Variable stoichiometry of the TatA component of the twin-arginine protein transport system observed by
            <i>in vivo</i>
            single-molecule imaging

Leake, Mark C.; Greene, Nicholas P.; Godun, R. M.; Granjon, Thierry; Buchanan, Grant; Chen, Shuyun; Berry, Richard M.; Palmer, Tracy; Berks, Ben C.

doi:10.1073/pnas.0806338105

Cited by 175 publications

(282 citation statements)

References 41 publications

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“…Custom-written imaging software automatically separated and quantified these components and was capable of detecting spots with a total intensity of 2,000 counts or more. The full width at half maximum of spots was typically 300-350 nm, consistent with a FliM ring of diameter ∼50 nm convolved with the point spread function of a single YPet molecule in our microscope of width 250-300 nm (12,15,16). (Fig.…”

Section: Resultsmentioning

confidence: 95%

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Signal-dependent turnover of the bacterial flagellar switch protein FliM

Delalez

Wadhams

Rosser

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

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179

181

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Most biological processes are performed by multiprotein complexes. Traditionally described as static entities, evidence is now emerging that their components can be highly dynamic, exchanging constantly with cellular pools. The bacterial flagellar motor contains ∼13 different proteins and provides an ideal system to study functional molecular complexes. It is powered by transmembrane ion flux through a ring of stator complexes that push on a central rotor. The Escherichia coli motor switches direction stochastically in response to binding of the response regulator CheY to the rotor switch component FliM. Much is known of the static motor structure, but we are just beginning to understand the dynamics of its individual components. Here we measure the stoichiometry and turnover of FliM in functioning flagellar motors, by using high-resolution fluorescence microscopy of E. coli expressing genomically encoded YPet derivatives of FliM at physiological levels. We show that the ∼30 FliM molecules per motor exist in two discrete populations, one tightly associated with the motor and the other undergoing stochastic turnover. This turnover of FliM molecules depends on the presence of active CheY, suggesting a potential role in the process of motor switching. In many ways the bacterial flagellar motor is as an archetype macromolecular assembly, and our results may have further implications for the functional relevance of protein turnover in other large molecular complexes.chemotaxis | single molecule | total internal reflection fluorescence | in vivo microscopy | molecular motor T he bacterial flagellar motor is one of the most complex biological nanomachines (1), ideal for investigating turnover within a multimeric complex. It is the result of the coordinated, sequential expression of about 50 genes (2) producing a structure that spans the cell membrane and rotates an extracellular filament extending several micrometers from the cell surface, at speed of hundreds of hertz. The motor is about is about 45 nm in diameter and is composed of at least 13 different proteins, all in different copy numbers. It is powered by a transmembrane ion flux (1, 3, 4) and consists of a core rotating against a ring of stator proteins (1, 5-7). The C ring, also called the "switch complex," is part of the rotor and localized to the cytoplasmic motor region. The response regulator CheY-P binds one of the C ring components, FliM, causing the rotor to switch rotational direction, thus making FliM the interface with the chemosensory pathway (8-11).Much is known of the static motor structure (5-7), but the dynamics and interactions of its constituents under natural conditions in living cells are poorly understood. Recent results showed that molecules of the stator protein MotB in the flagellar motor, fused to GFP, exchange with a membrane pool of freely diffusing MotB on a time scale of minutes (12)(13)(14). This observation raised the question of whether protein turnover is a general feature of molecular complexes or is a peculiarity of MotB.To ad...

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Section: Resultsmentioning

confidence: 95%

“…A home-built inverted TIRF microscope with 532 nm excitation wavelength was used, as described previously (12,15). Fluorescence emission was imaged at 50 nm∕pixel in frame-transfer mode at 25 Hz by a 128 × 128-pixel, cooled, back-thinned electron-multiplying charge-coupled device camera (iXon DV860-BI; Andor Technology).…”

Section: Methodsmentioning

confidence: 99%

Signal-dependent turnover of the bacterial flagellar switch protein FliM

Delalez

Wadhams

Rosser

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

179

181

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“…outside the motor complex) was found to be 0.0088 + 0.0026 mm 2 s -1 . Furthermore, in another study, Leake et al [23] monitored the mobility of the inner membrane protein TatA in the twin-arginine translocation (Tat) system while complexed to the other Tat proteins. By labelling TatA with YFP they found that the diffusion coefficient, when corrected for the curvature of the membrane, decreased with increasing size of the Tat complex, ranging from about 0.1 mm 2 s -1 for complexes with about 10 TatA-YFP molecules to less than 0.01 mm 2 s -1 for complexes of near 100 TatA-YFP molecules.…”

Section: (B) Inner Bacterial Membranementioning

confidence: 99%

Single-molecule imaging in live bacteria cells

Ritchie

Lill

Sood

et al. 2013

Phil. Trans. R. Soc. B

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Bacteria, such as Escherichia coli and Caulobacter crescentus , are the most studied and perhaps best-understood organisms in biology. The advances in understanding of living systems gained from these organisms are immense. Application of single-molecule techniques in bacteria have presented unique difficulties owing to their small size and highly curved form. The aim of this review is to show advances made in single-molecule imaging in bacteria over the past 10 years, and to look to the future where the combination of implementing such high-precision techniques in well-characterized and controllable model systems such as E. coli could lead to a greater understanding of fundamental biological questions inaccessible through classic ensemble methods.

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“…reorganization corresponds to TatA assembly rather than to TatA conformational change. To address this issue, we previously undertook direct imaging of YFP-labeled TatA (TatA-YFP) that had been expressed at native levels in living E. coli cells (31). Individual TatA complexes could be tracked by videorate fluorescence microscopy, and their oligomeric state could be estimated from their photobleaching traces.…”

Section: Significancementioning

confidence: 99%

Live cell imaging shows reversible assembly of the TatA component of the twin-arginine protein transport system

Alcock

Baker

Greene

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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170

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The twin-arginine translocation (Tat) machinery transports folded proteins across the cytoplasmic membrane of bacteria and the thylakoid membrane of chloroplasts. It has been inferred that the Tat translocation site is assembled on demand by substrate-induced association of the protein TatA. We tested this model by imaging YFP-tagged TatA expressed at native levels in living Escherichia coli cells in the presence of low levels of the TatA paralogue TatE. Under these conditions the TatA-YFP fusion supports full physiological Tat transport activity. In agreement with the TatA association model, raising the number of transport-competent substrate proteins within the cell leads to an increase in the number of large TatA complexes present. Formation of these complexes requires both a functional TatBC substrate receptor and the transmembrane proton motive force (PMF). Removing the PMF causes TatA complexes to dissociate, except in strains with impaired Tat transport activity. Based on these observations we propose that TatA assembly reaches a critical point at which oligomerization can be reversed only by substrate transport. In contrast to TatA-YFP, the oligomeric states of TatB-YFP and TatC-YFP fusions are not affected by substrate or the PMF, although TatB-YFP oligomerization does require TatC.

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Variable stoichiometry of the TatA component of the twin-arginine protein transport system observed by in vivo single-molecule imaging

Cited by 175 publications

References 41 publications

Signal-dependent turnover of the bacterial flagellar switch protein FliM

Signal-dependent turnover of the bacterial flagellar switch protein FliM

Single-molecule imaging in live bacteria cells

Live cell imaging shows reversible assembly of the TatA component of the twin-arginine protein transport system

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