Dissecting a new and vital checkpoint in SNARE recycling and plant growth

I seek, in this proposal, to address the fundamental question of how recycling of the proteins that drive secretory vesicle traffic is regulated post-fusion. SNARE proteins are central components of a well-defined mechanism for the delivery of vesicles carrying membrane and soluble cargo between compartments within cells and contribute to homeostasis and signaling in all eukaryotes. Cognate (Qa-, Qb-, Qc- and R-)SNARE proteins localize to vesicle and target membranes, and assemble in complex to drive membrane fusion. So-called Sec1/Munc18 (SM) proteins are known to regulate this process. SMs form clothespeg-like structures that 'clamp' and stabilize the SNAREs in complex during vesicle fusion. Post-fusion disassembly of the SNARE complex is essential to recycle the cognate SNARE proteins and maintain vesicle traffic. Disassembly is achieved by the NSF ATPase which binds the SNARE complex with the adaptor protein alpha-SNAP. Logic dictates that SM debinding is prerequisite for SNARE complex disassembly, but an understanding of how this process might be regulated is wholly absent. Indeed, most eukaryotes express only one or two alpha-SNAP and NSF proteins, yet maintain vesicle traffic via a large number of different SNARE-mediated trafficking pathways. Clearly, substantial coordination between trafficking pathways must occur to ensure NSF activity is effectively distributed.

This proposal builds on significant recent findings of my laboratory following our identification of SEC11 as the SM partner of SYP121 and the SNARE complexes it assembles. SYP121 and SYP122 are the two Qa-SNAREs that dominate in vesicle fusion at the plasma membrane of the plant model Arabidopsis. We found that manipulating SEC11 binding to SYP121 via a secondary site, previously thought to tether the SM prior to fusion, blocks vesicle traffic via both SYP121- and SYP122-mediated pathways, even though SEC11 does not interact with SYP122. The two Qa-SNAREs share other cognate (Qb-, Qc- and R-)SNAREs, leading us to observe that SEC11 binding to SYP121 via its secondary site is necessary, post-fusion, to promote SNARE disassembly and recycle these binding partners. In short, we have uncovered a previously unrecognized checkpoint and a new role for an SM protein in SNARE recycling post-fusion.

The findings offer the first opportunity to explore this, entirely novel function of an SM protein. Not only do they provide evidence of a previously unrecognized role for SM-SNARE binding, but they also support a new model for SM regulation of membrane traffic. My working hypothesis is that SEC11 debinding is a key checkpoint and serves as a molecular 'clutch' for disassembly of the SYP121 SNARE complex and its coordination with parallel trafficking pathways at the plasma membrane. I propose now to test various aspects of this hypothesis. I aim to fully characterise the binding of SEC11 with SYP121 and their association, post-fusion, with alpha-SNAP and NSF in disassembly of the SNARE complex. I propose also to examine the consequences of selectively manipulating SEC11-SYP121 interactions on vesicle traffic, cell expansion and growth. The multidisciplinary approach outlined here will further our understanding of SM function, SNARE recycling, and it is likely to provide a novel paradigm for understanding the coordination of vesicle traffic within eukaryotes.

This proposal builds on our findings of a previously unrecognized checkpoint and a role for an SM protein in SNARE recycling post-fusion. My laboratory recently identified in the model plant Arabidopsis a requirement for binding and debinding of the SNARE SYP121 by the SM protein SEC11 to facilitate traffic at the plasma membrane. SM proteins have been known for their role in preventing promiscuous interactions between non-cognate SNAREs and in promoting vesicle fusion by stabilizing SNARE complexes formed by cognate partners to accelerate fusion. Until now, however, any roles in regulating SNARE disassembly post-fusion have gone unrecognized. As the first substantive evidence of the importance of an SM in this process, our findings point to an entirely new paradigm for the regulation of vesicle traffic. The goals of this project are to understand the molecular basis for SM action post-fusion. We will use in vitro and in vivo analysis of protein-protein interactions, including studies designed to fully characterize the critical binding components and their motifs, in vitro methods to quantify binding and debinding kinetics, optobiological and pulse-chase methods to characterize the impacts on exo- and endocytosis in vivo, and stable transformations and complementations to validate the consequences for secretion, cell expansion and growth. The readouts in traffic via the parallel pathways to the plasma membrane are now genetically and functionally separable. Thus, our findings offer a unique opportunity to explore SM mechanics in these events. Furthermore, the proven potential for manipulating cell expansion and plant growth makes the SM-SNARE interactions an important target for agro-industrial applications.

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 developments by MRB in expanding the capacity for protein-protein interactions using split-ubiquitin and fluorescence assays and in optobiological methods for quantitative analysis of membrane traffic. 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 complex systems in vitro and in vivo. The research will feed into higher education 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 (e.g. Agrisera, Plant Bioscience) and research institutes (JHI, NIH 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.