Figure 5

DdRACK1 interacts with G proteins. (A) Yeast two-hybrid analyses. Y190 strain was co-transformed with DdRACK1 in pACT2, and Gβ, Gγ, Gα2, Gα4, and Gα8 in pAS2, respectively. β-galactosidase activity was assayed for colonies grown on Pre-SD + 3AT agar plates. pACTDdNCAP + pASDdNCAP is positive, pACTDdRACK1 + pAS is negative control. Images were taken between 1 to 6 hours of staining with the exception of Gα4 for which the photograph was taken after 24 hours. (B) (i), Co-precipitation assays to confirm DdRACK1 interaction with the Gβ, Gγ and Gα2 protein subunits. GFP-Gβ, Gγ-YFP bound to GFP-trap beads and Gα2-RFP bound to RFP-trap beads precipitated endogenous DdRACK1 (IP). GFP-trap beads incubated with AX2 lysates were used as control (Ctl). (ii), Gα4-RFP and Gα8-RFP bound to RFP-trap beads co-precipitated endogenous DdRACK1 (IP). GFP and RFP bound to beads served as controls. mAb K3-184-2 detected GFP-tagged proteins, mAb K73-875-7 detected RFP-tagged proteins, polyclonal antibodies detected DdRACK1. (C) GST pulldown experiments to confirm DdRACK1 interaction with Gα subunits 2, 4 and 8, and Gγ, respectively. DdRACK1 was detected with polyclonal antibodies. The Ponceau S stained membrane is shown below to reveal the proteins employed in the pulldown. (D) Growth and development of AX2, gβ null mutants (LW6) and LW6/GFP-DdRACK1 cells on a K. aerogenes lawn. Images taken after 3 days are shown. Scale bar, 0.5 mm. (E) Measurement of plaque diameter to determine size of plaques formed by AX2, LW6 and LW6/GFP-DdRACK1 cells over several days. The bar represents the mean and SD of ten independent experiments (***P < 0.001). (F) Phagocytosis was assayed using the strains from (E) and TRITC-labelled yeast. Approximately 200 cells from each strain were counted. The percentage of cells which had engulfed yeast after 30 min is shown in the graph (***P < 0.001).