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Synthesis and Processing of Macromolecules
The Tat Protein Export Pathway
Schematic representation of Sec and Tat signal peptides. Both Sec and Tat signal peptides show a recognizable tripartite structure with a polar (basic) n-region, a hydrophobic h-region, and a polar c-region that contains the recognition site for signal peptidase (shown as AxA in the figure, although generally residues at the -3 and -1 positions relative to the cleavage site are any amino acid with a small, neutral side chain; reference 16 ). The vast majority of Sec-targeting signal peptides in E. coli have a length between 15 (for the signal peptide of the lipoprotein CsgG) and 37 amino acids (FimO signal peptide). Some occasionally have longer length, for example, the unusually long signal peptides associated with autotransporter proteins such as Ag43 ( 17 , 18 ). However, the vast majority of E. coli Sec signal peptides are fewer than 24 amino acids long. Tat signal peptides have a conserved motif, S-R-R-x-F-L-K, that is found at the n-region/h-region boundary and are generally markedly longer than Sec signal peptides, varying in length in E. coli from 25 (YedY signal peptide) to 50 (YagT signal peptide) amino acids. Other differences include the fact that the h-regions of Tat signal peptides are less hydrophobic than Sec signals and that they often contain one or more basic residues in the c-region that are almost never found in Sec signal peptides and that act as a Sec-avoidance signal ( 19 , 20 ).
GFP as a reporter for export by the E. coli Tat pathway. Fluorescence light microscopy images of E. coli strain MC4100 (A) ( 47 ; tat +) and the cognate tatABCDE deletion strain DADE (B) ( 48 ) producing GFP fused to the TorA signal peptide from plasmid pRR-GFP ( 45 ). Halos of GFP are observed in a tat + strain, whereas only diffuse cytoplasmic fluorescence is seen when the Tat system is inactive. Note that arabinose-resistant isolates of the two strains were used. Subcellular fractionation reveals that the GFP detected in the tat + strain resides in the periplasmic compartment (data not shown). We thank Dr. Berengere Ize for providing the images.
Quality control and proofreading processes on the Tat pathway. Tat precursor proteins are believed to interact (a) either directly with the Tat machinery or (b) with the lipid bilayer before diffusing laterally toward the Tat channel ( 110 , 111 ). The Tat translocase itself is proposed to reject precursor proteins that are not folded (Tat Quality Control) ( 112 ). Complex proteins, such as cofactor-containing respiratory enzymes, undergo a second tier of quality control called “Tat Proofreading.” Here, the signal peptide is bound tightly by a target-specific cytoplasmic chaperone, shown in green (step 1). In some cases, a second molecule of the chaperone binds elsewhere on the apoenzyme. The redox cofactor is then loaded (step 2), and partner subunits may also bind where appropriate (not depicted) before the chaperones are released (step 3).
Molecular basis of the Tat proofreading process. (A) The primary sequence of the E. coli TorA signal peptide. The twin arginine motif is highlighted in dark blue, and the TorD-binding epitope is highlighted in cyan. The arrow indicates the signal peptidase I (LepB) cleavage site. (B) Crystal structure of the TorD homologue from Shewanella massilia (PDB ID no. 1N1C). The model shows an intertwined dimer of two protomers (blue and yellow). (C) Crystal structure of the DmsD protein from E. coli (PDB ID no. 3EPF). In this case the model shows a monomeric form of the protein. (D) The primary sequence of the E. coli NapA signal peptide. The twin arginine motif is highlighted in dark blue, and the NapD binding epitope is highlighted in cyan. The arrow indicates the LepB cleavage site. (E) A model of the high-resolution solution NMR structure of the E. coli NapD protein (PDB ID no. 2JSX). (F) A model of the NMR-derived solution structure of a complex between E. coli NapD and residues 1 to 35 of the NapA twin arginine signal peptide (PDB ID no. 2PQ4).
Genetic organization of the tatA and tatE loci in E. coli K-12 and S. enterica subsp. enterica serovar Typhimurium LT2. (A and C) Genetic context of the tatA operon in E. coli K-12 (A) and S. enterica LT2 (C). The distance, in base pairs, between genes is indicated. (B and D) The mRNA between tatC and tatD in E. coli (B) and S. enterica LT2 (D) can potentially fold into a stem-loop structure. (E and F) Genetic context of the tatE gene in E. coli K-12 (A) and S. enterica LT2 (C). The distance, in base pairs, between genes is indicated.
Predicted secondary structure and topological organization of proteins encoded by the E. coli tat genes.
Alternative topologies for the E. coli TatA protein. (A) The amino acid sequence of TatA is shown, with predicted secondary structure above. α-helical regions are represented as cylinders, and β-sheet is indicated as arrows. The essential glycine residue at the boundary between the transmembrane and amphipathic α-helices is boxed. (B to E) Alternative topological arrangements of the TatA protein proposed in different studies (see main text).
The Tat protein transport cycle. (Top) Under resting conditions the TatBC complex and tetrameric units of TatA exist as separate assemblies in the membrane. Step 1. A folded substrate protein docks at the TatBC complex, binding by virtue of its twin arginine signal peptide. Step 2. TatA tetramers assemble onto the substrate-bound TatBC complex to form the transport channel in a process requiring the proton motive force (Δp). Step 3. The substrate is transported across the membrane through a channel formed by TatA. It is not known whether the proton motive force is needed to drive this step. Step 4. The translocated substrate is released, its signal peptide is cleaved, and the TatABC complex dissociates to give the TatBC complex and separate multimers of TatA. Note that, for clarity, only one TatBC pair in the TatBC complex and only some of the necessary TatA molecules are depicted.
The known or likely E. coli and Salmonella Tat substrate proteins a
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