The Gβ protein Gpb1 blocks Tpk2-induced pseudohyphal differentiation.

<p><b>(A)</b> The <i>S</i>. <i>cerevisiae</i> diploid strain XPY5a/α, which had been transformed with <i>PbTPK2</i>^{<i>(1–583)</i>}, was transformed with a construct for the expression of PbGpb1-GFP and the transformants, selected on the basis of their green fluorescence, analysed for pseudohyphal growth in SLAD agar containing 50 μM (upper panel) or 200 μM (lower panel) ammonium sulfate. Single colonies from the agar plate were observed at 20x magnification in an Eclipse E-400 microscope. The scale bar is 50μm. Whilst control cells expressing PbTpk2 were able to form pseudohyphae, those co-transformed with <i>PbGPB1</i> could not. As a control the wild-type strain MLY61a/α was transformed with <i>PbGPB1</i> and shown to still produce pseudohyphae. <b>(B)</b> The <i>S</i>. <i>cerevisiae</i> haploid strain SGY446, which had been transformed with <i>PbTPK2</i>^{<i>(1–583)</i>}, so that it could grow at 37°C, was transformed with a construct for the expression of PbGpb1-GFP and the transformants, selected on the basis of their green fluorescence after growth at 25°C. The SGY446/<i>PbTPK2</i>^{<i>(1–583)</i>}/<i>PbGPB1-GFP</i> transformant was tested for growth at 37°C. In contrast to the control strain SGY446/<i>PbTPK2</i>^{<i>(1–583)</i>}, the SGY446/<i>PbTPK2</i>^{<i>(1–583)</i>}/<i>PbGPB1-GFP</i> transformant could not grow at 37°C. <b>(C)</b> A bar chart showing the relative <i>in vitro</i> kinase activity of <i>P</i>. <i>brasiliensis</i> Tpk2 (PKA) using kempeptide and PbGpb1 as substrates. Note that a slight excess of PbGpb1 causes about a 40% reduction in the kinase activity of PbTpk2 using kempeptide as substrate. <b>(D)</b> A bar chart showing <i>FLO11</i> transcript levels for the indicated transformants. The measured quantity of the <i>FLO11</i> mRNA in each of the treated samples is the relative abundance to the value for <i>actin</i>. The data represent the average of 3 measurements.