FIGURE 5:

Regulation of the RIM signaling pathway by casein kinase 1. (A) Constitutive Rim101 processing in casein kinase 1 mutant cells. OVY243 (WT) and OVY143 (yck1∆ yck2^ts) were grown at the indicated pH values and at a semipermissive temperature for yck2^ts (30ºC). Protein extracts were immunoblotted with anti-HA antibody to detect Rim101-HA. (B–F) Effect of phosphomimetic or phosphodeficient mutations in RIM8 on RIM signaling and Rim8 function. (B) Same experiment as in A with OVY244 (RIM8-17ST/A) and OVY245 (RIM8-17ST/D). (C) Y04414 (rim8∆) was transformed with the plasmid pLGn+3xNRE22D carrying a CYC-NRE-lacZ reporter and pFlag-Rim8 (WT), the mutant derivative pFlag-Rim8-17ST/A (17ST/A) or pFlag-Rim8-17ST/D (17ST/D), or the empty vector (∆). When indicated, cells were also cotransformed with pADH1-Rim21CT(L). Values are average β-galactosidase activity for four to seven transformants grown to mid log phase at pH 3 or 7. (D) Top, Y04414 (rim8∆) was transformed with pFlag-Rim8 or the mutant derivative pFlag-Rim8-17ST/A or pFlag-Rim8-17ST/D and grown to mid log phase (final pH 3.5), and protein extracts were immunoblotted with anti-Flag antibody. Bottom, Y00936 (rim101∆) was transformed with pHA-Rim8-17ST/A or pHA-Rim8-17ST/D and grown to mid log phase (final pH 3.5), and protein extracts were immunoblotted with anti-HA antibody. (E) Two-hybrid assay as in Figure 1C between GAD fusions to Rim21 C-tail (Rim21CT) or Vps23 and wild-type LexA-HA-Rim8 or the indicated mutant derivatives. (F) OVY264 (RIM8(17ST/A)-3GFP) and OVY265 (RIM8(17ST/D)-3GFP) were grown to mid log phase in YPD medium (final pH 5.8) and resuspended in SD medium at pH 4.5 or 7. Cells were examined by fluorescence and DIC microscopy. (G) Model for transduction of the pH signal from Rim21 to Rim8 (see the text for a description). Dashed arrows and arrowheads indicate weak interactions and phosphorylation events, respectively.