Refuelling the cell with energy

3D CGI image of mitochondria. Created by Andrew Parkes

Image above: Inside the human cell, showing the Golgi apparatus, the post office of the cell, (top left), the nucleus, where the DNA is stored (top right), the endoplasmic reticulum, where proteins are made (bottom left), and the mitochondrion (central), which can form large dynamic networks. 3D CGI image created by Andrew Parkes.

A group of scientists at the MRC Mitochondrial Biology Unit in Cambridge, led by Dr Edmund Kunji (Fellow in the Natural Sciences), have discovered how a key membrane protein, called the mitochondrial ADP/ATP carrier, transports adenosine triphosphate (ATP), the chemical fuel of the cell. This process is vital to keep us alive, every second of our lives, for all of our lives. This work will help us to understand how mutations can affect the function of these transport proteins, resulting in a range of neuromuscular, metabolic and developmental diseases.

We humans consist of 37 trillion cells, such as brain cells, nerve cells, skin cells, and muscle cells. Each cell has to generate its own cellular fuel, called ATP, to drive all of the energy-requiring processes. Nerve impulses, muscle contractions, DNA replication and protein synthesis are just some examples of the essential cellular processes that depend upon a supply of ATP. Most cells have special compartments, called mitochondria, where the vast majority of ATP is made. In fact, every day, we need our own body weight in ATP to fuel all of our cellular activities. Since we only have about 50 grams of ATP in our body, we need to remake it continually from the spent fuel ADP (adenosine diphosphate) and phosphate using an enzyme complex, called ATP synthase, which is located in mitochondria. In this way, every molecule of ATP is recycled roughly 1,300 times a day. In other words, each ATP molecule is used and remade every minute. For ADP to reach the enzyme complex, and for the product ATP to refuel the cell, both molecules have to cross a lipid membrane that surrounds the mitochondria. Since neither molecule can freely cross it, they require a protein to facilitate their transport. The mitochondrial ADP/ATP carrier is the membrane protein responsible for taking ADP in and ATP out of mitochondria.

The carrier cycles between two states; the cytoplasmic-open state in which a central binding site is accessible for binding of ADP, and the matrix-open state in which the binding site is accessible for binding of newly synthesized ATP.

A key question has been how the protein is able to convert between these two states, changing its shape to transport ADP and ATP specifically, without letting other small molecules or ions leak across the membrane, which would be very detrimental to the health of the cell.

The paper The molecular mechanism of transport by the mitochondrial ADP/ATP carrier, published in the journal Cell, describes the structure of the carrier trapped in the matrix-open state. To trap this highly dynamic protein in this state, a compound called bongkrekic acid was used. This compound was first isolated by Dutch physicians in Indonesia in the 1930s, who were investigating the deaths of people who had eaten fermented coconut cookies (tempe bongkrek), inadvertently colonized by a bacterium called Burkholderia gladioli variant cocovenenans. This pathological bacterium produces large amounts of bongkrekic acid, a lethal toxin more potent than cyanide, which binds to the ADP/ATP carrier and traps the protein in the matrix-open state, stopping it from working.

The researchers also produced nanobodies, fragments of llama antibodies, which were selected to bind specifically to the matrix-open state. The combination of bongkrekic acid and nanobody allowed the researchers to prepare enough of the protein to crystallise it and to determine its atomic structure. The structure shows that the protein is shaped like a basket, with a central cavity open ready to accept ATP from the inside of the mitochondrion. Bongkrekic acid is bound to the site where ATP is supposed to bind, meaning that the carrier can no longer function, explaining the toxic effect. Combined with earlier structures of the cytoplasmic-open state, this discovery shows that the carrier is incredibly dynamic, as it uses six moving parts to transport ADP or ATP across the membrane in a unique and carefully orchestrated way. The carrier works like a lock in a canal, as it has two gates and a central ADP/ATP binding site. These gates lie on either side of the membrane, and in each state, one gate is open and the other is closed. The binding site recognises ADP or ATP specifically, ensuring that only these molecules are allowed across the membrane.

The ADP/ATP carrier is just one member of a large family of related transport proteins that bring different food components in and out of mitochondria, and based on this discovery, the scientists believe that this mechanism is likely to work in a similar way for the whole transporter family. There are many diseases associated with their dysfunction and the structure provides significant insight into how disease mutations affect their molecular function.

This work was carried out by Jonathan Ruprecht and Edmund Kunji in the Mitochondrial Carrier research group at the MRC Mitochondrial Biology Unit, in collaboration with the group of Jan Steyaert at the Vrije Universiteit in Brussels, who produced the nanobodies. The work used experimental facilities at Diamond Light Source (Harwell, UK) and at the European Synchrotron Radiation Facility (Grenoble, France).

This long-term research project was funded by the Medical Research Council, UK (MRC), Instruct-ERIC (ESFRI), the Research Foundation-Flanders (FWO) and the Strategic Research Program (SRP) of the Vrije Universiteit of Brussels.