Role of the Sec1p/Munc18 (SM) protein Vps45p in SNARE complex assembly and bilayer fusion

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

Abstract

All eukaryotic cells (ranging from yeast to humans) contain numerous compartments, each surrounded by a lipid membrane. This compartmentalisation provides the basis for intracellular specialisation. For example, in order to dispose of unwanted components cells have developed degradative enzymes; it is essential that these are sequestered away from other cellular components to avoid destruction of valuable molecules. In addition, the cell has developed a complex assembly line of modifications that are added to proteins in a specific order as they travel to their final destination within the cell. This necessitates the accurate passage of molecules between compartments: a process known as membrane trafficking. Small portions of membrane bud off from one compartment to form transport vesicles that carry molecules to another compartment. Fusion of the two membranes results in the delivery of the contents of the vesicle to the target compartment. It is essential that these transport vesicles fuse with the appropriate target membrane. This specificity is controlled by the formation of specific SNARE complexes between proteins in the donor (vesicle) and target membranes. SNARE complex formation also provides the energy required for the fusion of the two lipid bilayers. This proposal is directed towards understanding how the formation of these SNARE complexes is regulated, concentrating on the role of a family of proteins known as SM proteins. I hypothesise that SM proteins are carried by the v(esicle)-SNARE on the transport vesicle to the target membrane where they activate the t(arget)-SNARE, ready to form SNARE complexes and drive membrane fusion. The aim of this study is to test this hypothesis and elucidate the molecular mechanism(s) by which SM proteins control SNARE-mediated membrane fusion.

Technical Summary

This work builds on a previous study by my lab, which demonstrated that, in addition to facilitating the entry of its cognate Syntaxin Tlg2p into functional SNARE complexes, the Sec1p/Munc18 (SM) protein, Vps45p also binds to the v-SNARE found in those complexes, Snc2p. This led us to propose the hypothesis that we aim to test in this proposal, namely that Vps45p is carried by Snc2p to coordinate activation of Tlg2p on the target membrane with the arrival of the transport vesicle. This work will further our understanding of the molecular mechanisms utilised by SM proteins to control SNARE complex assembly and membrane fusion. We will pursue two distinct, but complementary aims. Aim 1: To understand the role of Vps45p in SNARE complex assembly and bilayer fusion. We propose to use two separate assays to investigate the role of Vps45p in SNARE complex assembly, one looking at the kinetics of SNARE complex assembly and the other measuring the ability of SNARE complexes formed in vitro to drive bilayer fusion. We will use both of these assays to directly test our hypothesis that binding of Vps45p to Snc2p facilitates SNARE complex assembly. This aim will give insight into the molecular mechanisms utilised by Vps45p to control SNARE complex assembly and/or membrane fusion. Aim 2: To characterise the binding between Vps45p and Snc2p in vitro and in vivo. To understand the significance of the interaction between the SM protein Vps45p and the v-SNARE Snc2p, we will take advantage of the two-hybrid interaction that we have demonstrated to isolate mutant versions of Vps45p that are unable to bind Snc2p. We will then examine the effects of perturbing the SM/v-SNARE interaction on membrane trafficking in vivo by assessing the ability of these mutant versions of Vps45p to complement for the loss of wild-type Vps45p. We will also use the assays described in aim 1 to determine whether the binding of Vps45p to Snc2p is required for SNARE complex assembly and bilayer

Publications

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Bryant NJ (2011) SNARE proteins underpin insulin-regulated GLUT4 traffic. in Traffic (Copenhagen, Denmark)

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Furgason M (2009) The N-terminal peptide of the syntaxin Tlg2p modulates binding of its closed conformation to Vps45p in Proceedings of the National Academy of Sciences

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Kioumourtzoglou D (2018) Proximity Ligation Assay to Study the GLUT4 Membrane Trafficking Machinery. in Methods in molecular biology (Clifton, N.J.)

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Sadler JB (2015) mVps45 knockdown selectively modulates VAMP expression in 3T3-L1 adipocytes. in Communicative & integrative biology

 
Description 1. Identification of residues critical for each of the 3 binding modes that Vps45 uses to regulate its cognate SNARE proteins paved the way for us to dissect the function of these different binding modes. This was achieved using a combination of in silico (molecular modelling), in vitro (biochemistry and biophysics) and in vivo (genetics and cell biology) approaches.

2. Demonstration of evolutionary mechanisms of Sec1/Munc18 (SM) protein function in SNARE complex assembly. This was achieved by extrapolating findings from our studies with Vps45, Tlg2 and Snc2 from the yeast Saccharomyces cerevisiae, to two other (mammalian) systems; the orthologous mVps45/Syntaxin16 and the homologous Munc18c/Syntaxin4. This approach proved extremely productive to facilitate formulation of unifying hypotheses for mechanism of action of SM proteins.
Exploitation Route Our demonstration of evolutionarily conserved mechanisms of Sec1/Munc18 (SM) protein function in SNARE complex assembly will facilitate formulation of unifying hypotheses for mechanism of action of SM proteins.
Sectors Healthcare

 
Description Findings from this work contributed to our understanding of how SM proteins function to regulated SNARE complex assembly. My lab has subsequently been investigating how the SM protein Munc18c regulates insulin-regulated SNARE complex assembly in adipocytes. This process is defective in the disease states of insulin-resistance and Type2-diabetes and underscores the relevance of basic molecular sciences in societal battles against disease states. I have spoken about this at several outreach events including to people living with diabetes (organised in conjunction with Diabetes UK).
First Year Of Impact 2014