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

Lead Research Organisation: University of Glasgow
Department Name: School of Life Sciences

Abstract

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.

Technical Summary

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.

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 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.

Publications

10 25 50
 
Description This award has led to a number of findings, most important relating to how secretion is regulated at the plant plasma membrane. We can now show a difference between two major pathways for secretion to the membrane, a difference that is associated with a unique regulatory protein as well as control by interactions with a subset of ion channels. These findings define the first clear evidence of differential secretory control at a single membrane in a plant system. The findings also allow us to differentiate homeostatic and stress-related secretory activities in new ways that may have longer-term implications for understanding plant growth.
Exploitation Route possible implications for manipulation of secretion and plant cell growth in the longer term
Sectors Agriculture, Food and Drink,Education,Environment,Other

URL http://psrg.org.uk
 
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,
 
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,
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
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
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
URL http://psrg.org.uk