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David Owen

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.

 

Owen lab

Key papers:

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).

Kent, H.M., Evans, P.R., Schäfer, I.B., Gray, S.R., Sanderson, C.M., Luzio, J.P., Peden, A.A. and Owen, D.J. Structural basis of the intracellular sorting of the SNARE VAMP7 by the AP3 adaptor complex. Dev Cell 22, 979–988 (2012).

Jackson, L.P., Lewis, M., Kent, H.M., Edeling, M.A., Evans, P.R., Duden, R. and Owen, D.J. Molecular basis for recognition of dilysine trafficking motifs by COPI. Dev Cell 23, 1255–1262 (2012).

Miller, S.E., Sahlender, D.A., Graham, S.C., Höning, S., Robinson, M.S., Peden, A.A. and Owen, D.J. The molecular basis for the endocytosis of small R-SNAREs by the clathrin adaptor CALM. Cell 147, 1118–1131 (2011).

Jackson, L. P., Kelly, B. T., McCoy, A. J., Gaffry, T., James, L. C., Collins, B. M., Höning, S., Evans, P. R. and Owen, D. J. A large-scale conformational change couples membrane recruitment to cargo binding in the AP2 clathrin adaptor complex. Cell 141, 1220–1229 (2010).

Collins, B. M., McCoy, A. J., Kent, H. M., Evans, P. R. and Owen, D. J.Molecular Architecture and Functional Model of the Endocytic AP2 Complex. Cell 109, 523–535 (2002).

Professor David Owen

Wellcome Trust Principal Research Fellow

Professor of Structural and Molecular Biology

Department: Clinical Biochemistry

contact: djo30@cam.ac.uk

01223 762 643

 

Plain English

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.

 

Group members

Bernard Kelly · Lena Wartosch · Nathan Zaccai · Antoni Wrobel

Funding

Wellcome Trust