Investigating the role of molecular chaperones in the posttranslational targeting of tail-anchored proteins

Protein targeting is an essential component of cellular organisation, directing the specific localisation of biochemical processes and the maintenance of cell structures. The efficiency and specificity of targeting processes is critical, with defects resulting in disease states or impaired growth. For example, localisation of the prion protein is a key factor in the lethal brain diseases BSE and CJD. Knowledge of targeting mechanisms can also be exploited for biotechnological purposes. For example, plants can be genetically engineered to possess enzymes involved in the synthesis of plastic precursors, yet efficient accumulation of these precursors can only be achieved when the complete set of enzymes in the biosynthetic pathway is targeted to the chloroplast. My proposal uses a specialised class of proteins as a tool to understand fundamental issues of protein targeting. The majority of proteins are synthesised by ribosomes in the cytosol and then targeted to a specific compartment within the cell. The information specifying the destination is contained within the amino acid sequence of the protein, which is generally decoded by targeting factors present in the cytosol, and subsequently recognised at the target compartment. I am studying the targeting of a class of proteins termed tail-anchored (TA) proteins, in which a hydrophobic tail sequence tethers the protein to a specific membrane. TA proteins are found in many different intracellular membranes, where they play important roles in diverse cell processes, including biochemical pathways, protein targeting and the control of cell death. A key characteristic of TA proteins is that they are inserted into membranes after they have been fully synthesised and released from the ribosome i.e. posttranslational targeting. Whilst my previous studies have shown considerable overlap between molecular components involved in this posttranslational targeting pathway and the well-characterised cotranslational pathway, it is apparent that TA proteins use these components differently and use additional pathways. I have shown that the targeting of TA proteins is promoted by chaperones, which are best known for their roles in protein folding and preventing protein aggregation, but have more recently been shown to promote the posttranslational targeting of other classes of protein. It is possible that chaperones passively promote posttranslational protein targeting by maintaining signal sequences in a recognisable conformation. However, the involvement of specific chaperones with different targeting pathways indicates that chaperones may contribute to the actual specificity of targeting. I am hypothesising that chaperones provide specificity to the targeting process by recognising features of the tail-anchor, and form unique signatures through distinct combinations of chaperones. This raises a further question of how a chaperone complex could specify a targeting route. One mechanism could be recognition by a receptor at the target membrane, a notion that is supported by recent studies demonstrating the binding of chaperones to membrane receptors at intracellular membranes. I am hypothesising that chaperone interactions with their receptors generates protein targeting specificity. These studies will exploit recent advances in genome information to guide experimental approaches. Key issues will be addressed at a detailed molecular level through the use of purified components, an approach that is enabled by the nature of TA proteins and by techniques that I have recently developed. The findings will be placed in a cellular context by the genetic manipulation of plants cells. The outcome of this study will be to enhance our understanding of protein targeting as a fundamental cellular process. Ultimately, this will help to tackle diseases linked to protein mislocalisation, and create opportunities to engineer biochemical pathways for the production of valuable compounds.

Cellular organisation is dependent on accurate and efficient protein targeting. Most targeting pathways begin after precursor synthesis, and are dependent on cytosolic molecular chaperones. Current models suggest that chaperones simply prevent precursor aggregation, but there is growing evidence supporting the notion that they also determine the specificity of targeting. Molecular chaperones can adapt to perform a variety of specialised functions by combining with different cochaperones, suggesting a mechanism by which targeting specificity may be generated from complexes of generic chaperones and cochaperones. Furthermore, membrane translocases at the outer membranes of mitochondria and chloroplasts have been found to include chaperone receptors, which are capable of discriminating between different chaperones. I will test the hypothesis that chaperones bind signal sequences and form specific complexes, which are then recognised by membrane receptors at the destination organelle. The experimental system will exploit tail-anchored (TA) proteins, which are targeted to different organelles, despite possessing broadly similar targeting sequences. TA proteins are anchored to membranes by a C-terminal hydrophobic domain that doubles as the targeting sequence. Crucially, this tail-anchor permits targeting to be manipulated in tightly defined cell free assays using purified chaperones, enabling rigorous analysis and functional testing of chaperone-mediated mechanisms. Multiprotein complexes will be analysed by crosslinking, pulldown assays, and mass spectrometry. The role of membrane receptors will be investigated by selective knockout and reconstitution. Overall, this study will apply recent advances in our understanding of chaperones and their functional complexes to assess their role in selective posttranslational protein targeting. This will impact on our understanding of posttranslational targeting, TA proteins, and chaperone function.