Prof. Dr. Karin Schumacher
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.