How are signalling complexes assembled and regulated on cellular membranes

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Many cellular processes including growth, differentiation, and chemotaxis are controlled by the selective recruitment of large multi component protein complexes to lipid membranes. The correct recruitment of specific proteins to individual membrane compartments plays a major role in controlling the identity and function of different cellular organelles. The localisation of membrane binding proteins can be controlled by a number of factors including specific membrane and protein binding partners, as well as by more general features of the membrane including surface charge and curvature. Studying the molecular details of how proteins are enlisted, assembled, and regulated at membranes has remained a tremendous challenge due to the inherent difficulties of stabilising and reconstituting these complex protein membrane systems. **Research program objectives*Our goal is to identify the molecular details of membrane recruitment, assembly, and regulation of signalling enzymes using a cutting edge combination of novel proteomics based technology, specifically hydrogen deuterium exchange mass spectrometry, together with careful biophysical and biochemical characterisation. We will specifically target the Ras superfamily of small G proteins, as these proteins are membrane bound molecular switches that are able to selectively recruit a large number of protein effectors to membrane surfaces. This program aims to identify the mechanism of how they recruit and activate downstream effectors, specifically the phosphoinositide 3-kinase family of proteins, along with the mechanism of how the Ras superfamily members themselves are activated through the action of guanine nucleotide exchange factors (GEFs). Critical to this work will be the biochemical reconstitution of these proteins into protein membrane nanodiscs that will provide a stable platform for us to carry out detailed biochemical and structural studies. *In tandem with this approach we also plan to use the protein membrane nanodiscs as a platform to identify novel Ras superfamily binding proteins from cellular lysates using mass spectrometry. The major technical advantage of this system is that we can create nanodiscs with a variety of different lipid compositions, and hence are able to identify novel Ras superfamily binding partners that require the presence of both specific protein and membrane components. **This research is of broad impact as the development and application of novel proteomics based methodology in this proposal will be able to be applied to a wide variety of protein/membrane systems. The Ras supefamily of small G proteins constitute one of the most broadly utilised signalling pathways, yet many questions still remain in how they are regulated on membrane surfaces. Many enzymes that regulate or are regulated by Ras superfamily small GTPases undergo large conformational changes upon shifting from an inactive soluble form to an active membrane bound form. This program allows us to probe the molecular basis of this process, and will have wide ranging impact on the mechanism of how signalling complexes are assembled and regulated on membranes. Determining novel binding partners of small G protein signalling will also play a key role in advancing knowledge of fundamental cellular signalling pathways controlled by these signalling pathways.