cell lysates was detected with anti-Myc (WB, lysate), and the GST-fusion proteins were stained with Coomassie Brilliant Blue (CBB). (C) ³⁵S-labeled PI4Kβ was produced by an in vitro transcription translation reaction, and subjected to a GST pull-down assay with 5 μg of GST, GST-rab11Q70L, or GST-rab11S25N. ³⁵S-labeled PI4Kβ was detected by phosphorimaging. (D) ³⁵S-labeled PI4Kβ was produced by an in vitro transcription translation reaction and subjected to a GST pull-down assay with 5 μg of GST, GST-rab4, GST-rab5, or GST-rab11. Preloading was done with either GDP or GTPγS as indicated. Bound ³⁵S-labeled PI4Kβ was detected by phosphorimaging.