Prof. Dr. Karin Schumacher
The central vacuole is important for the storage of ions and metabolites and plays an important role during detoxification. Surprisingly, a double mutant lacking the two tonoplast-localized isoforms VHA-a2 and VHA-a3 is viable, indicating that activity of the V-PPase is sufficient for survival. However, the double mutant is growth retarded and shows severe changes ion and metabolite profiles. Vacuolar nitrate accumulation is reduced whereas nitrate assimilation is strongly increased providing a testable model fort he day-length dependent growth retardation. Moreover, the mutant shows symptoms of Ca2+-deficiency and increased sensitivity to Zn2+ that can both be explained by reduced vacuolar accumulation. Surprisingly, although vacuolar Na+-accumulation is supposed to be proton-dependent, salt sensitivity and accumulation are not affected in the vha-a2 vha-a3 mutant. In contrast, vha-a1 RNAi lines have increased salt sensitivity pointing to an important contribution of endosomal transporters for plant salt tolerance. Vacuolar Ca2+-transport contributes to the spatial and temporal characteristics of so-called [Ca2+]cyt-signatures and we have therefore established transgenic lines expressing Yellow CAMeleon 3.6 that allow high-resolution in vivo imaging of calcium dynamics. Furthermore, we investigate, how, in turn, the decoding network of Ca2+-dependent protein kinases regulates activity of the V-ATPase.
V-ATPase and vesicle trafficking
In animal cells, endocytic and secretory protein sorting take place in two distinct compartments, the trans-Golgi network (TGN) and the early endosome (EE). In contrast, we and others have demonstrated recently that in plants the TGN or a subdomain of it meets the criteria for an early endosome, indicating that biosynthetic and endocytic trafficking converge in this compartment. Furthermore, we provided evidence that acidification of the TGN/EE by the activity of a V-type H+-ATPase is required for both endocytic and secretory trafficking. In addition to many pH-dependent trafficking processes e.g. the dissociation of receptor-ligand complexes that take place in the lumen of endomembrane compartments, it has also been shown that membrane recruitment of cytosolic proteins required for vesicle formation can be dependent on luminal acidification. In mammalian cells, subunit a2 of the V-ATPase has been found to directly interact with an ARF and a ARF-GEF in a pH-dependent manner thus acting as a transmembrane pH-sensor for endosomal protein-trafficking. Our aim thus is to gain insight into the complex sorting processes taking place in the plant TGN/EE and to determine if the V-ATPase is directly involved in vesicle formation and protein trafficking.