Sec-independent Protein Translocation in Escherichia coli

279

385

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.

Section: Discussionmentioning

confidence: 99%

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

DeLisa

Tullman

Georgiou

2003

279

385

“…The number of TatA subunits was estimated either from the volume or based on transmembrane helix packing around the ring circumference. The first estimate is obtained by dividing the complex molecular mass by that of the His 6-tagged TatA subunit (10,730 Da), assuming that all residues are present in the observed volume. Alternatively, the number of TatA subunits is calculated from the average circumference (center of the ring wall), assuming a center-to-center distance of 10 Å for transmembrane helices.…”

Section: Discussionmentioning

confidence: 99%

“…In Escherichia coli, the minimal components of the Tat translocation system are the integral membrane proteins TatA, TatB, and TatC (7)(8)(9)(10)(11). Attempts to purify the Tat components from E. coli membranes have led to the identification of two distinct high-molecular-mass complexes, one corresponding to TatA and the other containing predominantly TatB and TatC (12)(13)(14)(15).…”

mentioning

confidence: 99%

The TatA component of the twin-arginine protein transport system forms channel complexes of variable diameter

Gohlke

Pullan

McDevitt

et al. 2005

Self Cite

243

274

The Tat system mediates Sec-independent transport of folded precursor proteins across the bacterial plasma membrane or the chloroplast thylakoid membrane. Tat transport involves distinct high-molecular-weight TatA and TatBC complexes. Here we report the 3D architecture of the TatA complex from Escherichia coli obtained by single-particle electron microscopy and random conical tilt reconstruction. TatA forms ring-shaped structures of variable diameter in which the internal channels are large enough to accommodate known Tat substrate proteins. This morphology strongly supports the proposal that TatA forms the proteinconducting channel of the Tat system. One end of the channel is closed by a lid that might gate access to the channel. On the basis of previous protease accessibility measurements, the lid is likely to be located at the cytoplasmic side of the membrane. The observed variation in TatA diameter suggests a model for Tat transport in which the number of TatA protomers changes to match the size of the channel to the size of the substrate being transported. Such dynamic close packing would provide a mechanism to maintain the membrane permeability barrier during transport.conical tilt reconstruction ͉ electron microscopy ͉ Tat protein transport ͉ three-dimensional structure ͉ twin-arginine signal peptide

“…The ⌬pH-dependent pathway operates independently of ATP and soluble factors, requiring only a thylakoid pH gradient. Recently, a Sec-independent export system was discovered in Escherichia coli , which is related to the thylakoid ⌬pH-dependent protein transport system of chloroplasts (12)(13)(14). The E. coli system involves the integral membrane proteins twin arginine translocation A (TatA), TatB, TatC, and TatE, and functions in the export of proteins that bind complex redox cofactors.…”

mentioning

confidence: 99%

An essential role of a TatC homologue of a ΔpH- dependent protein transporter in thylakoid membrane formation during chloroplast development in Arabidopsis thaliana

Motohashi

Nagata

Ito

et al. 2001

At least three transport systems function in targeting nuclearencoded chloroplast proteins to the chloroplast thylakoid membrane. One of these systems requires a thylakoid pH gradient and is named the ⌬pH-dependent protein transport system. A similar ⌬pH export system of Escherichia coli contains four components, twin arginine translocation A (TatA), TatB, TatC, and TatE. TatC is a major component of the ⌬pH-dependent protein transporter in E. coli and functions in the translocation of tightly folded proteins across membranes. We have isolated four transposon-inserted albino mutants named albino and pale green 2 (apg2) from Arabidopsis thaliana and showed that the transposons were inserted into different sites of a single gene. The APG2 gene product (named cpTatC) has sequence similarity with bacterial TatC and contains six putative transmembrane domains, including bacterial TatC proteins and a transit peptide in its N terminus. apg2 mutants showed albino phenotypes and could not grow in soil. The apg2 plastids were highly vacuolated, lacked internal membrane structures and lamellae of the thylakoid membrane, and contained many densely stained globule structures, like undifferentiated proplastids. Immunoblot analysis detected no thylakoid membrane proteins such as D1, light-harvesting complex, and OE23 in apg2 plastids, whereas soluble proteins such as rubisco large and small subunits were not decreased. These results indicate an essential role of cpTatC in chloroplast development, especially in thylakoid membrane formation.