A new function for the Arabidopsis thaliana SNARE SYP121

Eukaryotic cells maintain a compartmental cellular organization of membrane-enclosed organelles that communicate with each other through the exchange of trafficking vesicles. Members of a superfamily of membrane proteins, the so-called SNAREs, are essential for the necessary fusion of vesicle membranes to the membrane of target organelles. SNAREs are needed to overcome the energy barrier that prevents spontaneous membrane fusion events. A number of studies from the past decade indicated that SNARE proteins might fulfill a function beyond merging membranes. The mammalian plasma membrane SNARE Syntaxin1A was shown to directly interact with and through this interaction modify the activity of, for example, a calcium ion channel and a potassium ion channel. In its classical function as SNARE protein, Syntaxin1A mediates specialized vesicle fusion events such as synaptic transmission in neurons or secretion of insulin from pancreatic cells. These specialized vesicle fusion events require precise timing that is controlled by intracellular signaling events. These intracellular signaling events involve the coordinated action of members from different families of ion channels. Current models suggest that the dual functions of a SNARE protein in ion channel regulation and membrane fusion serve to fine-tune highly regulated vesicle fusion events. This thesis provides evidence for the first direct interaction between a SNARE protein and an ion channel from plants and suggests a function for this interaction in Arabidopsis potassium nutrition. Three different protein-protein interaction assays for full-length membrane proteins that comprised a yeast mating based split-ubiquitin assay, co-immunoprecipitation after expression in insect cells and bi-molecular fluorescence complementation after transient Arabidopsis root transformation, confirm that the Arabidopsis plasma membrane SNARE SYP121 interacts in vitro and in vivo with the Shaker ion channel subunit KC1. Furthermore, the interaction between KC1 and SYP121 is specific over the closest homologue of Syp121, namely SYP122. Shaker channels are plasma membrane proteins with four subunits that transport the essential macronutrient potassium in response to changes in membrane voltage. The KC1 subunit is unique among the Shaker channels. It can only act as a regulatory subunit that modifies channel properties when forming heterotetramers with other Shaker subunits such as AKT1, not as functional homotetramer. AKT1 is expressed predominantly in the root epidermis, i.e. root hairs, where it overlaps with the more broadly expressed KC1 and SYP121. Previous publications showed that a low external potassium concentration combined with high levels of ammonium that is used to block all root potassium uptake systems apart from AKT1, causes akt1 null mutants to display strongly reduced main root length as well as whole plant potassium content compared to wild type plants. It is shown here that the phenotype of both syp121 and kc1 null mutants is identical to the akt1 mutant under these growth conditions. The design of new antibodies against native AKT1 and KC1 and an optimized protocol for root plasma membrane protein enrichment and solubilisation allowed for the first time visualization of native Arabidopsis AKT1 protein. This technical advance made it possible to confirm that both Shaker channel subunits are present in equal amounts in the plasma membrane of roots cells from syp121 mutant and wild type plants. It is concluded that the potassium uptake phenotype of the syp121 mutant is not caused by the absence of channel proteins from the plasma membrane due to a disruption of the vesicle trafficking function of the SNARE SYP121. An alternative function for SYP121 in potassium nutrition that involves direct interaction with AKT1-KC1 heterotetrameric channels is supported by electrophysiological measurements after heterologous expression in Xenopus leavis oocytes. SYP121 modifies the voltage-dependent potassium uptake characteristics of AKT1-KC1 heterotetramers in a way most easily understood in context of a conformational change within the voltage sensing protein parts of the Shaker channel that are caused by the direct interaction with the SNARE protein. It is concluded that the identical potassium uptake phenotype of the akt1, kc1 and syp121 mutants is caused by the inability to form a functional tripartite complexes. As KC1 is able to form heterotetrameric channels with several different Shaker channel subunits, for example KAT1 that is highly expressed in guard cells, it is likely that this novel interaction between KC1 and SYP121 might modulate channel activities in different tripartite complexes to affect various cellular functions.