Signaling networks involving heterotrimeric G-proteins and phospholipids are fundamental to many cellular processes in eukaryotes. Oomycetes possess a family of twelve novel proteins called GPCR-PIPKs (GKs) that are composed of a N-terminal G-protein-coupled-receptor (GPCR) domain fused to a phosphatidylinositol phosphate kinase (PIPK) domain. Based on this structure GKs are anticipated to link G-protein and phospholipid signaling but their functions and biochemical activities are unknown. Previously we analyzed the function of PiGK4 in the potato late blight pathogen Phytophthora infestans by gene silencing and overexpression and showed its involved in spore development, sporangial cleavage, hyphal elongation and virulence (Hua, Meijer et al. 2013, Mol. Microbiol.). Microscopic analysis revealed that fluorescently tagged full length and truncated versions of PiGK4 localize to membranes surrounding certain cellular compartments. Since GKs are involved in developmental transitions in Phytophthora and GPCRs are major drug targets, GKs have potential as oomicide targets. To determine the enzymatic activity of PiGK4 we exploit the temperature sensitive yeast mutant mss4ts that lacks PI4P 5-kinase activity when grown at the restrictive temperature. Complementation is achieved with full-length PiGK4 but not with a truncated PiGK4 without GPCR domain. Apparently PiGK4 has the same enzymatic activity as yeast Mss4p but requires the GPCR domain to function, possibly by targeting the PIPK to its substrate which is likely a membrane associated phosphoinositide. Here we present a more detailed analysis of the function of the various conserved domains in PiGK4 by complementation assays in mss4ts using modified versions of PiGK4 generated by deleting and swapping domains.
|Title of host publication||Book of Abstracts 28th Fungal Genetics Conference|
|Publication status||Published - 2015|
|Event||28th Fungal Genetics Conference, Pacific Grove, CA, USA - |
Duration: 17 Mar 2015 → 22 Mar 2015
|Conference||28th Fungal Genetics Conference, Pacific Grove, CA, USA|
|Period||17/03/15 → 22/03/15|
van den Hoogen, D. J., Hua, C., Meijer, H. J. G., & Govers, F. (2015). Phytophthora infestans GPCR-PIPK GK4: a membrane localized receptor with PI4P5-kinase activity. In Book of Abstracts 28th Fungal Genetics Conference (pp. 125)