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Translocation reversal by the thylakoid twin-arginine translocase

Reference	BB/D009952/1
Principal Investigator / Supervisor	Professor Colin Robinson
Co-Investigators / Co-Supervisors
Institution	University of Warwick
Department	Biological Sciences
Funding type	Research
Value (£)	207,978
Status	Completed
Type	Research Grant
Start date	01/07/2006
End date	30/06/2009
Duration	36 months

Abstract

The twin-arginine translocation (Tat) system transports folded proteins across the thylakoid membrane. Over a decade of in vitro studies have pointed to a translocation mechanism that is unidirectional and delta pH-driven. We have carried out the first detailed in vivo study of this system and have made two completely unexpected findings. First, a large proportion of substrate (and indeed MOST of a GFP construct) is extensively transported across the thylakoid membrane, processed to the mature size by the lumen-facing processing enzyme but then returned to the stroma. Secondly, the entire process is delta pH-independent. The data clearly reflect the influence of factors that are only active in vivo and the first aim of this research is to understand the mechanism and physiological significance of this process. The second aim is to capitalise on another finding: mutation of the terminal processing site leads to the accumulation of intermediate-size substrate in a protected position within the thylakoid membrane, apparently within the Tat system. This may reflect a delayed reversal of translocation. We will use this intermediate to isolate and characterise the full Tat translocation complex for the first time.

Summary

Eukaryotic cells contain numerous different compartments that are bounded by tightly sealed biological membranes. Proteins destined for these compartments need to be transported across the membranes and this is carried out by a range of protein transport systems. These usually function by unfolding the protein and 'threading' it through a narrow pore in the membrane. However, a major protein translocase in the chloroplast thylakoid membrane, termed the twin-arginine translocation (Tat) system, uses a unique mechanism to transport large, fully-folded proteins across this membrane into the lumenal space. Previous studies using isolated membranes have shown that the system transports proteins with high efficiency, and invariably in one direction. After transport, the signal peptides on the transported proteins are removed in the lumen to create the mature protein. We have now discovered another highly unusual trait of this system; analysis of the same substrates in intact living cells shows that substrates are often transported only partially across the membrane, after which they are returned to the stroma and degraded. This is especially the case with a construct in which the signal peptide is linked to a 'foreign' protein, where almost all of the protein undergoes this process of 'translocation reversal'. This may reflect the operation of a quality control, or 'proofreading' activity, whereby only the correct type of folded structure is recognized and fully transported by the Tat apparatus. This is an unexpected and critical part of the system's activity and we propose to investigate the underlying causes and consequences. The second main aim is to capitalise on another finding of our work: when cleavage of the signal peptide is prevented, the substrate protein becomes trapped in the translocase during translocation (retrotranslocation is slowed down enormously). This gives us an opportunity to study the substrate in the process of being translocated, and to identifyand characterise the actual translocation pore for the first time. This, in turn, will lead to important mechanistic insights.

Committee	Closed Committee - Biochemistry & Cell Biology (BCB)
Research Topics	Plant Science
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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