The cAMP pathway is important for controlling the morphological switch to the pathogenic yeast form of -2

Abstract

<p><b>Copyright information:</b></p><p>Taken from "The cAMP pathway is important for controlling the morphological switch to the pathogenic yeast form of "</p><p></p><p>Molecular Microbiology 2007;65(3):761-779.</p><p>Published online 01 Aug 2007</p><p>PMCID:PMC2064555.</p><p>© 2007 The Authors Journal compilation © 2007 Blackwell Publishing Ltd</p>nd prey vectors were simultaneously transformed into yeast strain AH109 and plated out on SD/–Leu/–Trp for 3 days. Yeast colonies that grew on SD/–Leu/Trp were restreaked on SD/–Ade/–His/–Leu/–Trp and incubated for a further 3 days. The results growths indicate that pGAD- and pGAD- could, but pGAD- and pGAD- could not, directly interact with pGBK-. In a series of negative controls, pGBK- could not interact with pGAD-, pGADT7-T, pGAD- and pGAD-. B. Pull-down assays to demonstrate that Gpa1 and Gpb1 both interact with Gpb1. GST and GST-Cyr were purified from bacteria, loaded onto glutathione sepharose beads before incubation with translated S-Gpa1 and 10 mM nucleotide. After washing the beads, the proteins were eluted by the addition of 4× NuPAGE LDS sample buffer, followed by boiling at 90°C for 5 min, and separated on a 4–12% NuPAGE gel under denaturing conditions. Bound Gpa1 was detected as a gel band by autoradiography. Lanes 2, 4 and 6 establish that Gpa1 binds to immobilized Cyr1, but there was little difference in apparent affinity after incubation with GTP (lane 2), no nucleotide (lane 4) or GDP (lane 6). Negative controls, using immobilized GST, are shown in lanes 3, 5, 7, 10 and 13. A control using immobilized GST-Gpb1 to pull-down S-Gpa1, in the presence of 10 mM GDP, shows a more intense band, suggesting that Gpa1-GDP is bound with higher affinity to Gpb1 than to Cyr. Using translated S-Cyr (lane 11), an interaction with immobilized GST-Gpb1 (lane 12) was detected