Coordinating assembly and export of complex bacterial proteins

Abstract

The Escherichia coli twin-arginine protein transport (Tat) system is a molecular machine dedicated to the translocation of fully folded substrate proteins across the energy-transducing inner membrane. Complex cofactor-containing Tat substrates, such as the model (NiFe) hydrogenase-2 and trimethylamine N-oxide reductase (TorA) systems, acquire their redox cofactors prior to export from the cell and require to be correctly assembled before transport can proceed. It is likely, therefore, that cellular mechanisms exist to prevent premature export of immature substrates. Using a combination of genetic and biochemical approaches including gene knockouts, signal peptide swapping, complementation, and site-directed mutagenesis, we highlight here this crucial 'proofreading' or 'quality control' activity in operation during assembly of complex endogenous Tat substrates. Our experiments successfully uncouple the Tat transport and cofactor-insertion activities of the TorA-specific chaperone TorD and demonstrate unequivocally that TorD recognises the TorA twin-arginine signal peptide. It is proposed that some Tat signal peptides operate in tandem with cognate binding chaperones to orchestrate the assembly and transport of complex enzymes.

Figure 1

HybE is required for hydrogenase-2 assembly and activity. (A) Hydrogenase-2 activities of mutant strains. Strains MC4100 (‘parent'), FTD673 (‘ΔhybE'), RJ608 (‘ΔhybO'), and RJ603 (‘φtorA∷hybO'), which produce a HybO protein bearing the TorA signal peptide, were grown anaerobically in CR medium containing glycerol and fumarate. Washed whole cells were assayed for hydrogen∷BV oxidoreductase activity with units as μmol BV reduced/min/g cells. (B) Western blot analysis of the core hydrogenase-2 αβ dimer HybOC. Strains MC4100 (‘parent'), FTD673 (‘ΔhybE'), and RJ503 (‘ΔhybE, ΔtatC') were cultured anaerobically in CR medium supplemented with glycerol and fumarate. Cells were fractionated into periplasm (P), total membranes (M), and cytoplasm (C) proteins, separated by SDS–PAGE (14% w/v acrylamide), blotted, and challenged with an antihydrogenase-2 serum. The location of the hydrogenase-2 61 kDa α-subunit (HybC) and 35 kDa β-subunit (HybO) are indicated. (C) Western analysis of the hydrogenase-2 α-subunit HybC. Strains MC4100 (‘parent') and FTD673 (‘ΔhybE') were cultured in CR medium supplemented with glycerol and fumarate. Sphaeroplasts were prepared from whole cells (SP) and further fractionated into membrane (M) and cytoplasm (C). Proteins were separated by SDS–PAGE (10% w/v acrylamide), blotted, and challenged with an antihydrogenase-2 serum. The location of the precursor form of the α-subunit (‘pre-HybC') and the C-terminally processed mature form (‘mat-HybC') are indicated. The asterisks denote a nonspecific immunoreactive band.

Figure 2

Signal-swapping implicates TorD as a signal peptide chaperone. (A) Amino-acid sequences of the twin-arginine signal peptides from the E. coli hydrogenase-2 β-subunit (HybO) and the TMAO reductase (TorA). The twin-arginine motifs are boxed, the hydrophobic h-regions are shown in italics, and the signal peptidase-I cleavage sites are indicated by the arrows. (B) Hydrogenase-2 activities in mutant strains. Strains RJ606 (‘ΔhybA'), RJ607 φtorA∷hybO, ΔhybA, that produces a HybO protein bearing the TorA signal peptide, and RJ607-D (‘ΔtorD') were grown anaerobically in CR medium containing glycerol and fumarate. In addition, RJ607 was transformed with the pSU series of plasmids that constitutively overproduce TorD (‘torD⁺⁺'), DmsD (‘dmsD⁺⁺'), YcdY (‘ycdY⁺⁺'), or HybE (‘hybE⁺⁺'), and are grown under identical conditions. Washed whole cells were assayed for hydrogen∷BV oxidoreductase activity with units as μmol BV reduced/min/g cells. (C) Western analysis of TorD and DmsD. Strains RJ607 (φtorA∷hybO, ΔhybA), RJ607-D (‘ΔtorD'), and RJ607 transformed with plasmids overexpressing either torD (‘torD⁺⁺') or dmsD (‘dmsD⁺⁺') were cultured anaerobically in CR medium supplemented with glycerol and fumarate. Cells were harvested and resuspended to a concentration of 100 mg (wet weight)/ml (‘1 × '), and a sample diluted to 10 mg/ml (‘10 × '). Identical volumes of protein samples were separated by SDS–PAGE (14% w/v acrylamide), blotted, and challenged with either anti-TorD (top panel) or anti-DmsD (bottom panel) serum at 1:10 000 dilution. (D) Western analysis of the hydrogenase-2 αβ-dimer HybOC. Strains RJ606 (‘ΔhybA'), RJ607 (‘φtorA∷hybO'), RJ607-T (φtorA∷hybO, ΔhybA, ΔtatABC∷Kan^R; ‘+ ΔtatABC') and RJ607 following transformation with plasmid pSU-torD overexpressing torD (labelled ‘+ torD⁺⁺') were cultured anaerobically in the presence of glycerol and fumarate. Cells were fractionated in periplasm (P), total membranes (M), and cytoplasm (C), proteins separated by SDS–PAGE (14% w/v acrylamide), blotted, and challenged with an anti-hydrogenase-2 serum. The location of the hydrogenase-2 α-subunit (HybC) and β-subunit (HybO) are indicated. The asterisks denotes a nonspecific immunoreactive band.

Figure 3

TorD interacts with the TorA signal peptide. (A) Amino-acid sequences of N-terminal TorA fusions to the adenylate cyclase T18 fragment. All TorA-specific residues are shown in upper case and the twin-arginine motif is shown in upper bold case. Residues are numbered from the extreme N-terminus of clone 1. For the clone 2 sequence, residues derived from the lacZ′ gene on the vector are shown in lower case italics. Signal peptidase cleavage sites are indicated by the arrows. Fusion junctions with the T18 fragment are indicated by the asterisks. Sequences corresponding to the mature TorA protein (signal peptide cleaved) are shaded. (B) TorD interacts with the N-terminus of TorA. Interactions were measured between pT25 plasmid (‘−') or pT25-TorD expressing the TorD fusion (‘+') and either pUT18 or the library isolates clone 1 and clone 2 in the reporter strain BTH101. (C) TorD recognises the TorA signal peptide. Interactions were measured between pT25 plasmid (‘−') or pT25-TorD expressing the TorD fusion (‘+') and the engineered fusions to the complete signal peptide, pSig3, the signal peptide lacking n-region, pSig4, and 49 residues of the mature TorA protein, pMat1, in the reporter strain BTH101.

Figure 4

TorD is a cytoplasmic protein with a complex role in TorA assembly. (A) Western analysis of TorD. Strains MC4100 (‘parent') and FTD100 (‘ΔtorD') were cultured anaerobically in CR medium with glycerol and TMAO. Cells were fractionated into periplasm (P), total membranes (M), and cytoplasm (C), proteins separated by SDS–PAGE (14% w/v acrylamide), blotted, and challenged with anti-TorD serum. (B) TMAO reductase activity. Strains MC4100 (‘parent'), LCB628 (torA⁻), FTD100 (ΔtorD), FTD102 (ΔdmsD), FTD110 (ΔtorD, ΔdmsD), and FTD112 (ΔtorD, ΔdmsD, ΔycdY) were grown anaerobically in CR medium containing glycerol and TMAO. Washed intact cells were assayed for TMAO::BV oxidoreductase activity with units as μmol BV reduced/min/g cells. (C) TMAO reductase activity. Strains MC4100 (‘parent'), FTD100 (ΔtorD), RJ600 (φhybO∷torA) that produces a TorA protein bearing the HybO signal peptide, and RJ600-D (φhybO∷torA, ΔtorD∷Kan^R) were grown anaerobically in CR medium containing glycerol and TMAO. Whole cells were assayed.

Figure 5

Dissection of TorD activity. (A) Cartoon representation of the predicted structures of TorD-family proteins based on the crystal structure of a TorD dimer from S. massilia. An N-terminal domain (hatched) is separated from a C-terminal domain (shaded) by a short ‘hinge' region. ‘Domain swapping' results in the formation of the TorD homodimer. (B) Hydrogenase-2 activity. Strain RJ607 (φtorA∷hybO, ΔhybA) was grown anaerobically in CR medium containing glycerol and fumarate. In addition, the RJ607 strain was transformed with plasmids that constitutively overproduce TorD from pSU-torD (‘torD⁺⁺'), the TorD N-terminal domain from pUNI-NDOM (‘N-dom⁺⁺'), the TorD C-terminal domain from pSU-CDOM (‘C-dom⁺⁺'), or both separated domains together from plasmids pUNI-NDOM and pSU-CDOM (‘N+C⁺⁺'), and hydrogenase-2 activity assayed in intact cells. (C) TMAO reductase activity. Strain FTD112 (ΔtorD, ΔdmsD, ΔycdY) was grown anaerobically in CR medium containing glycerol and TMAO. In addition, the FTD112 strain was transformed with plasmids that constitutively overproduce TorD from pSU-torD (‘torD⁺⁺'), the TorD N-terminal domain from pUNI-NDOM (‘N-dom⁺⁺'), the TorD C-terminal domain from pSU-CDOM (‘C-dom⁺⁺'), or both separated domains together from plasmids pUNI-NDOM and pSU-CDOM (‘N+C⁺⁺'), and TMAO reductase activity assayed in intact cells.

Figure 6

Site-directed mutagenesis of the TorD protein. (A) Expression, production, and relative stability of mutant TorD proteins. The FTD100 strain (ΔtorD) was transformed with the original cloning vector pUNI-PROM (Amp^R), pUNI-torD (‘native'), and the four pUNI-torD derivatives expressing the mutant torD genes (as indicated). Cultures were grown anaerobically in CR medium supplemented with glycerol and TMAO. Cell pellets were harvested, washed, and resuspended to 100 mg (wet weight)/ml. Whole-cell proteins were separated by SDS–PAGE (14% w/v acrylamide), blotted, and challenged with anti-TorD. Identical proportions of cellular protein were loaded in each lane. (B) Hydrogenase-2 activity. Strain RJ607 (φtorA∷hybO, ΔhybA) was grown anaerobically in CR medium containing glycerol and fumarate. The RJ607 strain was transformed with plasmids that constitutively overproduce TorD from pUNI-torD (‘torD⁺⁺') or the four pUNI-torD derivatives expressing the mutant torD genes (as indicated). Whole cells were assayed for hydrogen∷BV oxidoreductase activity with units as μmol BV reduced/min/g cells. (C) TMAO reductase activity. Strain RJ600-D (φhybO∷torA, ΔtorD∷Kan^R) was grown anaerobically in CR medium containing glycerol and TMAO. The RJ600-D strain was transformed with plasmids that constitutively overproduce TorD from pUNI-torD (‘torD⁺⁺') or the four pUNI-torD derivatives producing TorD variants (as indicated). Whole cells were assayed for TMAO::BV oxidoreductase activity with units as μmol BV reduced/min/g cells.

Figure 7

A model for chaperone-mediated proofreading. An apoprotein bearing a twin-arginine signal peptide is released from the ribosome. Step 1: In order to prevent premature export, a proofreading chaperone binds to the twin-arginine signal peptide. In some cases, specific or general chaperones also bind to the mature portion of the protein at this stage. Step 2: Cofactor loading into the mature portion of the protein proceeds. At this stage, binding of partner subunits would also occur, if applicable. Step 3: All chaperones are released. Step 4: Targeting of the precursor to the Tat translocon proceeds. Step 5: Following Tat transport, the signal peptide is cleaved (‘N^*') and the mature protein is released to the periplasm. TatABC model is from Palmer et al (2004).