Protein-protein interactions in the early stages of chlorophyll biosynthesis

Photosynthesis is the most important process occurring on Earth whereby carbon dioxide and water is converted into carbohydrate using light energy harnessed from the sun by the green pigment chlorophyll. All green plants, algae and several species of bacteria can carry out photosynthesis and it is ultimately the basis of all life on the planet. Photosynthesis is a multi-step process involving many chemical reactions catalysed by specialised proteins known as enzymes and a large number of other proteins that act with chlorophyll as a 'light harvesting complex (LHC)' to absorb light. Chlorophyll, and its chemical precursors, can be highly dangerous when not bound to LHC proteins, causing oxidative damage to the cell. Chlorophyll biosynthesis must therefore be tightly controlled and regulated to prevent such a build-up. The first committed step of chlorophyll biosynthesis is carried out by the multi-subunit enzyme magnesium (Mg) chelatase that is made up of the H, I and D subunits. This is a key enzyme as it lies at the branch point of tetrapyrolle biosynthesis. Chlorophyll and haem are the end products of this pathway; haem is produced by the enzyme ferrochelatase and plays a crucial role in the process of respiration which generates energy for the cells needs. Depending on the cell's requirements the two branches of this pathway need to be balanced; too much haem and/or too little chlorophyll and magnesium chelatase activity will be stimulated, too much chlorophyll and/or too little haem and ferrochelatase activity will be stimulated. One way in which Mg chelatase is stimulated is by the addition of a protein known as GUN4. This protein was originally described in the higher plant Arabidopsis where it is involved in one of the communication pathways from the chloroplast to the nucleus. Signalling through this pathway is mediated by the chlorophyll biosynthetic intermediate magnesium protoporphyrin IX produced by magnesium chelatase. Interestingly, whilst GUN4 acts as an 'accelerator' on Mg chelatase it works as a 'brake' on the next enzyme in the chlorophyll biosynthetic pathway, Mg proto methyltransferase (ChlM). In contrast, the H subunit of Mg chelatase stimulates the latter. These stimulations and inhibitions must be the result of physical interactions between GUN4, the chelatase and the methyltransferase. We will use various techniques to investigate interactions between H, I, D, ChlM and GUN4 using the purified proteins and also directly in the model green algae Synechocystis. Determining how these proteins interact with each other will give us an important insight into how chlorophyll biosynthesis is controlled and regulated. This work is of fundamental importance as it could provide a potential way of improving photosynthetic yield in plants, particularly under stress conditions.

Mg chelatase lies at the branchpoint of the crucial haem and chlorophyll biosynthetic pathways, which provide all of the cofactors for the essential energy-yielding reactions in plants and cyanobacteria. Mg chelatase therefore is a prime candidate for pathway regulation. We will investigate interactions between Mg chelatase subunits and with the Gun4 and ChlM proteins from the beginning of the Chl pathway, both in vitro and in vivo: 1) Isolating complexes in vitro. The Mg chelatase HID complex will be isolated using affinity methods and analysed by mass spectrometry (MS). These techniques will also be used to isolate complexes involving Gun4 and ChlM. 2) Determining subunit stoichiometry. To quantify absolute amounts of 14N proteins in isolated complexes nanocapillary liquid chromatography and MS analysis will be used. The isolated complex will be spiked with known quantities of 15N labeled recombinant proteins. 3) Mapping interaction points. Affinity methods and mutagenesis will be used to identify interacting regions of the H and I subunits that form HID complexes. Isotopically labelled cross-linkers will be used in conjunction with MS for the study of intramolecular and intermolecular interactions in the HID complex. Sites of interaction of Gun4 with the HID complex will be mapped by exploiting the 3D structure of Gun4. 4) Structural studies. Scaling up of the affinity purification should yield enough of the complexes for low resolution structural methods such as negative stain single particle EM, AFM and single molecule spectroscopy. 5) In vivo formation of complexes. Pulldown assays using antibodies to the pathway enzymes will be used to trap tagged protein complexes from Synechocystis extracts. In vivo stoichiometry will be determined by growing cells in 15N so that all associated proteins are 15N-labelled and can be quantified by spiking with known amounts of 14N protein.