Assembly of human ATP synthase

Energy from the food we ingest is broken down by oxidative processes in our bodies. The net effect is to generate potential energy in the form of a voltage across the inner membranes of the mitochondria. The mitochondria are the cellular power-houses that generate the fuel required to provide the energy for biological processes such as muscular action, thought processes and replicating DNA and proteins. The fuel is provided in the form of the molecule adenosine triphosphate, known simply as ATP. Every day, each one of us generates 50-60 kg of ATP in the mitochondria of our cells to sustain our bodily activities. The ATP is produced in the mitochondria by millions of copies of a tiny molecular machine with a rotary action known as ATP synthase. The mitochondria are surrounded by two biological membranes and the ATP synthases are embedded in the inner one, with their synthetic heads pointing towards the inside making them resemble tiny mushrooms. In the membrane embedded region of the ATP synthase, the voltage across the membrane makes a rotor turn at about 100-200 rotations every second. The rotor is attached to a robust stalk which penetrates into the catalytic head, and the rotation of the stalk recombines spent fuel elements to form new ATP which is released into the mitochondria. The head and a static structure are joined by a second protein linkage called the peripheral stalk, which is required to prevent the head and rotor turning together. From the mitochondrion, the ATP is distributed around the cell by transport processes and, after release of energy from ATP, the spent fuel in the form of the molecules adenosine diphosphate (ADP) and inorganic phosphate is brought back to the mitochondria to be recombined into new ATP molecules. The rotary machines are both complex and fragile. They need to be put together when the mitochondria are made, and replaced when they break down. They are made from 29 proteins of 18 different kinds. The instructions for making all but two of these proteins resides in the cellular nucleus, and these proteins are made outside the mitochondrion and then imported to the inside. Here they are assembled into the ATP synthases together with the two other proteins that are made inside the mitochondrion with instructions from a small DNA molecule that resides there. We are studying how these complicated machines are assembled. We have discovered that the proteins are first assembled into specific preformed modules and then joined together to make the complete machine, rather like the simpler components of a car are assembled into the engine, gear-box and other modules before being made into the complete vehicle. The ATP synthase modules correspond to the catalytic head, the membrane part of the rotor and the peripheral stalk. The assembly of the membrane part of the rotor and that catalytic head require other proteins that are not part of the finished machines to help in the assembly process. We are studying the properties of these factors, partly because human mutations in at least two of them leads to disease. Finally, the completed machines pair up, linked together in their membrane domains, with the heads at about 90 degrees to each other, and the pairs associate into long rows and help give the inner membranes of the mitochondria their characteristic invaginated appearance. We want to know more about how this happens.

We will use CRISPR-Cas9 to disrupt nuclear encoded subunits of ATP synthase in human cells, and study the effects on the functioning of the ATP synthase and other components of the mitochondrial respiratory chain in a Seahorse XF analyzer. We will also study the impact on the assembly of the ATP synthase itself, and characterize the subunit compositions of the vestigial partially assembled ATP synthase complexes in mitochondria (or mitoplasts) and in purified vestigial complexes by SDS-PAGE, blue native- and clear native-gel electrophoresis, combined with mass spectrometric analyses of tryptic and chymotryptic digests of stained gel regions and protein bands. In addition, we will study the compositions of vestigial complexes by quantitative mass spectrometry of samples labelled in vivo with heavy isotopes (known as stable isotope labelling in cell culture, or SILAC). This approach allows not only the subunit compositions of vestigial complexes to be studied, but also detects other associated proteins, which can then be tested for their being assembly factors by disrupting their genes by CRISPR-Cas9. Where necessary (for example when there are no protein specific antibodies for purifying remaining vestigial complexes), we will introduce tagged subunits into subunit depleted HEK293 Flp-InTM T-RexTM cells by stably incorporating DNA encoding the tagged constructs into the Flp-In site, and induce controlled expression with doxycycline. We will also reintroduce mutated forms of IF1 into cell lines where the gene for IF1 has been disrupted, and characterize their effects by respiration measurements with a Seahorse instrument and by advanced light microscopy. With access to four confocal microscopes (Zeiss LSM880 and Apotome, Andor Dragonfly spinning disk and Nikon N-SIM instruments) we will be able to quantify changes in mitochondrial morphology e.g. branching or fragmentation, and with the N-SIM instrument visualise significant changes to the cristae ultrastructure.