Prof. Karin SchumacherCell Biology
Due to their sessile lifestyle, plants need to efficiently adapt their metabolic and developmental program to changing and often unfavourable environmental conditions. Adaptation often involves considerable fluctuations of metabolite and ion concentrations between tissues, cells and organelles that are mediated either by membrane transport or vesicular trafficking. Our lab has shown that in the model plant Arabidopis the V-ATPase, a highly conserved eukaryotic proton-pump, does not only fuel secondary active transport processes but also plays an important role in the regulation of endocytic and secretory trafficking.
Current research aims at understanding the regulatory networks that control V-ATPase activity as well as the identification of the biological interactions that depend on the activity of this complex proton-pump. Using Arabidopsis as our main model system, we employ genetics and cell biology as well as biochemistry and physiology to provide knowledge that could eventually lead to novel strategies for improved crop yield under stress conditions.
Regulation of V-ATPase activity
V-ATPases are multisubunit protein machines dedicated to proton-transport across eukaryotic membranes. How the two subcomplexes V1 and Vo are assembled from the individual subunits is largely unclear, but dedicated assembly factors for Vo have been identified in yeast. We have identified functional Arabidopsis orthologs of the ER-export chaperone VMA21p, the ER-resident VMA12p and its cytosolic interaction partner VMA22p. Characterization of these assembly actors has not only provided insight into the assembly mechanism but has also revealed that V-ATPase assembly is coupled to a novel form of functional ER quality control.
Reversible assembly of the two subcomplexes V1 and VO would provide a rapid and efficient way to adjust V-ATPase activity to changes in metabolite and environmental conditions. The presence of free V1 complexes in the cytosol indicates that this mechanism is operational in plants and we have thus established transgenic lines expressing functional fusions between V1- and Vo-subunits and suitable fluorescent proteins that allow to monitor and quantify complex assembly in vivo.
In Arabidopsis, like in all higher eukaryotes, most V-ATPase subunits are encoded by gene families. Using functional genomics tools we have investigated how this potential to form complexes with different enzymatic and regulatory properties is used and have identified redundant as well as tissue- and stress-regulated isoforms. Most importantly, we have shown that the the membrane-integral subunit VHA-a controls subcellular localization with VHA-a1 localized to the trans-Golgi network and VHA-a2 and VHA-a3 at the tonoplast. The differential localization of VHA-a creates the unique opportunity to specifically address the roles of the V-ATPase for vacuolar transport and vesicle trafficking (see below).
Like many other protein complexes the V-ATPase is subject to protein-protein interactions and regulatory modifications. We have identified a number of potential regulators of the V-ATPase and the best characterized so far is a protein-kinase that we have shown to interact with and phosphorylate VHA-C in vitro. Gain- and loss-of function alleles for this kinase have been identified and shown to affect signalling of an important stress hormone. Our current goal is to determine if the V-ATPase is a target of stress-induced hormone signaling.
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.