This project offers the opportunity to elucidate the structure and molecular interactions of a novel tetraspanin complex involved in autophagy. The tetraspanin superfamily includes 33 human proteins, and mediates the development of nervous and immune systems, infectious disease, fertilization and cell adhesion and motility. Their mechanisms remain opaque despite their importance as potential therapeutic targets for malaria, hepatitis C virus and HIV infection and progression of several tumour types, necessitating further structure-function analysis. The tetraspanin complexes involve four human proteins. The tetraspanin proteins link directly to a cytoplasmic adaptor protein called syntenin-1 which in turn interact with other cellular proteins (e.g. Alix). The syntenin-1 facilitates multiple interactions within tetraspanin-containing complexes thus serving as a critical molecular hub which regulates various cellular processes involving the endocytic vesicular trafficking (e.g. cell proliferation, migration). These pathways rely on ubiquitin-binding cellular proteins, which interact with syntenin-1 in a unique manner involving both C- and N-termini of the protein. However, our understanding of the mechanisms that control assembly of the tetraspanin-syntenin-1 complexes remains in its infancy, with no detailed structural information available which would allow us to intervene with their function in tetraspanin-driven pathological conditions. The project involves dissecting the role of syntenin-1 phosphorylation in regulation interactions involving tetraspanins and other cellular proteins, and, ultimately, illustrating the significance of these interactions in the trafficking of transmembrane proteins. We have already established that syntenin-1 is phosphorylated by Src and Ulk1 kinases and that the phosphorylation of syntenin-1 plays an important role in regulation of the assembly of the tetraspanin-syntenin-ubiquitin complex. These kinases also control the assembly of a new class of tetraspanin complexes involving several proteins implicated in autophagy. These novel observations identify tetraspanins and syntenin-1 as new components within network of autophagy pathways. Thus, the proposed project give an excellent opportunity to examine the structural-functional relationship between the newly identified molecular complexes and their phosphorylation-controlled dynamics and autophagy, one of the key physiological processes. Detailed structural characterisation of the tetraspanin-based molecular complexes will involved small-angle scattering of X-rays (SAXS), X-ray crystallography and nuclear magnetic resonance (NMR). Complementary to this, a mass spectrometry based approach will be employed to identify cellular proteins that bind both phosphorylated and non-phosphorylated forms of syntenin-1. We predict that phosphorylation of syntenin-1 will affect the ability of the protein to recruit ubiquitylated cellular partners (see above). The role of ubiquitination in the functional assembly of the tetraspanin-syntenin-1 complexes in protein trafficking and methods to disrupt this key interaction will be explored using confocal microscopy and life cell imaging. Purifying native transmembrane components remains a bottleneck for biological research, as all current methods rely on the use of detergents which dissociate protein and lipid complexes. We have developed a pioneering approach to purify functionally active complexes, which involves using polymer-based nanoparticles for extraction of transmembrane proteins together with their natural membrane environment. This preserves stability of the complexes and allows to perform functional studies. This new approach in conjunction with mass spectrometry will be utilised to comprehensively characterise protein-protein and protein-lipid interactions in the tetraspanin-syntenin-1 complex, and thereby provide an important insight into cellular processes that regulate its activity.