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Elucidating the function of the chloroplast protein import apparatus component Tic40

Reference	BB/C006348/1
Principal Investigator / Supervisor	Professor Paul Jarvis
Co-Investigators / Co-Supervisors
Institution	University of Leicester
Department	Biology
Funding type	Research
Value (£)	243,713
Status	Completed
Type	Research Grant
Start date	15/08/2005
End date	14/10/2008
Duration	38 months

Abstract

Our aim is to elucidate the function of the Arabidopsis chloroplast protein import apparatus component, atTic40. This protein is held in the inner envelope membrane by a single, N-terminal membrane span, and projects a soluble domain into the stroma. The stromal domain shares homology with mammalian co-chaperones, and so the protein is thought to play a co-chaperone role during import, perhaps as part of a chloroplast import motor. This, however, remains to be proven. We will investigate the function and structure of the atTic40 protein (1), and isolate the intact Tic complex to identify atTic40 interaction partners (2). (1) Our unpublished data demonstrate functional redundancy between atTic40 and the mammalian co-chaperone, Hip, with respect to a C-terminal Sti1 domain. We will assess if this redundancy extends to a central TPR domain, by conducting domain-swap experiments between atTic40 or Hip or the related co-chaperone, Hop. The in vivo activity of each fusion protein will be assess in transgenic complementation experiments using an atTic40 knockout mutant.These experiments will enable us to make predictions about atTic40 interaction partners, since interactions of the various Hip/Hop TPR domains with Hsp70, Hsp90 and/or Hsp 100 chaperones are well defined. Hip is known to possess both co-chaperone activity (it regulates Hsp70 function), and intrinsic chaperone activity (it binds unfolded proteins). We will conduct experiments to assess if atTic40 possesses similar co-chaperone and chaperone activities. First, we will assess the effects of recombinant atTic40 and Hip on ATP-binding and ¿hydrolysis by two different recombinant chaperones: mammalian Hsc70 (the interaction partner of Hip), and Arabidopsis Hsp93 (the putative interaction partner of atTic40). Second, we will assess the ability of recombinant atTic40 to prevent aggregation of denatured rhodanese (by measuring turbidity at 340 nm), and to mediate refolding of denatured luciferase (by measuring luciferase activity). We will attempt to map any detected atTic40 co-chaperone and chaperone activities to specific domains using deletion mutants. Since we will be generating large quantities of recombinant atTic40 stromal domain (which is highly soluble) for experiments described above, we will use the same recombinant protein to conduct a preliminary structural study of the protein. This will be conducted in collaboration with Peter Moody (Biochemistry Department, Leicester), an expert on X-ray chrystallography. (2) We will purify and characterise the Arabidopsis Tic complex using Tandem Affinity Purification (TAP), existing atTic40:TAP-expressing transgenic lines, and blue native (BN)-PAGE. This work will be constructed in collaboration with Peter Rehling (Freiberg, Germany), an expert on the TAP and BN-PAGE methods, and Kathryn Lilley (Cambridge Centre for Proteomics), who will identify Tic components by mass spectrometry. Our primary aim will be to identify novel components of the Tic complex, which may function with Tic40 as part of a chaperone/co-chaperone system. If we do identify novel components, these will be characterised in detail, using procedures similar to those recently used to characterise new components of the mitochondrial TIM23 complex. If we do not identify novel components, we will conduct a thorough study of the complex, including assessments of the stoichiometry and dynamic changes in response to Tic component knockout mutations. Since there is considerable disagreement in the literature concerning Tic complex composition, a thorough description of the complex will in itself constitute a significant result. As a precaution, in case problems are encountered when using atTic40:TAP to purify Tic complexes, we will generate new atTic110:TAP transgenic lines. Even if these lines are not required for Tic complex purification, they will nevertheless be useful, since they will allow us to purify complexes containing the atTic40:Hip/Hop fusions described in 1.

Summary

unavailable

Committee	Closed Committee - Biochemistry & Cell Biology (BCB)
Research Topics	Plant Science, Structural Biology
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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