Umesh K. Bageshwar

Two electrical potential-dependent steps are required for transport by the Escherichia coli Tat machinery.

The twin-arginine translocation (Tat) pathway in Escherichia coli transports fully folded and assembled proteins across the energy-transducing periplasmic membrane. In chloroplasts, Tat transport requires energy input only from the proton motive force. To elucidate the mechanism and energetics of bacterial Tat protein transport, we developed an efficient in vitro transport assay using TatABC-enriched inverted membrane vesicles and the physiological precursor pre-SufI. We report transport efficiencies of 60–80% for nanomolar pre-SufI concentrations. Dissipation of the pH gradient does not reduce pre-SufI transport efficiency. Instead, pre-SufI transport requires at least two electrical potential (Δψ)–dependent steps that differ in both the duration and minimum magnitude of the required Δψ. The data are consistent with a model in which a substantial Δψ of short duration is required for an early transport step, and in which a small Δψ of long duration is necessary to drive a later transport step.

Interconvertibility of Lipid- and Translocon-bound Forms of the Bacterial Tat Precursor pre-SufI

Signal peptides target protein cargos for secretion from the bacterial cytoplasm. These signal peptides contain a tri‐partite structure consisting of a central hydrophobic domain (h‐domain), and two flanking polar domains. Using a recently developed in vitro transport assay, we report here that a central h‐domain position (C17) of the twin arginine translocation (Tat) substrate pre‐SufI is especially sensitive to amino acid hydrophobicity. The C17I mutant is transported more efficiently than wild type, whereas charged substitutions completely block transport. Transport efficiency is well‐correlated with Tat translocon binding efficiency. The precursor protein also binds to non‐Tat components of the membrane, presumably to the lipids. This lipid‐bound precursor can be chased through the Tat translocons under conditions of high proton motive force. Thus, the non‐Tat bound form of the precursor is a functional intermediate in the transport cycle. This intermediate appears to directly equilibrate with the translocon‐bound form of the precursor.

Kinetics of Precursor Interactions with the Bacterial Tat Translocase Detected by Real-time FRET

Background: The Tat machinery transports folded proteins from the bacterial cytoplasm to the periplasm. Results: A Δψ is required for a Tat cargo to move away from the TatBC receptor complex. Conclusion: Cargo migration away from the TatBC complex requires a Δψ-dependent assembly step or conformational change. Significance: Cargo migration from the TatBC receptor is a major rate-limiting step of Tat transport. The Escherichia coli twin-arginine translocation (Tat) system transports fully folded and assembled proteins across the inner membrane into the periplasmic space. Traditionally, in vitro protein translocation studies have been performed using gel-based transport assays. This technique suffers from low time resolution, and often, an inability to distinguish between different steps in a continuously occurring translocation process. To address these limitations, we have developed an in vitro FRET-based assay that reports on an early step in the Tat translocation process in real-time. The natural Tat substrate pre-SufI was labeled with Alexa532 (donor), and the fluorescent protein mCherry (acceptor) was fused to the C terminus of TatB or TatC. The colored Tat proteins were easily visible during purification, enabling identification of a highly active inverted membrane vesicle (IMV) fraction yielding transport rates with NADH almost an order of magnitude faster than previously reported. When pre-SufI was bound to the translocon, FRET was observed for both Tat proteins. FRET was diminished upon addition of nonfluorescent pre-SufI, indicating that the initial binding step is reversible. When the membranes were energized with NADH, the FRET signal was lost after a short delay. These data suggest a model in which a Tat cargo initially associates with the TatBC complex, and an electric field gradient is required for the cargo to proceed to the next stage of transport. This cargo migration away from the TatBC complex requires a significant fraction of the total transport time.

Bacterial Sec Protein Transport Is Rate-limited by Precursor Length: A Single Turnover Study

An in vitro real-time single turnover assay for the Escherichia coli Sec transport system was developed based on fluorescence dequenching. This assay corrects for the fluorescence quenching that occurs when fluorescent precursor proteins are transported into the lumen of inverted membrane vesicles. We found that 1) the kinetics were well fit by a single exponential, even when the ATP concentration was rate-limiting; 2) ATP hydrolysis occurred during most of the observable reaction period; and 3) longer precursor proteins transported more slowly than shorter precursor proteins. If protein transport through the SecYEG pore is the rate-limiting step of transport, which seems likely, these conclusions argue against a model in which precursor movement through the SecYEG translocon is mechanically driven by a series of rate-limiting, discrete translocation steps that result from conformational cycling of the SecA ATPase. Instead, we propose that precursor movement results predominantly from Brownian motion and that the SecA ATPase regulates pore accessibility.

Position-dependent Effects of Polylysine on Sec Protein Transport

Background: The Sec machinery transports proteins from the bacterial cytoplasm to the periplasm. Results: Protein translocation kinetics are single exponential when multiple polylysine sites are simultaneously introduced into precursor proteins. Conclusion: Translocation through the SecYEG pore is not the rate-limiting step of transport. Significance: These data argue against the SecA motor model. The bacterial Sec protein translocation system catalyzes the transport of unfolded precursor proteins across the cytoplasmic membrane. Using a recently developed real time fluorescence-based transport assay, the effects of the number and distribution of positive charges on the transport time and transport efficiency of proOmpA were examined. As expected, an increase in the number of lysine residues generally increased transport time and decreased transport efficiency. However, the observed effects were highly dependent on the polylysine position in the mature domain. In addition, a string of consecutive positive charges generally had a more significant effect on transport time and efficiency than separating the charges into two or more charged segments. Thirty positive charges distributed throughout the mature domain resulted in effects similar to 10 consecutive charges near the N terminus of the mature domain. These data support a model in which the local effects of positive charge on the translocation kinetics dominate over total thermodynamic constraints. The rapid translocation kinetics of some highly charged proOmpA mutants suggest that the charge is partially shielded from the electric field gradient during transport, possibly by the co-migration of counter ions. The transport times of precursors with multiple positively charged sequences, or “pause sites,” were fairly well predicted by a local effect model. However, the kinetic profile predicted by this local effect model was not observed. Instead, the transport kinetics observed for precursors with multiple polylysine segments support a model in which translocation through the SecYEG pore is not the rate-limiting step of transport.

Identification, cDNA cloning, expression, crystallization and preliminary X-ray analysis of an exceptionally halotolerant carbonic anhydrase from Dunaliella salina

An extracellular alpha-type carbonic anhydrase (dCAII) from the salt-tolerant alga Dunaliella salina differs from its mesophilic counterparts in remaining active from zero to multimolar salt concentrations. To gain insight into the outstanding salt tolerance of dCAII, the enzyme was functionally overexpressed in Escherichia coli, purified by affinity chromatography and crystallized by the hanging-drop method. The crystals belonged to space group P2(1), with unit-cell parameters a = 47.0, b = 119.9, c = 58.5 A, beta = 94.2 degrees. Data from a single crystal were collected to 2.4 A resolution under cryogenic conditions (120 K) using an R-AXIS IV(++) detector mounted on a Rigaku RU-H3R rotating-anode generator. The asymmetric unit contains two molecules of the protein, which corresponds to V(M) = 2.65 A(3) Da(-1) and a solvent content of 52.7%.

A Hinged Signal Peptide Hairpin Enables Tat-Dependent Protein Translocation

The Tat machinery catalyzes the transport of folded proteins across the bacterial cytoplasmic membrane and the thylakoid membrane in plants. Using fluorescence quenching and cross-linking approaches, we demonstrate that the Escherichia coli TatBC complex catalyzes insertion of a pre-SufI signal peptide hairpin that penetrates about halfway across the membrane bilayer. Analysis of 512 bacterial Tat signal peptides using secondary structure prediction and docking algorithms suggest that this hairpin interaction mode is generally conserved. An internal cross-link in the signal peptide that blocks transport but does not affect binding indicates that a signal peptide conformational change is required during translocation. These results suggest, to our knowledge, a novel hairpin-hinge model in which the signal peptide hairpin unhinges during movement of the mature domain across the membrane. Thus, in addition to enabling the necessary recognition, the interaction of Tat signal peptides with the receptor complex plays a critical role in the transport process itself.

Effect of cargo size and shape on the transport efficiency of the bacterial Tat translocase

The Tat machinery translocates fully‐folded and oligomeric substrates. The passage of large, bulky cargos across an ion‐tight membrane suggests the need to match pore and cargo size, and therefore that Tat transport efficiency may depend on both cargo size and shape. A series of cargos of different sizes and shapes were generated using the natural Tat substrate pre‐SufI as a base. Four (of 17) cargos transported with significant (>20% of wild‐type) efficiencies. These results indicate that cargo size and shape significantly influence Tat transportability.