Postgraduate research opportunities Proximity Labelling proteins near glucose transporters

A major consequence of insulin action is a dramatic increase in the rate of glucose transport into fat and muscle. This is achieved through regulated trafficking of the facilitative glucose transporter GLUT4 from insulin-sensitive intracellular stores to the plasma membrane (PM). It is well established that that the insulin-regulated delivery of GLUT4 to the PM is dysfunctional in insulin-resistance and Type-2 diabetes. Patients with Type-2 diabetes exhibit impaired insulin-stimulated glucose transport in muscle and fat; many studies have supported the hypothesis that GLUT4 sorting and/or trafficking are impaired in these patients. We know very little about the proteins which interact with GLUT4 to control is distribution and trafficking in cells but hypothesise this involves protein interactions with GLUT4 in a ‘network’.

To study how spatially compartmentalized protein networks assemble into functionally integrated macromolecular complexes, a class of methods termed ‘proximity labelling’ (PL) has seen a surge in growth and utility. PL uses engineered enzymes (based on biotin ligases) fused to proteins of interest (in this case GLUT4) to selectively and covalently tag neighbouring proteins with biotin in living cells. After cell lysis, biotinylated proteins are captured by interaction with streptavidin beads and characterised using Mass Spec approaches (see figure)(Han, Li, and Ting 2018).

We shall use this approach to characterise the GLUT4 ‘interactome’ and test the role of identified proteins in the cell biology of insulin action.

Techniques used

• Cell Culture
• Molecular Biology
• Protein analysis
• Imaging
• Genome Editing/siRNA approaches

Further information

Han, S., J. Li, and A. Y. Ting. 2018. 'Proximity labelling: spatially resolved proteomic mapping for neurobiology', Curr Opin Neurobiol, 50: 17-23.

S.Morris, N.D.Geoghegan, J.B.A.Sadler, A.M. Koester, H.L.Black, M.Laub, L.Miller, L.F.Heffernan, J.C.Simpson, C.C.Mastick, J.Cooper, N.Gadegaard, N. J.Bryant and G.W.Gould. PeerJ (2020) In press. “Characterisation of GLUT4 trafficking in HeLa cells: comparable kinetics and orthologous trafficking mechanisms to 3T3-L1 adipocytes.”

N.J.Bryant and G.W.Gould. “Insulin-stimulated GLUT4 translocation – size isn’t everything!” Current Opinion in Cell Biology (2020). In press.