Export of active green fluorescent protein to the periplasm by the twin‐arginine translocase (Tat) pathway in <i>Escherichia coli</i>

Thomas, Joanna D.; Daniel, Richard A.; Errington, Jeff; Robinson, Colin

doi:10.1046/j.1365-2958.2001.02253.x

Cited by 267 publications

(283 citation statements)

References 26 publications

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“…In a strain expressing TorAss-YFP, most cells showed bright, uniform fluorescence, but some cells (about 10%) exhibited the same fluorescent haloes as those described in previous studies ( Fig. 7A4) (Santini et al, 2001;Thomas et al, 2001). We attributed the uniform fluorescence to overproduction of the fusion protein and inefficient export by the Tat system.…”

Section: Fusion Of Mmpa To Mrfp1 -An Effective Periplasmic Fluorescesupporting

confidence: 78%

A membrane metalloprotease participates in the sequential degradation of a Caulobacter polarity determinant

Chen

Viollier

Shapiro

2005

Molecular Microbiology

122

142

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SummaryCaulobacter crescentus assembles many of its cellular machines at distinct times and locations during the cell cycle. PodJ provides the spatial cues for the biogenesis of several polar organelles, including the pili, adhesive holdfast and chemotactic apparatus, by recruiting structural and regulatory proteins, such as CpaE and PleC, to a specific cell pole. PodJ is a protein with a single transmembrane domain that exists in two forms, full-length (PodJ L ) and truncated (PodJ S ), each appearing during a specific time period of the cell cycle to control different aspects of polar organelle development. PodJ L is synthesized in the early predivisional cell and is later proteolytically converted to PodJ S . During the swarmer-to-stalked transition, PodJ S must be degraded to preserve asymmetry in the next cell cycle. We found that MmpA facilitates the degradation of PodJ S . MmpA belongs to the site-2 protease (S2P) family of membraneembedded zinc metalloproteases, which includes SpoIVFB and YluC of Bacillus subtilis and YaeL of Escherichia coli . MmpA appears to cleave within or near the transmembrane segment of PodJ S , releasing it into the cytoplasm for complete proteolysis. While PodJ S has a specific temporal and spatial address, MmpA is present throughout the cell cycle; furthermore, periplasmic fusion to mRFP1 suggested that MmpA is uniformly distributed around the cell. We also determined that mmpA and yaeL can complement each other in C. crescentus and E. coli , indicating functional conservation. Thus, the sequential degradation of PodJ appears to involve regulated intramembrane proteolysis (Rip) by MmpA.

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Section: Fusion Of Mmpa To Mrfp1 -An Effective Periplasmic Fluorescesupporting

confidence: 78%

A membrane metalloprotease participates in the sequential degradation of a Caulobacter polarity determinant

Chen

Viollier

Shapiro

2005

Molecular Microbiology

122

142

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“…The observed efficiency of AP export is typical of native Tat substrates and of Tat fusions to heterologous proteins (14,29,32,40,41). If indeed the Tat translocator exports preferentially oxidized monomeric AP, as discussed above, then the enzymatically active protein remaining in the cytoplasm could represent dimerized protein that is incompatible with export.…”

Section: Discussionmentioning

confidence: 86%

“…13) before export and also proteins that can not be exported by the Sec pathway in a functional form, for unknown reasons (e.g., GFP; ref. 14). Processes such as cofactor incorporation or the assembly of protein subunits hinge on the formation of secondary or tertiary structure.…”

mentioning

confidence: 99%

Folding quality control in the export of proteins by the bacterial twin-arginine translocation pathway

DeLisa

Tullman

Georgiou

2003

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

278

385

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To examine the relationship between folding and export competence by the twin-arginine translocation (Tat) pathway we analyzed the subcellular localization of fusions between a set of eight putative Tat leader peptides and alkaline phosphatase in isogenic Escherichia coli strains that either allow or disfavor the formation of protein disulfide bonds in the cytoplasm. We show that export by the Tat translocator is observed only in strains that enable oxidative protein folding in the cytoplasm. Further, we show that other disulfide-containing proteins, namely single-chain Fv and heterodimeric F AB antibody fragments, are export-competent only in strains having an oxidizing cytoplasm. Functional, heterodimeric F AB protein was exported from the cytoplasm by means of a Tat leader peptide fused to the heavy chain alone, indicating that the formation of a disulfide-bonded dimer preceeds export. These results demonstrate that in vivo only proteins that have attained the native conformation are exported by the Tat translocator, indicating that a folding quality-control mechanism is intrinsic to the export process. The ability to export proteins with disulfide bonds and the folding proofing feature of the Tat pathway are of interest for biotechnology applications.

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“…7B), without fusing with any signal sequence or transmembrane peptide, it could not secrete out of the cells through the ER-Golgi pathway (Laukkanen et al, 1996). Instead, the soluble GFP in the ER cleaved from the FMDV-2A site was capable of translocation across the ER membrane to the cytosol (Thomas et al, 2001;Tanudji et al, 2002) and further diffusing into nuclei.…”

Section: Discussionmentioning

confidence: 99%

Development of Dengue type-2 virus replicons expressing GFP reporter gene in study of viral RNA replication

et al. 2012

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Insertion of green fluorescent protein (GFP) encoding-gene into virus genes has provided a valuable tool for flavivirus research. This study aimed to develop dengue virus (DENV) replicons expressing GFP reporter that would provide a fast in vitro system to analyze functional roles of specific DENV sequences in viral replication. Two classes of recombinant replicon constructs were generated; one was a RNA-launched replicon with a GFP gene directly inserted into a fulllength DENV genome (FL-DENV/GFP), and the other consisted of 4 types of DNA-launched DENV subgenomic replicons with GFP replacement at various structural genes (Δ-DENV/GFP). The FL-DENV/GFP resulted in GFP expression in transfected cells with no viable DENV being recovered from the transfection. The Δ-DENV/GFP constructs with partial structural gene deletion (ΔC-, ΔCprM/M-, ΔprM/M-, or ΔE-) expressed bright and long lasting GFP. The GFP expression intensity in living cells correlated well with the level of RNA replication. Various mutations in the 5′noncoding region of DENV-2 previously shown to be important genetic determinants for virus replication and mouse virulence were incorporated into the 5 different replicon constructs. Characterizations of 29 mutants demonstrated that these replicons can provide a useful platform for a quick and powerful in vitro system to analyze genetic determinants of DENV replication. These constructs can also be useful for development of vectors expressing foreign genes for various researches.

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Export of active green fluorescent protein to the periplasm by the twin‐arginine translocase (Tat) pathway in Escherichia coli

Cited by 267 publications

References 26 publications

A membrane metalloprotease participates in the sequential degradation of a Caulobacter polarity determinant

A membrane metalloprotease participates in the sequential degradation of a Caulobacter polarity determinant

Folding quality control in the export of proteins by the bacterial twin-arginine translocation pathway

Development of Dengue type-2 virus replicons expressing GFP reporter gene in study of viral RNA replication

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