Functional association of Loc1 and Puf6 with RNA helicase Dhh1 in translational regulation of Saccharomyces cerevisiae Ste12
Fig 5
Effects of phosphorylation mutations of Dhh1 on Ste12 translation and Dhh1-Puf6 interactions.
(A) Six mutations were introduced at the putative phosphorylation sites in the N-terminus of Dhh1. Mutated amino acids are indicated by gray boxes. (B) Ste12-HA protein levels in cells expressing various phosphorylation-mutant versions of Dhh1, as revealed by Western blot analysis. Protein extracts were prepared from the dhh1 deletion strain (JK400) carrying a DHH1 plasmid (pJI323), vector (pRS316), or mutant plasmid (pJI324-329). GAPDH was detected as a loading control. Graphs represent quantification of Ste12-HA to GAPDH ratio (n = 3 independent replicates). Values are mean ± SD. **p < 0.01. (C) Dhh1 phosphorylation was analyzed using an anti-phosphothreonine antibody. A wild-type BY4741 strain was transformed with DHH1-FLAG (pJI323), vector (pRS316), DHH1-T10A (pJI324), or DHH1-T16A (JI326) plasmids. Cell lysates were immunoprecipitated with anti-FLAG-conjugated agarose and probed with an anti-phosphor-threonine antibody. To detect Dhh1-FLAG, the membrane was re-probed with an anti-FLAG antibody. Graphs represent quantification of phosphorylated Dhh1 to Dhh1-FLAG ratio (n = 3 independent replicates). Values are mean ± SD. *p < 0.05. (D) Dhh1-Puf6 protein interactions, as revealed by co-immunoprecipitation analysis. Wild-type and PUF6-TAP were transformed with DHH1-FLAG (pJI330), DHH1-T16A (pJI331), or DHH1-T16E (pJI332). Cell lysates were immunoprecipitated with anti-FLAG-conjugated agarose in the absence (-) or presence (+) of RNase, and further probed with anti-PAP or anti-FLAG antibodies, respectively. (E) Dhh1-Loc1 protein interactions, as revealed by co-immunoprecipitation analysis. Wild-type and LOC1-TAP were transformed with DHH1-FLAG (pJI330), DHH1-T16A (pJI331), or DHH1-T16E (pJI332). Cell lysates were immunoprecipitated with anti-PAP-conjugated agarose in the absence (-) or presence (+) of RNase, and further probed with anti-PAP or anti-FLAG antibodies, respectively.