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

Lead Research Organisation: University of Glasgow
Department Name: College of Medical, Veterinary &Life Sci

Abstract

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.

Technical Summary

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.

Planned Impact

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.

Publications

10 25 50
 
Description This project centres on findings that a protein previously thought to be involved in an assembly for vesicle traffic to the plasma membrane also appears to have a role in the disassembly process as well. The findings are important, because the protein in question is recognised to play key roles across all eukaryotic organisms, including humans. The outcomes of the work therefore could well redefine the way we look at the processes of secretion and its regulation in a wide variety of functions and physiopathologies.

We can now place the sequence of traffic assemblies into context with the regulation of solute uptake in plants. This context is important, because the assembly can be shown to affect both processes through the mutual interactions.
Exploitation Route The findings are primarily of fundamental importance to understanding how plants regulate growth and coordinate solute uptake with vesicle traffic. In the longer term there may be opportunities to use the knowledge in enhancing biomass generation.
Sectors Agriculture, Food and Drink,Healthcare,Pharmaceuticals and Medical Biotechnology,Other

 
Title 2in1 vector systems 
Description Synthetic biology vectors for transient and stable transformation with quantitative visual reporting on cell-by-cell basis 
Type Of Material Technology assay or reagent 
Year Produced 2009 
Provided To Others? Yes  
Impact Multiple publications from my own research group and over 100 research groups worldwide Vector system distributions to more than 500 research groups worldwide 
URL http://psrg.org.uk
 
Title EZ-Rhizo 
Description Computer software tool for quantitative measurement and analysis of root growth/development 
Type Of Material Physiological assessment or outcome measure 
Year Produced 2010 
Provided To Others? Yes  
Impact Multiple publications from my own research group and research groups worldwide Online distribution has been accessed through the laboratory website with site views at a rate of >500 per month 
URL http://psrg.org.uk
 
Title Henry 
Description Software for electrophysiology and imaging data aquisition and analysis 
Type Of Material Technology assay or reagent 
Provided To Others? Yes  
Impact Multiple publications from my own research group and research groups worldwide Online distribution has been accessed through the laboratory website with site views at a rate of >500 per month 
URL http://psrg.org.uk
 
Title Multicistronic vector systems 
Description Synthetic biology vector systems for transient and stable transformation for expressing multiple, tagged proteins and for quantitative analysis of membrane traffic and transport 
Type Of Material Technology assay or reagent 
Year Produced 2010 
Provided To Others? Yes  
Impact Multiple publications from my own research group and over 100 research groups worldwide Vector system distributions to more than 500 research groups worldwide 
URL http://psrg.org.uk
 
Title OnGuard 
Description Systems biology software for quantitative modelling of cellular transport and homeostasis 
Type Of Material Physiological assessment or outcome measure 
Year Produced 2012 
Provided To Others? Yes  
Impact Multiple publications from my own research group and research groups worldwide Online distribution has been accessed through the laboratory website with site views at a rate of >500 per month 
URL http://psrg.org.uk
 
Title SUS vector systems 
Description Synthetic biological vector systems for protein-protein interaction screening 
Type Of Material Technology assay or reagent 
Year Produced 2010 
Provided To Others? Yes  
Impact Multiple publications from my own research group and over 100 research groups worldwide Vector system distributions to more than 500 research groups worldwide 
URL http://psrg.org.uk
 
Title Software tools for electrophysiology and imaging 
Description The laboratory continues to develop and refine software/hardware tools for data acquisition and analysis relevant to electrophysiology, single-cell imaging and analysis. These activities are long-standing and open-ended, and develop in line with the current research activities and needs of the laboratory. All software and related packages are made freely available to the research community through the laboratory website at psrg.org.uk 
Type Of Material Technology assay or reagent 
Provided To Others? Yes  
Impact The various software tools and packages have furthered the research activities of the laboratory since the 1990s and continue to provide key support and drivers for advancing much of current research. These tools and packages are disseminated, on average, to over 100 laboratories per year. 
URL http://psrg.org.uk
 
Title EZ-Rhizo 
Description Software for quantitative trait analysis and acquisition for root growth/development 
Type Of Material Database/Collection of data 
Year Produced 2010 
Provided To Others? Yes  
Impact Multiple publications from my own research group and research groups worldwide Online distribution has been accessed through the laboratory website with site views at a rate of >500 per month 
URL http://psrg.org.uk
 
Title Henry 
Description Software package for electrophysiology and imaging data acquisition and analysis 
Type Of Material Data handling & control 
Provided To Others? Yes  
Impact Multiple publications from my own research group and research groups worldwide Online distribution has been accessed through the laboratory website with site views at a rate of >500 per month 
URL http://psrg.org.uk
 
Title OnGuard 
Description Quantitative systems biology modelling of cellular transport and homeostasis 
Type Of Material Computer model/algorithm 
Year Produced 2012 
Provided To Others? Yes  
Impact Multiple publications from my own research group and research groups worldwide Online distribution has been accessed through the laboratory website with site views at a rate of >500 per month 
URL http://psrg.org.uk
 
Title SDM-assist 
Description Software for molecular primer design that enables introduction of silent markers for molecular cloning 
Type Of Material Data analysis technique 
Year Produced 2013 
Provided To Others? Yes  
Impact Multiple publications from my own research group and research groups worldwide Online distribution has been accessed through the laboratory website with site views at a rate of >500 per month 
URL http://psrg.org.uk
 
Description PBL 
Organisation Plant Bioscience Limited Technology
Country United Kingdom 
Sector Private 
PI Contribution IPR on ABA receptor technology and ABA signalling
Collaborator Contribution Funding related to IPR on ABA receptor technology and ABA signalling
Impact Multiple outcomes in publications and industrial contacts
 
Description PSG 
Organisation POSCO - South Korea
Country Korea, Republic of 
Sector Private 
PI Contribution Base support for meetings and exchange of materials
Collaborator Contribution Base support for meetings and exchange of materials
Impact Base support for meetings and exchange of materials
 
Title Software tools and packages for electrophysiology and imaging 
Description The laboratory continues to develop and refine software/hardware tools for data acquisition and analysis relevant to electrophysiology, single-cell imaging and analysis. These activities are long-standing and open-ended, and develop in line with the current research activities and needs of the laboratory. All software and related packages are made freely available to the research community through the laboratory website at psrg.org.uk 
Type Of Technology Software 
Impact The various software tools and packages have furthered the research activities of the laboratory since the 1990s and continue to provide key support and drivers for advancing much of current research. These tools and packages are disseminated, on average, to over 100 laboratories per year. 
URL http://psrg.org.uk
 
Description International online services 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact Prof. Blatt and members of his laboratory have contributed to various media events over the years, including online interview contributions (e.g. People behind the Science, a US-based media program)
Year(s) Of Engagement Activity Pre-2006,2006,2008,2011,2015,2016,2017,2018
 
Description Invited presentations 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact I regularly speak to audiences, from small groups (5-20) to large audiences (>1000) in a variety of settings. In addition to teaching and extramural activities associated with the university, I also speak on invitation to national and international groups a number of times each year and in a variety of settings, academic as well as public. I also reach audiences through short video presentations mounted on the web, these primarily via my laboratory website and the ASPB websites. Anyone reading this entry is welcome to visit these sites to learn more.

The impacts arising from my presentations are varied. For example, a common consequence of my speaking in academic settings is to attract potential researchers to visit my laboratory and, frequently, to interest potential collaborators and students/postdocs to my research group. At scientific meetings, my talks often attract interest also from researchers interested in the various tools and materials that my research has produced, including the various vector systems and software packages that I
Year(s) Of Engagement Activity Pre-2006,2006,2007,2008,2009,2010,2011,2012,2013,2014,2015,2016,2017,2018
URL http://psrg.org.uk
 
Description Schools and displays 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? Yes
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact As these were multiple events, this question is not informative or useful. Participants varied from numbers in the tens to several thousands

Extensive training of participating laboratory members as well as broad scope reach to schools and communities, in the case of the GCC science days to the west of Scotland and in the case of the IFPD activities to audiences within and outside the UK
Year(s) Of Engagement Activity 2010,2011,2012,2013,2014,2015,2016,2017,2018
URL http://psrg.org.uk
 
Description Teaching Tools 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? Yes
Geographic Reach International
Primary Audience Schools
Results and Impact The PI has supported the editor in developing these tools since their inception in 2009 and has contributed to recent tools relating to membranes and transport education

The Tool received an international award in 2010 for excellence in education and has an acknowledged takeup worldwide in over 3000 institutions
Year(s) Of Engagement Activity 2009,2010,2011,2012,2013,2014,2015,2016,2017,2018
URL http://psrg.org.uk
 
Description Teaching Tools 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? Yes
Geographic Reach International
Primary Audience Schools
Results and Impact The PI has supported the editor in developing these tools since their inception in 2009 and has contributed to recent tools relating to membranes and transport education

The Tool received an international award in 2010 for excellence in education and has an acknowledged takeup worldwide in over 3000 institutions
Year(s) Of Engagement Activity 2009,2010,2011,2012,2013,2014,2015,2016,2017,2018
URL http://psrg.org.uk