Life depends on processes of energy conversion, in which energy obtained from sunlight, or from catalysing chemical reactions such as the breakdown of food molecules, is converted into energy in forms that can be used to power the biochemistry of the cell. Some key biological energy conversion processes take place in membranes and involves the transport of electrons from donors to acceptors, powered by either light (photosynthesis) or chemical energy (respiration). These processes require a set of membrane components including protein complexes and smaller molecules that can transport electrons in the membrane. The components involved are now well understood, but their organisation and interactions in the intact membrane are much less understood. In some cases there are alternative possible routes for electron transport. Electron transport routes are regulated by physiological factors, and have strong effects on the physiology of the cell. It seems clear that the pathways of electron transport must depend somehow on the organisation of the electron transport components in the membrane. However, it remains unclear on what length scales electron transport takes place (i.e. how far in the membrane do electrons travel from their initial donor to their final acceptor) and on what length scales complexes might be organised in order to control electron transport pathways. Knowing the answers to these questions could give us powerful new tools for controlling biological energy conversion, allowing the engineering or organisms suitable for more efficient biofuel production, for example. We are using a cyanobacterium (a kind of photosynthetic bacterium) as a model organism. In cyanobacteria both photosynthesis and respiration occur in a complex membrane system inside the cell called the thylakoid membranes. Our initial approach to understanding the control of electron transport routes in cyanobacteria has used techniques in which we generate mutants in which particular electron transport proteins are fused to a fluorescent protein. We can then use a fluorescence microscope to see the distribution of electron transport proteins in intact membranes in living cells. A disadvantage of fluorescence microscopy is that it has relatively low resolution, i.e. we cannot see the organisation of electron transport components at molecular scales. However, the technique has allowed us to observe that the distribution of electron transport complexes is very heterogeneous on the scales that we can observe. Under some conditions particular electron transport components are concentrated into distinct patches in the membrane. We can show that the distribution of complexes into patches, or otherwise, is under physiological control, and we can relate this to the physiological control of electron transport. Our aims in this project are to understand better how the membrane patches are formed, what they contain, how they are organised and what effects they have on electron transport. Initially we will use an extension of our current fluorescent labelling techniques to get a more complete picture of the composition of the patches in living cells. This will be combined with studies to test hypotheses for the ways in which the distribution of complexes in the membrane are under physiological control. We will then use biochemical techniques to isolate the membrane patches and determine their full composition, combined with the use of electron microscopy to get higher-resolution information on the organisation of protein complexes in the membrane. At the end of the study we expect to understand much better how electron transport in the membrane is controlled. We expect our studies on a cyanobacterium to act as an exemplar for studies of biological electron transport at the membrane scale in other organisms, and to provide new ideas for the control of the organisation and function of biological membranes in general.

Technical Abstract:
The thylakoid membranes of cyanobacteria contain both photosynthetic and respiratory electron transport complexes. This allows the possibility of multiple electron transport routes, including routes in which electrons are transferred from respiratory donors to photosynthetic acceptors, and vice versa. The routes taken by electrons are crucial for cellular physiology, since they control both the redox balance of the cell and its main means of energy conversion. The question we wish to address in this proposal is what controls the probability of the different possible electron pathways. The problem in cyanobacteria is a specific example of a more general problem in bioenergetic membranes. Our current results strongly suggest that routes of electron transport in the cyanobacterium Synechococcus 7942 (and in other organisms) are controlled by lateral heterogeneity in the membrane on the sub-micron scale. This heterogeneity is on a large enough scale to be visualisable by GFP-tagging and fluorescence microscopy. In this project we will further define the composition and structure of zones in the Synechococcus membrane in which respiratory complexes are concentrated, initially by further fluorescent tagging and confocal microscopy, then using biochemical approaches and electron microscopy. We will use spectroscopic techniques to determine the effects that membrane heterogeneity has on electron transport pathways. We will investigate the signal transduction mechanisms that regulate the lateral distribution of complexes, in terms of the initial triggering signals and in terms of the downstream factors that lead to re-organisation of the membrane. We will combine this with an investigation of the dynamic behaviour of quinone electron carriers in bioenergetic membranes, in order to get a complete picture of the way in which electron transport pathways in an intact bioenergetic membrane relate to the mobility and distribution of the electron carriers.

Potential Impact:
Please refer to lead application