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Cambridge Institute for Medical Research

 

Structural biology of transport vesicle and organelle biogenesis

General audience summary:
Protein cargo is moved to the correct locations within cells in special transport vehicles or 'vesicles'. The formation of these transport vesicles, and packaging of proteins inside them, is a carefully orchestrated process. We want to understand how cargo is selected for transport, including the general signals that specify transport of many cargo proteins with widely varying function as well as those specifying selection of highly specialised cargo such as SNARE proteins (which ultimately allow the transport vesicles to deliver their general cargoes to the correct destination). We aim to understand how this works through detailed study of protein structure, followed by analysis of how different mutations affect the transport and function of proteins in test tube assays and in cells. As membrane traffic goes awry in both neurological and immune disease, this work has important implications for our understanding of both normal development and disease.

Strategic CIMR themes:Membrane Trafficking, Organelle Biology 

Funding: Wellcome Trust

Research Group members: Luther Davis (joint with Prof JP Luzio), Sally Gray (joint with Prof JP Luzio), Veronica Kane Dickson (joint with Prof JP Luzio), Jonathan Kaufman, Natalya Leneva, Bernard Kelly, Nathan Zaccai

 

Research

Structural biology of transport vesicle and organelle biogenesis

Uncovering the structural and molecular basis of membrane traffic along the endocytic pathway.

Transmembrane proteins are moved between organelles in transport vesicles, a process that is essential for eukaryotic cells. Defects in vesicle trafficking pathways lead to a wide range of pathophysiological effects. We take a combined structural and functional analyses approach to dissecting the mechanisms that control this process.

Cargo is sorted into the curved membrane of a forming vesicle and once membrane deformation is completed, the vesicle buds from the donor membrane. Subsequently it is transported to and then fuses with its target membrane. The protein coats that surround a transport vesicle possess self-assembly, membrane deformation and cargo recognition functions. Cargo selection is mediated by the direct binding of coat components to determinants in the cytosolic portions of transmembrane cargo. In endocytic clathrin-coated vesicles (CCVs), the most commonly used recognition motif determinants are YxxΦ and ExxxLL, which are recognised by the AP2 clathrin adaptor. Membrane deformation is achieved through a combination of the insertion of helices into the membrane bilayer and molecular crowding. In endocytic CCVs, the clathrin adaptor CALM plays a central role in this process along with AP2 and membrane-sculpting BAR-domain-containing proteins.

SNAREs are membrane-embedded proteins, which provide specificity and energy to transport vesicle-organelle fusion events. Appropriate SNAREs must be actively sorted into transport vesicles to allow the vesicles to fuse with their desired target organelle and also to return SNAREs that are required for subsequent vesicle transport events to their correct location. These recognition events, which occur in parallel with standard cargo selection, are mainly mediated by direct and highly specific recognition of the folded regions of SNAREs by vesicle coat components. In collaboration with other groups in CIMR and elsewhere, we investigate the structures and functions of proteins that control transport vesicle–organelle and organelle–organelle fusion through regulating SNARE-mediated membrane fusion activity, SNARE localization and membrane tethering events. One example of such a study is our work on the SNARE VAMP7 and its binding partner the retromer-coat-associated protein VARP.

Publications

Key publications: 

Kovtun O, Kane Dickson V, Kelly BT*, Owen DJ* and Briggs JAG*. Architecture of the AP2/clathrin coat on the membranes of clathrin-coated vesicles. Science Advances 6 (30), eaba8381 (2020)
*Co-corresponding authors

Wrobel AG, Kadlecova Z*, Kamenicky J, Yang JC, Herrmann T, Kelly BT, McCoy AJ, Evans PR, Martin S, Müller S, Salomon S, Sroubek F, Neuhaus D, Höning S*, Owen DJ*. Temporal Ordering in Endocytic Clathrin-Coated Vesicle Formation via AP2 Phosphorylation. Dev. Cell 19;50(4):494-508.e11 (2019)
*Co-corresponding authors

Kovtun O, Leneva N, Bykov YS, Ariotti N, Teasdale RD, Schaffer M, Engel BD, Owen DJ*, Briggs JAG*, Collins BM*. Structure of the membrane-assembled retromer coat determined by cryo-electron tomography. Nature 561(7724):561-564 (2018)
*Co-corresponding authors

Ma L, Umasankar PK, Wrobel AG, Lymar A, McCoy AJ, Holkar SS, Jha A, Pradhan-Sundd T, Watkins SC, Owen DJ & Traub LM. Transient Fcho1/2⋅Eps15/R⋅AP-2 Nanoclusters Prime the AP-2 Clathrin Adaptor for Cargo Binding. Dev. Cell 37(5):428-43 (2016).

Miller SE, Mathiasen S, Bright NA, Pierre F, Kelly BT, Kladt N, Schauss A, Merrifield CJ, Stamou D, Höning S & Owen DJ.  CALM regulates clathrin-coated vesicle size and maturation by directly sensing and driving membrane curvature. Dev. Cell 3, 163–175 (2015).

Kelly BT, Graham SC, Liska N, Dannhauser PN, Höning S, Ungewickell EJ & Owen DJ. AP2 controls clathrin polymerization with a membrane-activated switch. Science 345, 459–463 (2014).

Hesketh GG, Pérez-Dorado I, Jackson LP, Wartosch L, Schäfer IB, Gray SR, McCoy AJ, Zeldin OB, Garman EF, Harbour ME, Evans PR, Seaman MN, Luzio JP & Owen DJ. VARP is recruited on to endosomes by direct interaction with retromer, where together they function in export to the cell surface. Dev. Cell 29, 591–606 (2014).

Jackson LP, Kelly BT, McCoy AJ, Gaffry T, James LC, Collins BM, Höning S, Evans PR & Owen DJ. A large-scale conformational change couples membrane recruitment to cargo binding in the AP2 clathrin adaptor complex. Cell 141, 1220–1229 (2010).

Professor of Structural and Molecular Biology
Wellcome Trust Principal Research Fellow

Contact Details

djo30@cam.ac.uk
01223 762 643
Not available for consultancy

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