Investigating the Role of the Ribosome in Membrane Protein Biogenesis

Living organisms are made up of cells. Cells require barriers or membranes to physically separate themselves from the environment and hence maintain their integrity. Internally, cells contain compartments which are again bounded by membranes. Such internal compartments within the cells allow processes which would otherwise interfere with each other to occur simultaneously. They also allow the generation of energy which powers the rest of the cell. Although the presence of membranes acting as barriers is essential, if the membrane is completely impermeable the cell cannot survive as molecules such as nutrients cannot enter the cell and likewise waste products can not be expelled. To overcome this problem, membranes contain large molecules called proteins, which allow the movement of specific molecules from one side of the membrane to the other. They allow a cell to tightly control what enters and leaves the cell. Membrane proteins can also act as signal relays, sensing molecules or processes occurring outside the cell and then triggering events inside the cell in response. The importance of these membrane proteins is highlighted in many diseases where a single membrane protein is defective and can no longer permit movement of a specific molecule. A good example would be cystic fibrosis where a protein that allows movement of chloride ions fails to function leading to the disease. Having seen that membrane proteins play such important functions and that when they are absent or fail to work properly the consequences for the cell are severe. It is therefore important to understand how such protein are made in the cell, particularly as some diseases including cystic fibrosis are caused when the membrane protein is not made correctly. Proteins are made by large complex machines termed ribosomes. Not only does the ribosome make the protein but it can also interact with the machinery that correctly places, or inserts, the membrane protein into the membrane. The two machineries are organised such that while the protein is still being made it starts to be inserted in to the membrane. Ribosomes make a vast array of proteins yet only around a third of them are membrane proteins. A key question of this project is to understand how the ribosome knows that it is making a membrane protein and therefore that it needs to be placed in the membrane. As new proteins are made they move from a place deep inside the ribosome through a narrow tunnel to the surface of the ribosome, where they can be released to the rest of the cell. Parts of this tunnel inside the ribosome can 'sense' when a membrane protein is being made and so the machinery that inserts the membrane proteins can be alerted that a membrane protein is on the way. This project aims to understand how the ribosome manages to do this, which bits of the ribosome are needed and what happens if it doesn't work properly. By understanding how this processes occurs normally it will help us to understand what can go wrong and how this can cause disease. The sensing of the new protein inside the tunnel within the ribosome may also be important for other types of proteins that need different machineries to help them be made correctly and so the project will also investigate what other processes in the cell fail to happen when the ribosome tunnel no longer works properly. Overall the project will help us to understand more clearly the detailed workings of the ribosome, the machine which synthesizes all proteins in the cell, and how it can respond to different types of proteins as they are being made to ensure they are then further processed correctly.

The ribosome is a fascinatingly complex machine that synthesises proteins. However, it is clear that it has functions beyond translation. In particular the fate of newly synthesised polypeptide chains is influenced by recruitment of factors to the ribosome in response to features within specific nascent chains. Nascent chains moves through an aqueous tunnel(exit tunnel) from the site of peptide bond formation to the ribosome surface (exit site). Tunnel wall components are implicated in recognising features of nascent chains, triggering recruitment of factors to the exit site, which then process the nascent chains as they emerge from the tunnel. A paradigm for this mode of action is provided by secretory and membrane proteins. Targeting factors are recruited to the ribosome and deliver the ribosome and nascent chain to the endoplasmic reticulum (ER). Secretory and membrane proteins are targeted to the same translocation channel (translocon) at the ER membrane. Importantly, the presence of a Trans-Membrane domain (TM) inside the exit tunnel of the ribosome leads to reprogramming of the translocon permitting subsequent insertion of the TM into the bilayer. Ribosomal protein Rpl17 forms a restriction inside the exit tunnel and contacts the translocon near the exit site. Furthermore, TM regions interact intimately with Rpl17 and triggering recruitment of additional factors to the translocon, suggesting Rp17 is a signal-relay. We will dissect the function of Rpl17 in vivo, to test its role in membrane protein integration and elucidate its mode of action. We will employ a multifaceted approach using yeast as a model organism. We will combine targeted and unbiased approaches to generate mutants of rpl17 with membrane protein biogenesis defects and employ in vitro assays to dissect the molecular basis of their phenotypes. Our genetic approach will also allow us to ask if the exit tunnel and in particular, Rpl17 functions in processes beyond membrane protein biogenesis.

Wherever possible we will attempt to maximize the impact of the work generated from grant to the non-acadamic sector by: Engaging the public via the media to place our basic research findings in an understandable context Seek to consider commercialisation opportunities of IP arising from the work especially in the areas of ribosome and novel antibiotics and the over-expression of membrane proteins in yeast. Exploiting existing public engagement programme to host 6th form students in lab for summer placements, which would be supervised by the post-doctoral fellow. Presenting at international conferences to the yeast community attended by a broad audience.