Regulation of membrane fusion by a novel Sec1/Munc18-associated protein

SNARE proteins are central components of a well-defined mechanism for the delivery of vesicles carrying membrane and soluble cargo between compartments within eukaryotic cells. Vesicle traffic contributes to neurotransmitter release in nerves, to cell wall delivery and budding in yeast, and is essential for cellular homeostasis, growth and development in plants. Cognate SNARE proteins localise to vesicle and target membranes, and assembly of functional SNARE complexes is sufficient to drive membrane fusion. SNAREs also bind other protein partners, specialised to align vesicle fusion within certain physiological roles. SNARE interactions with ion channels in animals appear to facilitate electrical signalling and neuroendocrine secretion, and we have shown that interaction between the Arabidopsis plasma membrane SNARE SYP121 and the ion channel subunit KC1 affects transport of the osmotically-active K+ ion. An obvious potential corollary of this is that binding between SNAREs and ion channels is important in regulating vesicle fusion. Despite the fundamental nature of the process, we have little understanding of the molecular mechanisms that couple cellular volume with osmotic solute transport in plants or other walled eukaryotic cells. Indeed, how walled cells regulate transport of osmotically-active solutes (especially of K+) in parallel with cell volume - whether reversible as in guard cells or during irreversible expansive growth - remains a matter of considerable debate. This proposal builds on significant recent findings we have made following on our identification of the SYP121-KC1 protein complex and its role in K+ channel regulation: (1) we have identified the site on the SNARE protein SYP121 that binds the channel subunit KC1, and (2) we have demonstrated that KC1 competes with the Sec1/Munc18 (SM) protein KEULE for binding to SYP121. SM proteins are key regulators of membrane vesicle fusion in all eukaryotes, but their precise role in controlling SNARE-mediated membrane fusion remains unclear and a topic of intense research. Our findings point to a link between SM protein function and a SNARE-channel interaction. Not only do they offer the first evidence of a mechanism that directly couples osmotic solute transport with vesicle traffic to control cell expansion in walled eukaryotes, but they also support a new model for SM-regulated membrane traffic. Our working hypothesis is that the SNARE-K+ channel interaction of SYP121 and KC1 serves as a 'molecular governor', analogous to the mechanical invention James Watt employed in moderating the turnover rate of his steam engines, to coordinate vesicle traffic (and cell expansion) with uptake of the osmotically-active K+ ion. We now propose to test various aspects of this hypothesis. We aim to fully characterize the binding of SYP121 to KC1 and KEULE in order to build up a molecular map of the interactions. We also propose to examine the consequences of selectively disrupting the interactions between SYP121, KC1 and KEULE on SNARE complex assembly, membrane traffic and channel-mediated K+ transport. Our multidisciplinary approach will not only further our understanding of the link between osmotic solute transport and control of cell volume, but is also likely to provide a novel paradigm for linking membrane traffic with other physiological processes.

This proposal builds on our discovery of a novel SNARE protein complex that is essential for K+ channel regulation in Arabidopsis. We have recently identified a unique SNARE binding site for the channel subunit, KC1, on the Qa-SNARE SYP121 and shown that it competes with the Sec1/Munc18 (SM) protein KEULE. SM proteins are key regulators of membrane vesicle fusion in all eukaryotes, and our findings point to a link, previously unrecognized, between the SNARE-channel interaction and SM protein function. As the first substantive evidence of a direct connection between osmotic solute transport and vesicle traffic, our findings point to an entirely new model for the regulation of vesicle traffic during cell expansion in walled eukaryotes. Our working hypothesis is that SNARE-K+ channel interaction serves as a molecular 'governor' to coordinate channel-mediated uptake of the osmotically-active K+ ion with cell expansion. The goals of this project are to fully understand the molecular basis for the interactions between SYP121, KC1 and KEULE. Understanding the dynamic relationship between these three proteins will enable us to investigate how their interactions impinge upon SM protein function and feed into cell turgor and volume control.

This proposal is for fundamental research developing new concepts at the core of ideas emerging within the international cell biology community. The research should stimulate thinking about these topics and help facilitate a paradigm shift in approach. These studies will also extend recent development by MRB of a novel split-ubiquitin 'bridge' (SUB) assay for high-throughput, multicomponent molecular interaction analysis. Thus, the research is expected to benefit fundamental researchers as well as industry through conceptual developments as well as the introduction of new technologies for the analysis of multicomponent systems. The research will feed into higher education training programmes through research training at the postgraduate and postdoctoral levels. Finally it will help guide future efforts in applications to agricultural/industrial systems. MRB has established links with industrial/technology transfer partners (Agrisera, Dualsystems, Plant Bioscience) and research institutes (SCRI and JIC) to take advantage of these developments. Further details of these, and additional impacts will be found in Part 1 of the Case for Support and in the attached Impact Plan.