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The chloroplast twin arginine translocation (cpTat) system transports highly folded precursor proteins into the thylakoid lumen using the protonmotive force as its only energy source. Hcf106, as one of the core components of the cpTat system, is part of the precursor receptor complex and functions in the initial precursor-binding step. Hcf106 is predicted to contain a single amino terminal transmembrane domain followed by a Pro-Gly hinge, a predicted amphipathic α-helix (APH), and a loosely structured carboxy terminus. Hcf106 has been shown biochemically to insert spontaneously into thylakoid membranes. To better understand the membrane active capabilities of Hcf106, we used solid-state NMR spectroscopy to investigate those properties of the APH. In this study, synthesized peptides of the predicted Hcf106 APH (amino acids 28-65) were incorporated at increasing mol.% into 1-palmitoyl-2-oleoyl-sn-glycero-phosphocholine (POPC) and POPC/MGDG (monogalactosyldiacylglycerol; mole ratio 85:15) multilamellar vesicles (MLVs) to probe the peptide-lipid interaction. Solid-state (31)P NMR and (2)H NMR spectroscopic experiments revealed that the peptide perturbs the headgroup and the acyl chain regions of phospholipids as indicated by changes in spectral lineshape, chemical shift anisotropy (CSA) line width, and (2)H order SCD parameters. In addition, the comparison between POPC MLVs and POPC/MGDG MLVs indicated that the lipid bilayer composition affected peptide perturbation of the lipids, and such perturbation appeared to be more intense in a system more closely mimicking a thylakoid membrane., (© 2013.)

The chloroplast twin arginine translocation system transports highly folded precursor proteins across the thylakoid using the protonmotive force as its only energy source. Hcf106 and another thylakoid protein, cpTatC compose the precursor receptor complex. Hcf106 is predicted to contain a single amino terminal transmembrane domain (TMD) followed by a Pro-Gly hinge, an amphipathic α-helix, and a loosely structured carboxyl terminus. Hcf106 has been shown biochemically to insert spontaneously into thylakoid membranes; however, how this occurs is not understood. To investigate how Hcf106 inserts itself into the membrane unassisted, solid-state NMR spectroscopy was used to investigate the membrane activity of the TMD. A synthetic peptide of the Hcf106 TMD was incorporated into multilamellar vesicles made of 100% 1-palmitoyl-2-oleoyl-sn-glycero-phosphocholine (POPC) or 85%:15% ratio with monogalactosyl diacylglycerol (POPC/MGDG) to probe peptide-lipid interaction. Solid-state (31)P NMR and (2)H NMR spectroscopic techniques were used to reveal peptide perturbations of the phospholipid membranes. Changes in spectral lineshape, chemical shift anisotropy width, (31)P T1 relaxation time and SCD order parameters demonstrated that the Hcf106 TMD peptide interacted with the phospholipids. Furthermore, the comparison between POPC and POPC/MGDG multilamellar vesicles indicated that lipid bilayer composition affected the peptide-lipid interaction with the peptide interacting preferentially with vesicles that more closely mimic the thylakoid., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

Proteins destined for the thylakoid lumen of chloroplasts must cross three membranes en route. The chloroplast twin arginine translocation (cpTat) system facilitates the transport of about one-half of all proteins that cross the thylakoid membrane in chloroplasts. Known mechanistic features of the cpTat system are drastically different from other known translocation systems, notably in its formation of a transient complex to transport fully folded proteins utilizing only the protonmotive force generated during photosynthesis for energy. However, key details, such as the structure and composition of the translocation pore, are still unknown. One of the three transmembrane cpTat components, Tha4, is thought to function as the pore by forming an oligomer. Yet, little is known about the topology of Tha4 in thylakoid, and little work has been done to detect precursor-Tha4 interactions, which are expected if Tha4 is the pore. Here, we present evidence of the interaction of the precursor with Tha4 under conditions leading to transport, using cysteine substitutions on the precursor and Tha4 and disulfide bond formation in pea (Pisum sativum). The mature domain of a transport-competent precursor interacts with the amphipathic helix and amino terminus of functional Tha4 under conditions leading to transport. Detergent solubilization of thylakoids post cross linking and blue-native polyacrylamide gel electrophoresis analysis shows that Tha4 is found in a complex containing precursor and Hcf106 (i.e. the cpTat translocase). Affinity precipitation of the cross-linked complex via Tha4 clearly demonstrates that the interaction is with full-length precursor. How these data suggest a role for Tha4 in cpTat transport is discussed.

Twin arginine transport (Tat) systems transport folded proteins using proton-motive force as sole energy source. The thylakoid Tat system comprises three membrane components. A complex composed of cpTatC and Hcf106 is the twin arginine signal peptide receptor. Signal peptide binding triggers assembly of Tha4 for the translocation step. Tha4 is thought to serve as the protein-conducting element, and the topology it adopts during transport produces the transmembrane passageway. We analyzed Tha4 topology and conformation in actively transporting translocases and compared that with Tha4 in nontransporting membranes. Using cysteine accessibility labeling techniques and diagnostic protease protection assays, we confirm an overall N(OUT)-C(IN) topology for Tha4 that is maintained under transport conditions. Significantly, the amphipathic helix (APH) and C-tail exhibited substantial changes in accessibility when actively engaged in protein transport. Compared with resting state, cysteines within the APH became less accessible to stromally applied modifying reagent. The APH proximal C-tail, although still accessible to Cys-directed reagents, was much less accessible to protease. We attribute these changes in accessibility to indicate the Tha4 conformation that is adopted in the translocase primed for translocation. We propose that in the primed translocase, the APH partitions more extensively and uniformly into the membrane interface and the C-tails pack closer together in a mesh-like network. Implications for the mode by which the substrate protein crosses the bilayer are discussed.

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.

Chloroplasts contain several thousand different proteins, of which more than 95% are encoded on nuclear genes, synthesized in the cytosol as precursor proteins, and imported into the organelle. The major pathways for import and routing have been described; a general import apparatus in the chloroplast envelope and several ancestral translocases in the thylakoid membranes. In this update we focus on some interesting and emerging areas: the Tat translocase, which operates in parallel with the Sec system but transports folded proteins; different routes to the envelope membranes, which promises an understanding of the ways the Tic apparatus sorts transmembrane domains (TMDs) and may also uncover developmental relationships between envelope and thylakoids; and novel routes for proteins into chloroplasts including delivery from the secretory system.

The Tat (twin arginine translocation) systems of thylakoids and bacteria transport fully folded protein substrates without breaching the permeability barrier of the membrane. Two components of the thylakoid system, cpTatC and Hcf106, compose a precursor-bound receptor complex. The third component, Tha4, assembles with the precursor-bound receptor complex for the translocation step and is thought to compose at least part of the protein-conducting channel. Here, we used two different cross-linking approaches to explore the organization of Tha4 in the translocase. These cross-linking techniques showed that transition to an active protein transport state resulted in an alignment of the Tha4 amphipathic helix and C-terminal tail domains to form Tha4 oligomers. Oligomerization required functional Tha4, a twin arginine signal peptide, and an active cpTatC-Hcf106 receptor complex. The spectrum of oligomers obtained was independent of the mature folded domain of the precursor. We propose a trapdoor mechanism for translocation whereby aligned oligomers of Tha4 amphipathic helices fold into the membrane to allow formfitting passage of precursor proteins.

The thylakoid Tat system employs three membrane components and the pH gradient to transport folded proteins. The translocase is signal-assembled, i.e. a receptor complex containing cpTatC and Hcf106 binds the precursor protein, and upon membrane energization, Tha4 is recruited to the precursor-receptor complex to effect translocation. We developed a two-step complementation assay to examine the implied central role of Tha4 in translocation. The first step results in the inactivation of endogenous Tha4 with specific antibodies. The second step involves integrating exogenous Tha4 and presenting the system with precursor protein. We verified this approach by confirming the results obtained recently with the Escherichia coli Tha4 ortholog TatA, i.e. that the carboxyl terminus is dispensable and the amphipathic helix essential for transport. We then investigated a conserved Tha4 transmembrane glutamate in detail. Substitution of glutamate 10 with alanine, glutamine, and even aspartate largely eliminated the ability of Tha4 to complement transport, whereas a conservative substitution elsewhere in the transmembrane domain was without effect. Chemical cross-linking assays showed that the mutated Tha4s failed to be recruited to the receptor complex under transport conditions, indicating a role for the transmembrane glutamate in translocase assembly. This assay promises an avenue into understanding the role of Tha4 in both the assembly and translocation steps of the Tat translocase.