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Colin Robinson

Publications and source records attributed to Colin Robinson.

At least 37 records · Page 2Linked to original sources
PubMed2005

Evidence for interactions between domains of TatA and TatB from mutagenesis of the TatABC subunits of the twin-arginine translocase.

The twin-arginine translocation (Tat) system transports folded proteins across the bacterial plasma membrane. Three subunits, TatA, B and C, are known to be involved but their modes of action are poorly understood, as are the inter-subunit interactions occurring within Tat complexes. We have generated mutations in the single transmembrane (TM) spans of TatA and TatB, with the aim of generating structural distortions. We show that substitution in TatB of three residues by glycine, or a single residue by proline, has no detectable effect on translocation, whereas the presence of three glycines in the TatA TM span completely blocks Tat translocation activity. The results show that the integrity of the TatA TM span is vital for Tat activity, whereas that of TatB can accommodate large-scale distortions. Near-complete restoration of activity in TatA mutants is achieved by the simultaneous presence of a V12P mutation in the TatB TM span, strongly implying a direct functional interaction between the TatA/B TM spans. We also analyzed the predicted amphipathic regions in TatA and TatB and again find evidence of direct interaction; benign mutations in either subunit completely blocked translocation of two Tat substrates when present in combination. Finally, we have re-examined the effects of previously analyzed TatABC mutations under conditions of high translocation activity. Among numerous TatA or TatB mutations tested, TatA F39A alone blocked translocation, and only substitutions of P48 and F94 in TatC blocked translocation activity.

Amino Acid Sequence↗
PubMed2004

Mechanisms of protein import and routing in chloroplasts.

The vast majority of the approximately 3000 different proteins required to build a fully functional chloroplast are encoded by the nuclear genome and translated on cytosolic ribosomes. As chloroplasts are each surrounded by a double-membrane system, or envelope, sophisticated mechanisms are necessary to mediate the import of these nucleus-encoded proteins into chloroplasts. Once inside the organelle, many chloroplast proteins engage one of four additional protein sorting mechanisms that direct targeting to the internal thylakoid membrane system.

Cell Nucleus↗
PubMed2004

The Escherichia coli twin-arginine translocation apparatus incorporates a distinct form of TatABC complex, spectrum of modular TatA complexes and minor TatAB complex.

The Tat system transports folded proteins across bacterial plasma and plant thylakoid membranes. To date, three key Tat subunits have been identified and mechanistic studies indicate the presence of two types of complex: a TatBC-containing substrate-binding unit and a separate TatA complex. Here, we used blue-native gel electrophoresis and affinity purification to study the nature of these complexes in Escherichia coli. Analysis of solubilized membrane shows that the bulk of TatB and essentially all of the TatC is found in a single 370kDa TatABC complex. TatABC was purified to homogeneity using an affinity tag on TatC and this complex runs apparently as an identical band. We conclude that this is the primary core complex, predicted to contain six or seven copies of TatBC together with a similar number of TatA subunits. However, the data indicate the presence of an additional form of Tat complex containing TatA and TatB, but not TatC; we speculate that this may be an assembly or disassembly intermediate of the translocator. The vast majority of TatA is found in separate complexes that migrate in blue-native gels as a striking ladder of bands with sizes ranging from under 100 kDa to over 500 kDa. Further analysis shows that the bands differ by an average of 34 kDa, indicating that TatA complexes are built largely, but possibly not exclusively, from modules of three or four TatA molecules. The range and nature of these complexes are similar in a TatC mutant that is totally inactive, indicating that the ladder of bands does not stem from ongoing translocation activity, and we show that purified TatA can self-assemble in vitro to form similar complexes. This spectrum of TatA complexes may provide the flexibility required to generate a translocon capable of transporting substrates of varying sizes across the plasma membrane in a folded state.

Arginine↗
PubMed2004

Tat-dependent protein targeting in prokaryotes and chloroplasts.

The twin-arginine translocation (Tat) system operates in the chloroplast thylakoid and the plasma membranes of a wide range of bacteria. It recognizes substrates bearing cleavable signal peptides in which a twin-arginine motif almost invariably plays a key role in recognition by the translocation machinery. These signal peptides are surprisingly similar to those used to specify transport by Sec-type systems, but the Tat pathway differs in fundamental respects from Sec-type and other protein translocases. Its key attribute is its ability to translocate large, fully folded (even oligomeric) proteins across tightly sealed membranes. To date, three key tat genes have been characterised and the first details of the Tat system are beginning to emerge. In this article we review the salient features of Tat systems, with an emphasis on the targeting signals involved, the substrate specificities of Tat systems, our current knowledge of Tat complex structures and the known mechanistic features. Although the article is focused primarily on bacterial systems, we incorporate relevant aspects of plant thylakoid Tat work and we discuss how the plant and bacterial systems may differ in some respects.

Amino Acid Sequence↗
PubMed2004

A homolog of Albino3/OxaI is essential for thylakoid biogenesis in the cyanobacterium Synechocystis sp. PCC6803.

YidC/OxaI play essential roles in the insertion of a wide range of membrane proteins in Eschericha coli and mitochondria, respectively. In contrast, the chloroplast thylakoid homolog Albino3 (Alb3) facilitates the insertion of only a specialized subset of proteins, and the vast majority insert into thylakoids by a pathway that is so far unique to chloroplasts. In this study, we have analyzed the role of Alb3 in the cyanobacterium Synechocystis sp. PCC6803, which contains internal thylakoids that are similar in some respects to those of chloroplasts. The single alb3 gene (slr1471) was disrupted by the introduction of an antibiotic cassette, and photoautotrophic growth resulted in the generation of a merodiploid species (but not full segregation), indicating an essential role for Alb3 in maintaining the photosynthetic apparatus. Thylakoid organization is lost under these conditions, and the levels of photosynthetic pigments fall to approximately 40% of wild-type levels. Photosynthetic electron transport and oxygen evolution are reduced by a similar extent. Growth on glucose relieves the selective pressure to maintain photosynthetic competence, and under these conditions, the cells become completely bleached, again indicating that Alb3 is essential for thylakoid biogenesis. Full segregation could not be achieved under any growth regime, strongly suggesting that the slr1471 open reading frame is essential for cell viability.

Arabidopsis Proteins↗
PubMed2003

Consensus structural features of purified bacterial TatABC complexes.

The twin-arginine translocation (Tat) system transports folded proteins across bacterial plasma membranes and the chloroplast thylakoid membrane. Here, we investigate the composition and structural organization of three different purified Tat complexes from Escherichia coli, Salmonella typhimurium and Agrobacterium tumefaciens. First, we demonstrate the functional activity of these Tat systems in vivo, since expression of the tatABC operons from S.typhimurium or A.tumefaciens in an E.coli tat null mutant strain resulted in efficient Tat-dependent export of an E.coli cofactor-containing substrate, TMAO reductase. The three isolated, affinity-tagged Tat complexes comprised TatA, TatB and TatC in each case, demonstrating a strong interaction between these three subunits. Single-particle electron microscopy studies of all three complexes revealed approximately oval-shaped, asymmetric particles with maximal dimensions up to 13 nm. A common feature is a number of stain-excluding densities surrounding more or less central pools of stain, suggesting protein-lined pores or cavities. The characteristics of size variation among the particles suggest a modular form of assembly and/or the recruitment of varying numbers of TatBC/TatA units. Despite low levels of sequence homology, the combined data indicate structural and functional conservation in the Tat systems of these three bacterial species.

Agrobacterium tumefaciens↗
PubMed2003

Quantitative export of a reporter protein, GFP, by the twin-arginine translocation pathway in Escherichia coli.

The Tat system mediates the transport of folded proteins across the bacterial cytoplasmic membrane. To study the properties of the Escherichia coli Tat-system, we used green fluorescent protein (GFP) fused to the twin-arginine signal peptide of TMAO reductase (TorA). In the presence of arabinose, low levels of this protein rapidly saturate the translocase and cause the accumulation of inactive, membrane-bound TorA-GFP; fluorescence microscopy also showed active TorA-GFP to be distributed throughout the cytoplasm. However, the efficiency of export can be massively increased by alteration of the growth conditions, and further increased by overexpression of the tatABC genes. Under these conditions, the levels of GFP in the periplasm are raised over 20-fold and the export efficiency nears 100%. These results show that the Tat-system is relatively inactive under some growth conditions and the data suggest that the system may be applicable for the larger-scale export of heterologous proteins.

Cell Membrane↗
PubMed2003

Identification of key regions within the Escherichia coli TatAB subunits.

The twin-arginine translocation (Tat) system catalyzes the transport of folded proteins across the bacterial plasma membrane or the chloroplast thylakoid membrane. In Escherichia coli and most other species, three important tat genes have been identified but the structure and mechanism of this system are poorly understood; the role and location of TatA are particularly unclear. In this report we have used site-specific mutagenesis to probe the significance of conserved features of the related TatA/B subunits. We find that an apparent 'hinge' region between the transmembrane (TM) span and an adjacent amphipathic region is important in both proteins, in that substitution of turn-inducing residues inhibits the export of a natural Tat substrate. Surprisingly, large-scale mutagenesis of the conserved amphipathic regions of TatA and TatB leads only to minor effects on Tat-dependent export suggesting that this particular feature is not central to the translocation mechanism. This domain is, however, critical for the translocation process and we identify Gly/Pro residues in these regions of TatA/B that are essential for efficient export.

Amino Acid Sequence↗
PubMed2003

DmsD is required for the biogenesis of DMSO reductase in Escherichia coli but not for the interaction of the DmsA signal peptide with the Tat apparatus.

The DmsD protein is essential for the biogenesis of DMSO reductase in Escherichia coli, and binds the signal peptide of the DmsA subunit, a Tat substrate. This suggests a role as a guidance factor to target pre-DmsA to the translocase. Here, we have analysed the export of fusion proteins in which the DmsA and TorA signal peptides are fused to green fluorescent protein. Both chimeras are efficiently exported to the periplasm in wild-type E. coli cells and we show that their export efficiencies are essentially identical in a mutant lacking DmsD. An authentic Tat substrate, TMAO reductase, is also efficiently exported in the dmsD mutant. The data indicate that DmsD carries out a critical role in DMSO reductase biogenesis/assembly but is not required for the functioning of the DmsA signal peptide.

Carrier Proteins↗
PubMed2003

Fluoride profiles in premolars after different durations of water fluoridation in Ho Chi Minh City, Vietnam.

In order to evaluate the uptake of fluoride (F) into tooth structures following water fluoridation in Ho Chi Minh City, F concentrations were measured from the enamel surface through the enamel-dentine junction (EDJ) to dentine close to the pulp in premolars. Calcification had been completed in all the teeth before fluoridation. Samples were obtained from fluoridated areas of the City ([F]: 0.7 parts/10(6)) after 3 years (eight samples), 6 years (eight samples) and 8 years (nine samples). Samples were also taken from regions outside the fluoridated water supply of the City in 1998 after 8 years of water fluoridation. An abrasive microsampling method was used to determine the profiles of fluoride and phosphorus concentrations. In enamel surfaces, F concentrations tended to increase with increasing periods of fluoridation. In dentine close to the pulp, F concentrations also increased statistically significantly with the increasing duration of fluoridation of water (between 6 and 3 years: P=0.006; between 8 and 3 years: P=0.001; between 8 years and 8 years without F: P=0.0001). It was concluded that F concentrations in enamel and dentine had tended to increase gradually with the duration of water fluoridation in Ho Chi Minh City. The increase was most obvious in dentine near the junction with the pulp.

Adolescent↗
PubMed2003

Membrane-specific targeting of green fluorescent protein by the Tat pathway in the cyanobacterium Synechocystis PCC6803.

The transport and sorting of extracytoplasmic proteins in cyanobacteria is made complex by the presence of a highly differentiated membrane system. Proteins destined for the periplasm and thylakoid lumen are initially transported by Sec- and Tat-type pathways but little is known of the mechanisms that ultimately direct them to the correct destinations. We have generated a Synechocystis PCC6803 transformant that expresses a fusion protein comprising the Tat-specific targeting signal of Escherichia coli TorA linked to green fluorescent protein (GFP). Immunoblotting indicates the presence of mature-size GFP but no precursor form, demonstrating that efficient translocation has taken place. Confocal microscopy and immunogold electron microscopy reveal GFP to be almost exclusively located in the periplasm, with almost no protein evident in the thylakoid network. These data point to the operation of highly effective sorting pathways for soluble proteins in this cyanobacterium. The observed sorting of the GFP suggests that either (a) the Tat apparatus is located only in the plasma membrane or (b) the TorA-GFP is targeted across either membrane but the GFP is subsequently directed to the periplasm, perhaps by a default sorting pathway to this compartment.

Bacterial Proteins↗
PubMed2003

An Escherichia coli twin-arginine signal peptide switches between helical and unstructured conformations depending on the hydrophobicity of the environment.

The Tat system catalyzes the transport of folded globular proteins across the bacterial plasma membrane and the chloroplast thylakoid. It recognizes cleavable signal peptides containing a critical twin-arginine motif but little is known of the overall structure of these peptides. In this report, we have analyzed the secondary structure of the SufI signal peptide, together with those of two nonfunctional variants in which the region around the twin-arginine, RRQFI, is replaced by KKQFI or RRQAA. Circular dichroism studies show that the SufI peptide exists as an unstructured peptide in aqueous solvent with essentially no stable secondary structure. In membrane-mimetic environments such as SDS micelles or water/trifluoroethanol, however, the peptide adopts a structure containing up to about 40% alpha-helical content. Secondary structure predictions and molecular modelling programs strongly suggest that the helical region begins at, or close to, the twin-arginine motif. Studies on the thermal stability of the helix demonstrate a sharp transition between the unstructured and helical states, suggesting that the peptide exists in one of two distinct states. The two nonfunctional peptides exhibit almost identical spectra and properties to the wild-type SufI peptide, indicating that it is the arginine sidechains, and not their contribution to the helical structure, that are critical in this class of peptide.

Arginine↗
PubMed2003

FtsH is involved in the early stages of repair of photosystem II in Synechocystis sp PCC 6803.

When plants, algae, and cyanobacteria are exposed to excessive light, especially in combination with other environmental stress conditions such as extreme temperatures, their photosynthetic performance declines. A major cause of this photoinhibition is the light-induced irreversible photodamage to the photosystem II (PSII) complex responsible for photosynthetic oxygen evolution. A repair cycle operates to selectively replace a damaged D1 subunit within PSII with a newly synthesized copy followed by the light-driven reactivation of the complex. Net loss of PSII activity occurs (photoinhibition) when the rate of damage exceeds the rate of repair. The identities of the chaperones and proteases involved in the replacement of D1 in vivo remain uncertain. Here, we show that one of the four members of the FtsH family of proteases (cyanobase designation slr0228) found in the cyanobacterium Synechocystis sp PCC 6803 is important for the repair of PSII and is vital for preventing chronic photoinhibition. Therefore, the ftsH gene family is not functionally redundant with respect to the repair of PSII in this organism. Our data also indicate that FtsH binds directly to PSII, is involved in the early steps of D1 degradation, and is not restricted to the removal of D1 fragments. These results, together with the recent analysis of ftsH mutants of Arabidopsis, highlight the critical role played by FtsH proteases in the removal of damaged D1 from the membrane and the maintenance of PSII activity in vivo.

Bacterial Proteins↗
PubMed2002

Functional genomic analysis of the Bacillus subtilis Tat pathway for protein secretion.

Protein secretion from Bacillus species is a major industrial production tool with a market of over $1 billion per year. However, standard export technologies, based on the well-characterised general secretory (Sec) pathway, are frequently inapplicable for the production of proteins. The recently discovered twin-arginine translocation (Tat) pathway offers additional potential to transport proteins. Here we review the use of functional genomic and proteomic approaches to explore the Tat pathway of Bacillus subtilis. The properties of Tat pathway components and the twin-arginine signal peptides that direct proteins into this pathway are discussed. Where appropriate, a comparison is made with Tat systems from other organism, such as Escherichia coli. Recent findings with the latter organism in particular provide proof-of-principle that the Tat pathway can be exploited for the production of Sec-incompatible proteins.

Amino Acid Sequence↗
PubMed2002

Novel approach reveals localisation and assembly pathway of the PsbS and PsbW proteins into the photosystem II dimer.

A blue-native gel electrophoresis system was combined with an in organello import assay to specifically analyse the location and assembly of two nuclear-encoded photosystem II (PSII) subunits. With this method we were able to show that initially the low molecular mass PsbW protein is not associated with the monomeric form of PSII. Instead a proportion of newly imported PsbW is directly assembled in dimeric PSII supercomplexes with very fast kinetics; its negatively charged N-terminal domain is essential for this process. The chlorophyll-binding PsbS protein, which is involved in energy dissipation, is first detected in the monomeric PSII subcomplexes, and only at later time points in the dimeric form of PSII. It seems to be bound tighter to the PSII core complex than to light harvesting complex II. These data point to radically different assembly pathways for different PSII subunits.

Chloroplasts↗
PubMed2002

Essential cytoplasmic domains in the Escherichia coli TatC protein.

The twin-arginine translocation (Tat) system mediates the transport of proteins across the bacterial plasma membrane and chloroplast thylakoid membrane. Operating in parallel with Sec-type systems in these membranes, the Tat system is completely different in both structural and mechanistic terms, and is uniquely able to catalyze the translocation of fully folded proteins across coupled membranes. TatC is an essential, multispanning component that has been proposed to form part of the binding site for substrate precursor proteins. In this study we have tested the importance of conserved residues on the periplasmic and cytoplasmic face of the Escherichia coli protein. We find that many of the mutations on the cytoplasmic face have little or no effect. However, substitution at several positions in the extreme N-terminal cytoplasmic region or the predicted first cytoplasmic loop lead to a significant or complete loss of Tat-dependent export. The mutated strains are unable to grow anaerobically on trimethylamine N-oxide minimal media and are unable to export trimethylamine-N-oxide reductase (TorA). The same mutants are completely unable to export a chimeric protein, comprising the TorA signal peptide linked to green fluorescent protein, indicating that translocation is blocked rather than cofactor insertion into the TorA mature protein. The data point to two essential cytoplasmic domains on the TatC protein that are essential for export.

Amino Acid Sequence↗
PubMed2002

Unique structural determinants in the signal peptides of "spontaneously" inserting thylakoid membrane proteins.

A series of thylakoid membrane proteins, including PsbX, PsbY and PsbW, are synthesized with cleavable signal peptides yet inserted using none of the known Sec/SRP/Tat/Oxa1-type insertion machineries. Here, we show that, although superficially similar to Sec-type signal peptides, these thylakoidal signal peptides contain very different determinants. First, we show that basic residues in the N-terminal domain are not important, ruling out electrostatic interactions as an essential element of the insertion mechanism, and implying a fundamentally different targeting mechanism when compared with the structurally similar M13 procoat. Second, we show that acidic residues in the C-domain are essential for the efficient maturation of the PsbX and PsbY-A1 peptides, and that even a single substitution of the -5 Glu by Val in the PsbX signal peptide abolishes maturation in the thylakoid. Processing efficiency is restored to an extent, but not completely, by the highly hydrophilic Asn, implying that this domain is required to be hydrophilic, but preferably negatively charged, in order to present the cleavage site in an optimal manner. We show that substitution of the PsbX C-domain Glu residues by Val leads to a burial of the cleavage site within the bilayer although insertion is unaffected. Finally, we show that substitution of the Glu residues in the lumenal A2 loop of the PsbY polyprotein leads to a block in cleavage on the stromal side of the membrane, and present evidence that the PsbY-A2 signal peptide is required to be relatively hydrophilic and unable to adopt a transmembrane conformation on its own. These data indicate that, rather than being merely additional hydrophobic regions to promote insertion, the signal peptides of these thylakoid proteins are complex domains with uniquely stringent requirements in the C-domain and/or translocated loop regions.

Amino Acid Sequence↗