Neuronal exocytosis is catalyzed by the SNARE protein syntaxin-1A1. Syntaxin-1A is clustered in the plasma membrane at sites where synaptic vesicles undergo exocytosis2,3. However, how syntaxin-1A is sequestered is unknown. Here, we show that syntaxin clustering is mediated by electrostatic interactions with the strongly anionic lipid phosphatidylinositol-4,5-bisphosphate (PIP2). We found with super-resolution STED microscopy on the plasma membrane of PC12 cells that PIP2 is the dominant inner-leaflet lipid in ~73 nm-sized microdomains. This high accumulation of PIP2 was required for syntaxin-1A sequestering, as destruction of PIP2 by the phosphatase synaptojanin-1 reduced syntaxin-1A clustering. Furthermore, co-reconstitution of PIP2 and the C-terminal part of syntaxin-1A in artificial giant unilamellar vesicles resulted in segregation of PIP2 and syntaxin-1A into distinct domains even when cholesterol was absent. Our results demonstrate that electrostatic protein-lipid interactions can result in the formation of microdomains independent of cholesterol or lipid phases.
A 2-year Free Air CO 2 Enrichment (FACE) experiment was conducted with winter wheat. It was investigated whether elevated atmospheric CO 2 concentration (e[CO 2 ]) inhibit nitrate assimilation and whether better growth and nitrogen acquisition under e[CO 2 ] can be achieved with an ammonium-based fertilization as it was observed in hydroponic culture with wheat. Under e[CO 2 ] a decrease in nitrate assimilation has been discussed as the cause for observed declines in protein concentration in C 3 cereals. Wheat was grown under ambient [CO 2 ] and e[CO 2 ] (600 ppm) with three levels (deficiency, optimal, and excessive) of nitrate-based fertilization (calcium ammonium nitrate; CAN) or with optimal ammonium-based fertilization. Ammonium fertilization was applied via injection of an ammonium solution into the soil in the 1st year and by surface application of urea combined with nitrification inhibitors (UNI) in the 2nd year.Results showed that ammonium-based fertilization was successfully achieved in the 2nd year with respect to nitrification control, as soil ammonium concentration was considerably higher over the growing season for UNI fertilized plots compared to optimal CAN plots. Also, stem nitrate concentration, flag leaf nitrate reductase activity, and transcript levels were lower in UNI fertilized plants compared to optimal CAN. Regarding the e[CO 2 ] effect on nitrate reductase activity and transcript levels, no alteration could be observed for any nitrogen fertilizer treatment.
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