Coated vesicle adaptors
Proteins are transported between the various organelles of the cell by vesicles, which bud from one membrane and fuse with another. The formation of these vesicles and the selection of the right sort of cargo are dependent on coat proteins. Several types of coated vesicles have been described, the best characterised of which are the clathrin-coated vesicles (CCVs). The coats on CCVs consist primarily of clathrin, adaptor protein (AP) complexes, and 'alternative' adaptors. Our working hypothesis is that for each trafficking pathway, there are a number of different adaptors, each of which is recruited independently onto the appropriate membrane. Once on the membrane, the various adaptors would work together to package different types of cargo into the newly forming vesicle.
We are using several approaches to look for novel adaptors and other components of the trafficking machinery, including proteomic analyses of subcellular fractions, genome-wide siRNA library screening, insertional mutagenesis, and a new method we developed for rapidly inactivating proteins, called 'knocksideways'. Current projects include: establishing the functions of AP-1 and other adaptors in differentiated cells; matching up machinery and cargo proteins; investigating how clathrin and adaptors are hijacked by the HIV-1-encoded protein Nef; determining why mutations in the non-clathrin adaptors AP-4 and AP-5 cause hereditary spastic paraplegia; and exploring the evolution of adaptors.
Hirst, J., J.R. Edgar, T. Esteves, F. Darios, M. Madeo, J. Chang, R.H. Roda, A. Dürr, M. Anheim, C. Gellera, J. Li, S. Züchner, C. Mariotti, G. Stevanin, C. Blackstone, M.C. Kruer, and M.S. Robinson. Loss of AP-5 results in accumulation of aberrant endolysosomes: defining a new type of lysosomal storage disease. Hum. Mol. Genet. 24, 4984-4996 (2015).
Hirst, J., Schlacht, A., Norcott, J. P., Traynor, D., Bloomfield, G., Antrobus, R., Kay, R. R., Dacks, J. B. and Robinson, M. S. Characterization of TSET, and ancient and widespread membrane trafficking complex. eLife 3: e02866 (2014).
Borner, G.H.H., Hein, M.Y., Hirst, J., Edgar, J.R., Mann, M., and Robinson, M.S. Fractionation profiling: a fast and versatile approach for mapping vesicle proteomics and protein-protein interactions. Mol. Biol. Cell. 25: 3178-3194 (2014).
Hirst, J., Borner, G. H. H., Edgar, J., Hein, M. Y., Mann, M., Buchholz, F., Antrobus, R. and Robinson, M. S. Interaction between AP-5 and the hereditary spastic paraplegia proteins SPG11 and SPG15. Mol. Biol. Cell 24, 2558–2569 (2013).
Kozik, P., Hodson, N. A., Sahlender, D. A., Simecek, N., Soromani, C., Wu, J., Collinson, L. M. and Robinson, M. S. A human genome-wide screen for regulators of clathrin-coated vesicle formation reveals an unexpected role for the V-ATPase. Nature Cell Biol. 15, 50–60 (2013).
Borner, G.H.H., Antrobus, R., Hirst, J., Bhumbra, G. S., Kozik, P., Jackson, L. P., Sahlender, D. A. and Robinson, M. S. Multivariate proteomic profiling identifies novel accessory proteins of coated vesicles. J. Cell Biol. 197, 141–160 (2012).
Hirst, J. et al. Distinct and overlapping roles for AP-1 and GGAs revealed by the 'knocksideways' system. Curr. Biol. 22, 1711–1716 (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).
Hirst, J., Francisco, G.C., Sahlender, D.A., Seaman, M.N.J., Dacks, J.B. and Robinson, M. S. The fifth adaptor protein complex. PLoS Biol. 9, e1001170 (2011).
Robinson, M.S.*, Sahlender, D. A. and Foster, S. D. Rapid inactivation of proteins by rapamycin-induced rerouting to mitochondria. Dev. Cell 18, 324–331 (2010).