Visualising membrane transport
Cryo electron microscopy is capable of mapping the conformational space of molecular machines under turnover conditions. I will present new mechanistic insight in glutamate transport, revealed by cryo electron microscopy. Glutamate transporters of the SLC1 family are secondary active transporters that harness cation gradients across the plasma membrane as the driving force to pump the neurotransmitter L-glutamate from the synaptic cleft into neuronal and glial cells. The transporters are homotrimeric and operate via a so-called elevator mechanism, where the transport domain of each monomer can move trough the membrane to carry substrates from one side of the membrane to the other. Using cryo electron microscopy we have visualized the prokaryotic glutamate transporter homologue GltTk embedded into lipid nanodiscs. By titrating in substrate, we observed dramatic changes in the conformational ensemble, corresponding to turnover. Our data reveal symmetrical and asymmetric states of the elevator domains in the homotrimeric protein, and indicate that the transport domain of each subunit moves as independent entity. The structures also show pronounced membrane deformations in some of the asymmetric states, suggesting that lipid bilayer properties may influence transport kinetics. Together with classical transport assays and single molecule FRET measurements using proteoliposomes, a comprehensive picture of the mechanism of transport is emerging.
Biochemistry department University of Groningen, The Netherlands