Interactions of Tat pathway-related chaperone proteins

The twin-arginine transport pathway is a protein-targeting system dedicated to the transmembrane translocation of fully folded proteins. In bacteria, proteins are targeted to a membrane-embedded Tat translocase by specialized N-terminal twin-arginine signal peptides bearing an SRRXFLK amino acid motif. Complex cofactor-containing Tat substrates, such as DmsA and TorA, acquire their redox cofactors prior to export from the cell and have to be correctly assembled before transport can proceed. It is therefore likely that cellular mechanisms exist to prevent premature export of immature substrates. In this study, we demonstrate that the Tat pathway related chaperones DmsD and TorD specifically bind to the signal sequences of their corresponding substrates, i.e., ssDmsA and ssTorA, respectively. We propose that this interaction may orchestrate the availability of the target protein to the membrane-embedded Tat translocase. Introduction The twin arginine transport system is present in plant chloroplasts and many prokaryotes. In Escherichia coli, it was first identified as a transporter of redox enzymes that require cofactor insertion and assembly in the cytoplasm prior to transport (1). Later, a broader range of proteins was added to the list of Tat substrates. This list also varies among different organisms. Similar to Sec substrates, Tat substrates bear a transport-essential N-terminal signal sequence that consists of a positively charged N-region, a hydrophobic H-region and a short polar C-region. However, Tat signal sequences bear several additional features (2): their Nregions are usually longer; a consensus S/TRRxFLK motif is found at the boundary between Nand H-regions, which contains the highly conserved name-giving RR-pair; the H-regions of Tat signals are less hydrophobic than the ones of Sec signals; and finally the C-regions of Tat signals usually contain at least one positively charged amino acid, which is sometimes referred to as the “Sec-avoidance motif”. The E. coli Tat translocon consists of TatA/TatE, TatB, and TatC proteins (3). There is currently no explicit evidence for the presence of essential factors additional to TatA/EBC that are productive for transport. However, there are instances where cytoplasmic chaperones play a pivotal role along the Tat pathway (for review see (4)). Recent evidence demonstrates E. coli DnaK to be directly involved in the Tat pathway (5, 6). But the involvement of other general E. coli chaperones, like GroEL, SlyD, etc in the Tat pathway remains obscure owing to their largely overlapping functions. In general, it is envisioned that chaperones (or chaperone-like proteins) may participate in folding and (or) proofreading. It also remains possible that Tat pathway-related chaperones exists to protect the signal sequences from unwanted interactions and (or) assist their targeting to the inner membrane. Interaction of Tat chaperone 35 In addition to the known general chaperones, a class of substrate-specific “redox enzyme maturation proteins (REMPs)” has been identified ( reviewed in (7)). DmsD, one of the best characterized REMPs, has been shown to bind to the Tat signal peptide of its cognate redox enzyme, DmsA (8). Later, other REMPs have been shown to possess similar cognate signal peptide binding nature. According to this finding, REMPs are proposed to constitute parts of the Tat pathway. However, their role is currently under debate. Initially, it was proposed that the role of DmsD is to direct DmsA to the Tat translocon (9). However, more recent data show that DmsD does not function as a targeting factor (10). Instead, it has been proposed to perform a proofreading function (11), by binding to the DmsA signal sequence and rendering it unavailable for export until DmsA cofactor attachment has been completed. In a separate study, binding of TorD to its cognate TorA signal sequence is demonstrated to act in protection against proteolytic degradation that will hamper export of TorA (12). The work presented here aims to shed some light on the nature of the interactions between Tat substrates and REMPs. Most previous studies only employ synthetic signal peptides as substrates to study in vitro interactions involving Tat pathway-related chaperones. For interaction experiments we used artificial substrates based on the green fluorescent protein (GFP) fused to different signal sequences (13). This is the first instance where intact preproteins are presented as the substrates, aiming at a more native behavior of the signal peptide. Materials and Methods Plasmids and bacterial strains: To express GFP-based artificial substrates, vector pTYB11 (New England Biolabs) has been used to fuse a cleavable 55 kDa Intein tag to various signal peptides and then to GFP (Table 1). To express TorD and DmsD, E. coli torD and dmsD genes were amplified from the genome and cloned with N-terminal His tag fusions into pET16b vector (Invitrogen). The E. coli strains used in this study are MC4100 (araD139 (lacIPOZYA-arfF) U169 rpsl thi) and BL21(DE3)* cells. Table 1: List of plasmids used in the study Plasmid Reference Protein of interest pTYB11-T1 (13) ssTorA-GFP with self cleavable Intein tag pTYB11-T2 This work ssTorA(KKK)-GFP with self cleavable Intein tag pTYB11-D1 (13) ssDmsA-GFP with self cleavable Intein tag pET16b-T This work N-terminally His-tagged TorD pET16b-D This work N-terminally His-tagged DmsD Protein expression and purification: For large-scale purification, the intein-fused signal GFPs were expressed in E. coli BL21(DE3)* cells and column purified as described in our previous work (13). His-tagged TorD and DmsD were also expressed in E. coli BL21(DE3)* cells and column purified as recommended in the manual, “The Qiaexpressionist” (Qiagen). Analysis of TorD localization: Cells expressing TorD were harvested from 1 litre cultures grown in LB medium for 12 h at 37C. The harvested cell pellet was suspended in a 10-fold (w/v) excess of buffer A (20 mM Hepes, pH 7.6, 100mM NaCl, 3mM EDTA). The cell suspension was passed twice at 16,000 p.s.i. through a French Pressure cell and centrifuged at 10,000 rpm for 20 min to remove intact cells, particulate, and protein aggregates. Next, the clarified cell free extract was centrifuged at 45,000 rpm 138,000 x g for 90 min to separate the cytoplasmic and membrane fractions. The membrane fractions were resuspended and thoroughly washed in a 20-fold (w/v) excess of buffer A by 5 min of homogenization using a hand-held Wheaton 5-ml homogenizer. The membrane suspensions were centrifuged at 140,000 x g for 35 min to obtain the soluble and washed membrane fractions. Washed membranes were resuspended by homogenization in a 10-fold (w/v) excess of buffer A. All centrifugations were performed at 4 °C, and an aliquot of each fraction was kept. SDS-PAGE (12%) was performed using 50 μg of total protein from the fractions of interest: the cytoplasmic, membrane, and washed membrane fractions. Western blotting employing anti His5 antibody was performed to detect TorD in various fractions. SPR measurements: SPR measurements were performed on a Biacore 2000 system (Biacore AB, Uppsala, Sweden) at 25°C and a flow rate of 10 l/min. All solutions were freshly prepared, degassed, and filtered through 0.22 μm pores. NTA chips providing affinity for Ni-ions were used to study interaction between preprotein and REMPs. In each cycle on the NTA chip, 2 mM NiCl2 was injected to reach 50 RUs, followed by injection of His-tagged protein with a constant flow of buffer A , supplemented with 0.002% P20, to immobilize up to 2000 RUs. The chip was regenerated with 250 mM EDTA after each binding assay before the next cycle. Sensorgrams were run in duplicate. L1 chips were used to study protein interactions with membranes. Small unilamellar vesicles (SUVs) were generated from lipids mimicking the E. coli inner membrane composition as described in our previous work (13). SUVs were spread and immobilized on L1 sensor chips (13). After completion of an experiment, the surface of the chip was regenerated by washing with 40 mM N-octyl -D-glucopyranoside.