Ingo Heilmann
Prof. Ingo Heilmann
Institute of Biochemistry and Biotechnology/Cellular Biochemistry
Kurt-Mothes-Straße 3
06120 Halle (Saale)
phone: +49 (0) 345-55 24840
ingo.heilmann@biochemtech.uni-halle.de
Katharina Gerth
PhD student
The protein-protein interaction network of plant PI4P 5-kinases controlling polar tip growth
The regulatory phospholipid, phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2), exerts various effects on polar tip growth in plant cells. Two types of PI4P 5-kinases can be distinguished that produce PtdIns(4,5)P2 with distinct roles, controlling actin dynamics or apical secretion, respectively. We hypothesize that the two types of PI4P 5-kinases each generate PtdIns(4,5)P2 that is then channelled toward specific downstream targets to enable the exclusive effects.
The channeling of signaling molecules is not well understood. PI4P 5-kinase isoforms contain variable protein regions whose reciprocal swapping between isoforms with divergent function conferred the capabilities to act in the context of controlling actin-dynamics or of apical secretion on the respective other isoforms. We hypothesize further that the different isoforms of PI4P 5-kinases are recruited to functional plasma membrane domains by interacting with specific protein partners via their variable domains.
It is the goal of the proposed work to begin to unravel the molecular mechanisms of differential PI4P 5-kinase membrane-recruitment. Two PI4P 5-kinase isoforms have been selected for this study, that exert clear-cut differential effects on actin-dynamics and secretion, respectively, when overexpressed in tobacco pollen tubes. Protein partners interacting with variable domains of these enzymes will be identified. The identification of interacting proteins of relevant PI4P 5-kinases will be achieved using a membrane-associated yeast-two-hybrid (Y2H) approach. Results will be verified using coimmunoprecipitation and bimoleular fluorescence complementation. Positively identified and verified interactors will be recombinantly expressed in E. coli and tested for binding to phospholipids, including PtdIns(4,5)P2, using lipid overlay-assays and liposome-binding tests. Functional channeling of PtdIns(4,5)P2 according to the protein-protein interactions will be addressed by targeted suppression of the putative PtdIns(4,5)P2-pools.