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

Lead Research Organisation: Sheffield Hallam University
Department Name: Faculty of Health and Wellbeing

Abstract

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.

Technical Summary

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.
 
Description Bioinformatics analysis has been completed using the position of the tail-anchor as the main criterion for the search. This has resulted in over 500 candidates in Arabidopsis; a substantial proportion can be assigned with reasonable confidence to a specific organelle, and crucially include many examples with evidence for chloroplast and mitochondrial localisation. Plasmids containing the cDNA for potential TA proteins have been obtained from ABRC and recloned in suitable expression plasmids or joined to a promoter by overlap extension PCR. These constructs have been successfully used for cell free targeting assays and expression in tobacco as YFP-tagged fusions. The outcome is the identification of 29 TA proteins located in chloroplasts, some of which were completely uncharacterised prior to this investigation.



Targeting assays to chloroplasts have been successfully developed, with rigorous optimisation of preparation conditions from pea leaves. Mitochondria have been prepared from maize coleoptiles, and have been successfully combined with chloroplasts to perform competitive targeting assays. Proteins with known localisation have served as controls to validate these assays. This work was supported by visits to Dr Caroline Bowsher (University of Manchester). ER membranes have also been included in these assays to permit competition between any combination of these organelles.



We collaborated with Anne-Marie Harrison (Sheffield Hallam University) to analyse the sequence information in TA proteins and correlate this to subcellular localisation where data exists. Results were used to write a program for the analysis of TA proteins with unknown localisation, and therefore predict their localisation. The reliability of this tool was tested on the training data set and found to perform with 55-80% reliability, and also successfully predicted the localisation of OEP61 to chloroplasts. A key prediction is that Arabidopsis plastids possess 138 TA proteins, most of which are uncharacterised.



Bacterial expression has been optimised to produce plant chaperones in the Hsp70 and Hsp90 (Hsp81) families, and also to produce Toc64 and a range of variants of OEP61. These chaperones are functional in binding assays using polyHis tag pulldown. We have also confirmed these interactions and measured the kinetics of interaction using ellipsometry, demonstrating that OEP61 exhibits differing affinity towards different Hsp70 isoforms. Furthermore, these chaperones have been shown to stimulate protein targeting in cell free assays.



TA protein precursors known to localise chloroplasts have been shown to interact with chloroplast chaperone receptors, and this is dependent on ATP levels, so is most likely to involve an indirect interaction via the chaperone. The interaction between receptor and chaperone can be shown to be mediated by the C-terminus of the chaperone and the TPR clamp domain of the receptor, thereby confirming a true regulatory interaction. The exact nature of the interaction between the receptor and the precursor needs to be determined, and may also involve direct contacts. We can also show that the non-membrane portion of OEP61 is able to inhibit the targeting of precursors to the chloroplast.



Proteolysis of organelles has been performed and analysed using competitive targeting assays. The results show that receptors do play a role in the selectivity of protein targeting. In addition, we have also found that the presence of targeting factors is not necessary for the accurate targeting of most precursors, and therefore targeting specificity appears to be controlled primarily by processes at the membrane surface.
Exploitation Route Ellipsometry analysis could now be used to for drug screening with membrane protein targets. Ellipsometry analysis has been developed using chloroplasts to enable the measurement of protein-protein interactions at a membrane surface. This has now been successfully applied to mammalian cells and the ability to detect interactions of GPCRs with ligands targeted by pharmaceuticals has been successfully demonstrated (published)
Sectors Agriculture/ Food and Drink,Manufacturing/ including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology

 
Description Invited speaker at international conferences (3) 
Form Of Engagement Activity Scientific meeting (conference/symposium etc.)
Part Of Official Scheme? No
Type Of Presentation Workshop Facilitator
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact April 2013: EMBO Meeting on Protein Translocation, Dubrovnik, Croatia. "Specificity of tail-anchored membrane protein targeting in plants"



July 2012: SEB Main Meeting, Salzburg, Austria. "Subcellular targeting of tail-anchored membrane proteins in plants"



Sept 2008: ENPER conference (European plant membrane research), Lecce, Italy. " The role of molecular chaperones in posttranslational protein targeting"

no actual impacts realised to date
Year(s) Of Engagement Activity 2008
 
Description Invited speaker at international conferences (3) 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate Students
Results and Impact April 2013: EMBO Meeting on Protein Translocation, Dubrovnik, Croatia. "Specificity of tail-anchored membrane protein targeting in plants"






"

no actual impacts realised to date
Year(s) Of Engagement Activity 2013
 
Description Invited speaker at international conferences (3) 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate Students
Results and Impact July 2012: SEB Main Meeting, Salzburg, Austria. "Subcellular targeting of tail-anchored membrane proteins in plants"

no actual impacts realised to date
Year(s) Of Engagement Activity 2012
 
Description Invited speaker at international conferences (3) 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate Students
Results and Impact Sept 2008: ENPER conference (European plant membrane research), Lecce, Italy. " The role of molecular chaperones in posttranslational protein targeting

no actual impacts realised to date
Year(s) Of Engagement Activity 2008