Structure of TatA Paralog, TatE, Suggests a Structurally Homogeneous Form of Tat Protein Translocase That Transports Folded Proteins of Differing Diameter

The chloroplast Twin arginine translocation (Tat) pathway uses three membrane proteins and the proton gradient to transport folded proteins across sealed membranes. Precursor proteins bind to the cpTatC-Hcf106 receptor complex, triggering Tha4 assembly and protein translocation. Tha4 is required only for the translocation step and is thought to be the protein-conducting component. The organization of Tha4 oligomers was examined by substituting pairs of cysteine residues into Tha4 and inducing disulfide cross-links under varying stages of protein translocation. Tha4 formed tetramers via its transmembrane domain in unstimulated membranes and octamers in membranes stimulated by precursor and the proton gradient. Tha4 formed larger oligomers of at least 16 protomers via its carboxy tail, but such C-tail clustering only occurred in stimulated membranes. Mutational studies showed that transmembrane domain directed octamers as well as C-tail clusters require Tha4's transmembrane glutamate residue and its amphipathic helix, both of which are necessary for Tha4 function. A novel double cross-linking strategy demonstrated that both transmembrane domain directed- and C-tail directed oligomerization occur in the translocase. These results support a model in which Tha4 oligomers dock with a precursor–receptor complex and undergo a conformational switch that results in activation for protein transport. This possibly involves accretion of additional Tha4 into a larger transport-active homo-oligomer.

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Substrate-triggered position-switching of TatA and TatB is an essential step in the Escherichia coli Tat protein export pathway

10.1101/113985 ◽

2017 ◽

Cited By ~ 3

Author(s):

Johann Habersetzer ◽

Kristoffer Moore ◽

Jon Cherry ◽

Grant Buchanan ◽

Phillip Stansfeld ◽

...

Keyword(s):

Escherichia Coli ◽

Binding Sites ◽

Protein Transport ◽

Transmembrane Helix ◽

Tat Protein ◽

Disulfide Crosslinking ◽

Additional Sequence ◽

Tat System ◽

Folded Proteins ◽

Related Proteins

AbstractThe twin arginine protein transport (Tat) machinery mediates the translocation of folded proteins across the cytoplasmic membrane of prokaryotes and the thylakoid membrane of plant chloroplasts. The Escherichia coli Tat system comprises TatC and two additional sequence-related proteins, TatA and TatB. Here we use disulfide crosslinking and molecular modelling to show there are two binding sites for TatA/B proteins on TatC. TatA and TatB are each able to occupy both sites if they are the only TatA/B protein present. However, under resting conditions the sites are differentially occupied with TatB occupying the ‘polar cluster’ site while TatA binds adjacently at the TatC transmembrane helix 6 binding site. When the Tat system is activated by the overproduction of a substrate, TatA and TatB switch their binding sites. We propose that this substrate-triggered positional exchange is a key step in the assembly of an active Tat translocase.

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Assembling the Tat protein translocase

eLife ◽

10.7554/elife.20718 ◽

2016 ◽

Vol 5 ◽

Cited By ~ 34

Author(s):

Felicity Alcock ◽

Phillip J Stansfeld ◽

Hajra Basit ◽

Johann Habersetzer ◽

Matthew AB Baker ◽

...

Keyword(s):

Structural Model ◽

Protein Translocation ◽

Transmembrane Helix ◽

Tat Protein ◽

Polar Cluster ◽

Evolution Analysis ◽

Protein Interfaces ◽

Multiple Copies ◽

Folded Proteins ◽

Combine Sequence

The twin-arginine protein translocation system (Tat) transports folded proteins across the bacterial cytoplasmic membrane and the thylakoid membranes of plant chloroplasts. The Tat transporter is assembled from multiple copies of the membrane proteins TatA, TatB, and TatC. We combine sequence co-evolution analysis, molecular simulations, and experimentation to define the interactions between the Tat proteins of Escherichia coli at molecular-level resolution. In the TatBC receptor complex the transmembrane helix of each TatB molecule is sandwiched between two TatC molecules, with one of the inter-subunit interfaces incorporating a functionally important cluster of interacting polar residues. Unexpectedly, we find that TatA also associates with TatC at the polar cluster site. Our data provide a structural model for assembly of the active Tat translocase in which substrate binding triggers replacement of TatB by TatA at the polar cluster site. Our work demonstrates the power of co-evolution analysis to predict protein interfaces in multi-subunit complexes.

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STUDIES ON THE AROMATISATION OF NEUTRAL STEROIDS IN PREGNANT WOMEN

Acta Endocrinologica ◽

10.1530/acta.0.0650069 ◽

1970 ◽

Vol 65 (1) ◽

pp. 69-83 ◽

Cited By ~ 5

Author(s):

H. Vokal ◽

D. F. Archer ◽

N. Wiqvist ◽

E. Diczfalusy

Keyword(s):

Pregnant Women ◽

Placental Perfusion ◽

Homogeneous Form ◽

Urine Specimens

ABSTRACT The following steroids, [7α-3H]5-androstene-3β,16α,17β-triol and [4-14C] 5-androstene-3β,16β,17β-triol were biosynthesized and their metabolism was studied in two subjects at midgestation, following placental perfusion in situ. Among the metabolites isolated in a radiochemically homogeneous form, exclusively 3H-labelled 16α,17β-dihydroxy-4-androsten-3-one was isolated from the extracts of placentas and perfusates. Exclusively 14C-labelled 16β,17β-dihydroxy-4-androsten-3-one was isolated from the placentas and perfusates and 16-epioestriol (1,3,5(10)-oestratriene-3,16β,17β-triol) from the placentas, perfusates and urine specimens. The following compounds contained both 3H and 14C-label: oestriol (placentas and urine specimens) and 5β-androstane-3α,16α,17β-triol (urine specimens). The 3H/14C-ratio of oestriol isolated from the urine specimens was much lower than that of urinary 5β-androstane-3α,16α,17β-triol, or that of the oestriol isolated from the placentas. The 3H/14C-ratio of the oestriol isolated from the urine 2–4 days following the perfusion was lower than that of the perfused material. It is concluded that a considerable amount of the 16-epioestriol secreted by the placenta is gradually converted to oestriol by the maternal organism. A limited conversion occurs also in the placenta.

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