Analysis of subunit organisation and conformational flexibility in the vacuolar ATPase molecular motor by electron microscopy

The vacuolar ATPase is a large protein complex found embedded in the lipid membranes of virtually all eukaryotic cells where it uses energy from the consumption of ATP to fuel the movement of protons across the otherwise impermeable membrane. Its biological role is therefore as an acid pump, a function which is fundamental to a large variety of processes such as maintaining the correct pH in the kidney, bone metabolism and recycling and transport of proteins within the cell. Despite the fundamental importance of the V-ATPase to biological systems, there remain many outstanding questions about its structure, particularly about the organisation of its many protein components, its operating mechanism and the control of that mechanism. The V-ATPase is essentially made up of two molecular-scale motors linked together- a three-step ATP-driven motor coupled to a second, membrane-bound motor which acts as the proton pump. Energy from ATP is transmitted to the proton pump part as rotational movement- ATP turnover causes rotation of a central 'shaft' protein in the V-ATPase which in turn is attached to a ring of small proteins within the membrane-embedded proton pump part. Rotation of the central shaft therefore also causes rotation of this ring, and its movement past a second fixed protein structure within the membrane is thought to be the key step in proton pumping. The whole process is fast, smooth and highly efficient, with virtually no losses from the energy conversion processes. The ring structure of the proton pump motor may contain as many as 10 or as few as 6 proton carrier units. This mismatch between the two motor parts presents a puzzle: how does the V-ATPase link the consumption of a fixed number of ATPs per cycle (3) to the transport of an apparently variable number of protons per cycle, whilst using the same basic structure and mechanism? A current theory suggests that this would be possible if the three ATP-requiring steps in one part of the ATPase are not synchronised with specific proton pumping steps in the membrane-embedded part, but instead the energy released at each ATP consuming step is 'buffered' by storage in an elastic spring or flexible rod structure. The energy stored in these structures would then drive the proton pump. Several of the protein components of the V-ATPase could be candidates for this structure, but direct observation of flexing or stretching has not been made, and the theory remains unproven. The aim of this project is to use state-of-the-art techniques in electron microscopy to capture images of individual molecules of the V-ATPase, and then to use computational methods to combine these images and produce the first complete, accurate and detailed 3-dimensional representation of this large and complex protein. By attaching antibody molecules that recognise specific polypeptide components of the V-ATPase we will be able to work out the position of each of these components, telling us more about their role within the whole protein complex. Using this approach we will be able to see the individual structures linking the two motor units. Developing this further, we can use inhibitor compounds and rapid freezing to lock the V-ATPase in a particular stage in its active cycle and look for differences in its structure that might suggest stretching or flexing of some component parts. This will allow us to test the validity of current theories about the V-ATPase mechanism.

The vacuolar H+-ATPase (V-ATPase) is a multi-subunit membrane protein complex that plays a central role in regulating pH in virtually all eukaryotic cells. At the plasma membrane it contributes to the regulation of cytoplasmic pH by extruding protons, and within the cell it maintains the acidity of intracellular compartments such as lysosomes and Golgi-derived vesicles. V-ATPase dysfunction correlates with certain disease states and its involvement in bone metabolism and tumour metastasis continue to make it a focus for drug development. Although the V-ATPase shares a core structure and basic rotational mechanism with the better-characterised F1F0-ATPase, many questions remain unanswered regarding the organisation and function of the many subunits that are unique to the V-type pump. Particular questions also relate to how the V-ATPase mechanism accommodates the symmetry mismatch between its ATP hydrolysing and ion translocating domains and about the structural effects of interactions with other proteins that are known to control V-ATPase activity. In this study we aim to acquire images of single molecules of the V-ATPase using cryo-electron microscopy of frozen, unstained specimens and apply image processing techniques to produce the first complete 3-dimensional model of the V-ATPase at a resolution of 10 A or less. This level of resolution will be sufficient to visualise secondary structure elements within the complex. Support from antibody labelling studies in negative stain will allow us to map the positions of individual subunits and to characterise interactions with cytoplasmic enzymes such as aldolase that appear to regulate the V-ATPase. We also aim to compare inactive V-ATPase to enzyme trapped at a specific stage in its rotational cycle and look for structural variations that would be consistent with elastic stretching or flexing of component subunits, providing a test of emerging theories about the mechanisms of energy transfer in the bi-domain ATPases.