The Chloroplast Twin Arginine Transport (Tat) Component,Tha4, Undergoes Conformational Changes Leading to Tat Protein Transport

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.     

细菌蛋白质Tat转运系统的研究进展

蛋白质Tat转运系统不同于细菌中普遍存在的Sec转运系统,而与植物叶绿体中蛋白质转运的ΔpH依赖系统相似.通过Tat系统转运的蛋白质底物含有特征性的双精氨酸保守序列核心S/T-R-R-x-F-L-K的信号肽,其h区的疏水性低,c区有由高赖氨酸、高精氨酸构成的避开Sec系统信号,信号肽和成熟蛋白质的组成对蛋白质的转运都有影响.TatA、TatB、TatC和TatE四种蛋白质参与了大肠杆菌的Tat转运系统.被转运的底物蛋白质绝大多数为与细菌厌氧呼吸有关的含氧化还原辅因子的酶,并以折叠形式转运.     

Oligomers of Tha4 organize at the thylakoid Tat translocase during protein transport

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.     

利用荧光蛋白对大肠杆菌蛋白质Tat转运系统的研究

探讨了荧光蛋白作为报告蛋白用于蛋白质转运系统研究的可行性 ,结果表明海葵红色荧光蛋白聚集在细胞质内 ,不能转运至周质空间。而水母绿色荧光蛋白在Tat信号肽和Tat转运酶的共同作用下 ,以折叠形式转运至周质空间。通过荧光定量分析表明信号肽保守序列中的双精氨酸是保证绿色荧光蛋白转运及转运效率所必需的 ,且第二个精氨酸比第一个精氨酸更为重要。同时 ,揭示了Tat信号肽需要一定的高级结构才能行使功能 ;Tat信号肽不仅引导蛋白质的转运 ,而且也参与蛋白质的折叠。因此 ,绿色荧光蛋白是非常理想的报告蛋白 ,可用于研究Tat系统 ,但是海葵红色荧光蛋白易于聚集而不适合于此目的。     

Substrate-gated docking of pore subunit Tha4 in the TatC cavity initiates Tat translocase assembly

The twin-arginine translocase (Tat) transports folded proteins across tightly sealed membranes. cpTatC is the core component of the thylakoid translocase and coordinates transport through interactions with the substrate signal peptide and other Tat components, notably the Tha4 pore-forming component. Here, Cys–Cys matching mapped Tha4 contact sites on cpTatC and assessed the role of signal peptide binding on Tha4 assembly with the cpTatC–Hcf106 receptor complex. Tha4 made contact with a peripheral cpTatC site in nonstimulated membranes. In the translocase, Tha4 made an additional contact within the cup-shaped cavity of cpTatC that likely seeds Tha4 polymerization to form the pore. Substrate binding triggers assembly of Tha4 onto the interior site. We provide evidence that the substrate signal peptide inserts between cpTatC subunits arranged in a manner that conceivably forms an enclosed chamber. The location of the inserted signal peptide and the Tha4–cpTatC contact data suggest a model for signal peptide–gated Tha4 entry into the chamber to form the translocase.     

Direct Interaction between a Precursor Mature Domain and Transport Component Tha4 during Twin Arginine Transport of Chloroplasts

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.The thylakoid membrane of plant chloroplasts possesses two systems working in parallel for the transport of soluble proteins across the bilayer and into the lumen, namely the chloroplast secretory system and the chloroplast twin arginine translocation (cpTat) system (Müller and Klösgen, 2005; Cline and Theg, 2007; Cline and Dabney-Smith, 2008; Albiniak et al., 2012). For both systems, proteins destined for the thylakoid lumen are encoded by nuclear genes, cytoplasmically translated as higher molecular mass precursor proteins containing targeting sequences, and imported into the chloroplast. However, lumen-targeting sequences on precursors directed to the cpTat system contain obligate twin Arg residues on the amino-proximal side of the hydrophobic core, and the precursors are transported in folded conformations. Both systems require energy to drive the translocation process, but the cpTat system relies solely on the transmembrane potential generated by the protonmotive force (PMF) of photosynthesis, whereas the secretory system also relies on ATP hydrolysis (Cline and Theg, 2007). It is estimated that roughly one-half of the lumen proteins contain twin-Arg signal peptides (Peltier et al., 2004; Sun et al., 2004), several of which are involved in photosynthetic processes, such as the 23-kD subunit of the oxygen evolving complex of PSII (OE23; Ifuku et al., 2011), OE17 (Yi et al., 2006), and subunit T of PSII (Kapazoglou et al., 1995) and subunit N of PSI (Haldrup et al., 1999), making the cpTat system a vital pathway for higher plants.Twin arginine translocation (Tat) systems are also found in bacterial plasma membrane, and both thylakoid and bacteria serve as model systems for studies on the Tat pathway mechanism, each providing insight into different aspects of the mechanism and demonstrating important differences between the two. The cpTat machinery contains three membrane-bound components, Tha4, Hcf106, and cpTatC, with homologous proteins TatA, TatB, and TatC in bacteria, respectively. Tha4 and Hcf106 share sequence and structural homology. They both contain an N-terminal single-transmembrane region followed by a hinge region that connects to an amphipathic α-helix and a divergent C-terminal tail. However, they have distinct functions in the cpTat pathway (Sargent et al., 1999; Dabney-Smith et al., 2003). Hcf106 is largely found in complex with cpTatC, together composing the Tat receptor complex in the thylakoid that migrates as an approximately 700-kD complex by blue native (BN)-PAGE, whereas Tha4 is found as a separate homooligomeric complex of approximately 400 kD or less by BN-PAGE. cpTatC contains six transmembrane regions with the N and C termini on the stromal face of the membrane and serves as the initial receptor of the signal peptide in the receptor complex.Translocation occurs in a cyclical fashion. Precursor binds to the cpTatC-Hcf106 receptor complex in an energy-independent manner. Then, in the presence of the PMF, which mainly consists of ∆pH in isolated thylakoids, Tha4 assembles with the precursor-bound receptor complex to form the active translocase. At this point, transport occurs. After transport, Tha4 dissociates from the receptor complex, thus resetting the system for subsequent rounds of translocation (Mori et al., 1999; Cline and Mori, 2001; Mori and Cline, 2002). This regulated assembly of Tha4 and its tight correlation to transport of the precursor suggests that Tha4 has a critical role in the translocation step.Several models of the Tat translocase propose that Tha4 (TatA) serves as the protein-conducting channel. Several characteristics support this hypothesis, including a regulated assembly mechanism, the requirement for Tha4 only at the translocation step (Cline and Mori, 2001), the molar excess of Tha4 over cpTatC and Hcf106 (Mori et al., 2001; Celedon and Cline, 2012), oligomerization of Tha4 at the translocase (Dabney-Smith and Cline, 2009), and observations of channel-like structures of the Escherichia coli TatA in detergent extracts or even in vivo in E. coli cells (Gohlke et al., 2005; Sargent et al., 2006; Berthelmann et al., 2008). However, none of these studies demonstrate a direct interaction between precursor and Tha4 (TatA).Studies on the E. coli Tat system demonstrate weak cross links between TatA and precursor but did not follow the interaction during active transport, as the UV-inducible cross linking occurred after transport (Maurer et al., 2010). The question still remains how Tha4 (TatA) is directly involved in the translocation event itself. If Tha4 serves the role of protein-conducting channel, one would expect that as the precursor passes through the channel it would interact with Tha4. To test this hypothesis, we have employed an alternative cross-linking strategy involving disulfide exchange cross linking by generating Cys-containing variants of both precursor and Tha4 in pea (Pisum sativum). This method allows probing of the interactions between precursor and Tha4 in the steps immediately prior to and during the transport of precursor, unlike other cross-linking methods employed previously. Through one-to-one disulfide bond formation between single Cys residues placed throughout the mature domain of pOE17 and Tha4, we determined that Tha4 is in direct contact with full-length precursor after its binding the receptor and immediately prior to or during transport across the membrane. BN-PAGE demonstrated that Cys-substituted Tha4 was able to relocate into the approximately 700-kD complex in the presence of Cys-substituted precursor, demonstrating that direct interaction between the two occurs as part of the active translocase. Moreover, site-specific Cys mutations allow us to determine, to our knowledge for the first time, the region of Tha4 in contact with precursor during transport. How these data affect current models for protein transport by the cpTat pathway are discussed.     

Requirement of a Tha4-conserved transmembrane glutamate in thylakoid Tat translocase assembly revealed by biochemical complementation

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.     

Mechanistic Aspects of Folded Protein Transport by the Twin Arginine Translocase (Tat)

The twin arginine translocase (Tat) transports folded proteins of widely varying size across ionically tight membranes with only 2–3 components of machinery and the proton motive force. Tat operates by a cycle in which the receptor complex combines with the pore-forming component to assemble a new translocase for each substrate. Recent data on component and substrate organization in the receptor complex and on the structure of the pore complex inform models for translocase assembly and translocation. A translocation mechanism involving local transient bilayer rupture is discussed.     

C-Terminal Helical Domains of Dengue Virus Type 4 E Protein Affect the Expression/Stability of prM Protein and Conformation of prM and E Proteins

Background

The envelope (E) protein of dengue virus (DENV) is the major immunogen for dengue vaccine development. At the C-terminus are two α-helices (EH1 and EH2) and two transmembrane domains (ET1 and ET2). After synthesis, E protein forms a heterodimer with the precursor membrane (prM) protein, which has been shown as a chaperone for E protein and could prevent premature fusion of E protein during maturation. Recent reports of enhancement of DENV infectivity by anti-prM monoclonal antibodies (mAbs) suggest the presence of prM protein in dengue vaccine is potentially harmful. A better understanding of prM-E interaction and its effect on recognition of E and prM proteins by different antibodies would provide important information for future design of safe and effective subunit dengue vaccines.

Methodology/Principal Findings

In this study, we examined a series of C-terminal truncation constructs of DENV4 prME, E and prM. In the absence of E protein, prM protein expressed poorly. In the presence of E protein, the expression of prM protein increased in a dose-dependent manner. Radioimmunoprecipitation, sucrose gradient sedimentation and pulse-chase experiments revealed ET1 and EH2 were involved in prM-E interaction and EH2 in maintaining the stability of prM protein. Dot blot assay revealed E protein affected the recognition of prM protein by an anti-prM mAb; truncation of EH2 or EH1 affected the recognition of E protein by several anti-E mAbs, which was further verified by capture ELISA. The E protein ectodomain alone can be recognized well by all anti-E mAbs tested.

Conclusions/Significance

A C-terminal domain (EH2) of DENV E protein can affect the expression and stability of its chaperone prM protein. These findings not only add to our understanding of the interaction between prM and E proteins, but also suggest the ectodomain of E protein alone could be a potential subunit immunogen without inducing anti-prM response.     

C-Terminal Protein Characterization by Mass Spectrometry: Isolation of C-Terminal Fragments from Cyanogen Bromide-Cleaved Protein

A sample preparation method for protein C-terminal peptide isolation from cyanogen bromide (CNBr) digests has been developed. In this strategy, the analyte was reduced and carboxyamidomethylated, followed by CNBr cleavage in a one-pot reaction scheme. The digest was then adsorbed on ZipTip_C18 pipette tips for conjugation of the homoserine lactone-terminated peptides with 2,2′-dithiobis (ethylamine) dihydrochloride, followed by reductive release of 2-aminoethanethiol from the derivatives. The thiol-functionalized internal and N-terminal peptides were scavenged on activated thiol sepharose, leaving the C-terminal peptide in the flow-through fraction. The use of reversed-phase supports as a venue for peptide derivatization enabled facile optimization of the individual reaction steps for throughput and completeness of reaction. Reagents were replaced directly on the support, allowing the reactions to proceed at minimal sample loss. By this sequence of solid-phase reactions, the C-terminal peptide could be recognized uniquely in mass spectra of unfractionated digests by its unaltered mass signature. The use of the sample preparation method was demonstrated with low-level amounts of a whole, intact model protein. The C-terminal fragments were retrieved selectively and efficiently from the affinity support. The use of covalent chromatography for C-terminal peptide purification enabled recovery of the depleted material for further chemical and/or enzymatic manipulation. The sample preparation method provides for robustness and simplicity of operation and is anticipated to be expanded to gel-separated proteins and in a scaled-up format to high-throughput protein profiling in complex biological mixtures.     

Dynamic Localization of Tat Protein Transport Machinery Components in Streptomyces coelicolor

The Tat pathway transports folded proteins across the bacterial cytoplasmic membrane and is a major route of protein export in the Streptomyces genus of bacteria. In this study, we have examined the localization of Tat components in the model organism Streptomyces coelicolor by constructing enhanced green fluorescent protein (eGFP) and mCherry fusions with the TatA, TatB, and TatC proteins. All three components colocalized dynamically in the vegetative hyphae, with foci of each tagged protein being prominent at the tips of emerging germ tubes and of the vegetative hyphae, suggesting that this may be a primary site of Tat secretion. Time-lapse imaging revealed that localization of the Tat components was highly dynamic during tip growth and again demonstrated a strong preference for apical sites in growing hyphae. During aerial hypha formation, TatA-eGFP and TatB-eGFP fusions relocalized to prespore compartments, indicating repositioning of Tat components during the Streptomyces life cycle.     

Protein transport by the bacterial Tat pathway

The twin-arginine translocation (Tat) system accomplishes the remarkable feat of translocating large – even dimeric – proteins across tightly sealed energy-transducing membranes. All of the available evidence indicates that it is unique in terms of both structure and mechanism; however its very nature has hindered efforts to probe the core translocation events. At the heart of the problem is the fact that two large sub-complexes are believed to coalesce to form the active translocon, and ‘capturing’ this translocation event has been too difficult. Nevertheless, studies on the individual components have come a long way in recent years, and structural studies have reached the point where educated guesses can be made concerning the most interesting aspects of Tat. In this article we review these studies and the emerging ideas in this field. This article is part of a Special Issue entitled: Protein trafficking and secretion in bacteria. Guest Editors: Anastassios Economou and Ross Dalbey.     

Structures of the Gasdermin D C-Terminal Domains Reveal Mechanisms of Autoinhibition

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Process of Protein Transport by the Type III Secretion System

The type III secretion system (TTSS) of gram-negative bacteria is responsible for delivering bacterial proteins, termed effectors, from the bacterial cytosol directly into the interior of host cells. The TTSS is expressed predominantly by pathogenic bacteria and is usually used to introduce deleterious effectors into host cells. While biochemical activities of effectors vary widely, the TTSS apparatus used to deliver these effectors is conserved and shows functional complementarity for secretion and translocation. This review focuses on proteins that constitute the TTSS apparatus and on mechanisms that guide effectors to the TTSS apparatus for transport. The TTSS apparatus includes predicted integral inner membrane proteins that are conserved widely across TTSSs and in the basal body of the bacterial flagellum. It also includes proteins that are specific to the TTSS and contribute to ring-like structures in the inner membrane and includes secretin family members that form ring-like structures in the outer membrane. Most prominently situated on these coaxial, membrane-embedded rings is a needle-like or pilus-like structure that is implicated as a conduit for effector translocation into host cells. A short region of mRNA sequence or protein sequence in effectors acts as a signal sequence, directing proteins for transport through the TTSS. Additionally, a number of effectors require the action of specific TTSS chaperones for efficient and physiologically meaningful translocation into host cells. Numerous models explaining how effectors are transported into host cells have been proposed, but understanding of this process is incomplete and this topic remains an active area of inquiry.     

The C-Terminal Domains of Adenovirus Serotype 5 Protein IX Assemble into an Antiparallel Structure on the Facets of the Capsid

Adenovirus serotype 5 protein IX (pIX) has two domains connected by a flexible linker. Three N-terminal domains form triskelions on the capsid facets that cement hexons together, and the C-terminal domains of four monomers form complexes toward the facet periphery. Here we present a cryoelectron microscopy structure of recombinant adenovirus with a peptide tag added to the C terminus of pIX. The structure, made up by several C termini of pIX, is longer at both ends than the wild-type protein, and Fabs directed against the tag bind to both ends of the oligomer, demonstrating that the pIX C termini associate in an antiparallel manner.     

Regulation of Long-Term Plasticity Induction by the Channel and C-Terminal Domains of GluN2 Subunits

Conventional long-term potentiation (LTP) and long-term depression (LTD) are induced by different patterns of synaptic stimulation, but both forms of synaptic modification require calcium influx through NMDA receptors (NMDARs). A prevailing model (the “calcium hypothesis”) suggests that high postsynaptic calcium elevation results in LTP, whereas moderate elevations give rise to LTD. Recently, additional evidence has come to suggest that differential activation of NMDAR subunits also factors in determining which type of plasticity is induced. While the growing amount of data suggest that activation of NMDARs containing specific GluN2 subunits plays an important role in the induction of plasticity, it remains less clear which subunit is tied to which form of plasticity. Additionally, it remains to be determined which properties of the subunits confer upon them the ability to differentially induce long-term plasticity. This review highlights recent studies suggesting differential roles for the subunits, as well as findings that begin to shed light on how two similar subunits may be linked to the induction of opposing forms of plasticity.     

The Lateral Gate of SecYEG Opens during Protein Translocation

The SecYEG translocon of Escherichia coli mediates the translocation of preproteins across the cytoplasmic membrane. Here, we have examined the role of the proposed lateral gate of the translocon in translocation. A dual cysteine cross-linking approach allowed the introduction of cross-linker arms of various lengths between adjoining aminoacyl positions of transmembrane segments 2b and 7 of the lateral gate. Oxidation and short spacer linkers that fix the gate in the closed state abolished preprotein translocation, whereas long spacer linkers support translocation. The cross-linking data further suggests that SecYEG lateral gate opening and activation of the SecA ATPase are coupled processes. It is concluded that lateral gate opening is a critical step during SecA-dependent protein translocation.Translocation of preproteins across the cytoplasmic membrane in Escherichia coli is mediated by the Sec translocase (for a recent review see Ref. 1). Preproteins targeted for secretion contain a signal sequence that is removed upon translocation. Their synthesis and translocation are uncoupled events (2), and directly after synthesis at the ribosomes, preproteins are targeted post-translationally to the Sec translocase by the molecular chaperone SecB (3). SecB transfers the preprotein to the motor protein SecA bound at the SecYEG pore complex (4, 5). SecA utilizes cycles of ATP binding and hydrolysis to bind and release the translocating protein resulting in its stepwise translocation across the membrane (6–8). In addition, the proton motive force facilitates translocation when the preprotein is released by SecA (6, 9). Various models for SecA-mediated translocation have been proposed wherein SecA functions as a power-stroke device (10) or as a directed molecular ratchet wherein SecA controls the opening and closure of the pore (11). Another view is that SecA thrusts deep into the SecYEG channel during translocation (12, 13). In a recent study on the co-crystallization of the Thermotoga maritima SecA with SecYEG, it was suggested that a two-helix finger from the helical scaffold domain of SecA inserts into the cytoplasmic domain of SecY, utilizing cycles of ATP hydrolysis to push the substrate into the SecY pore (14).The translocation pore consists of three integral membrane proteins SecY, SecE, and SecG as subunits (15), and this organization is universally conserved in all three kingdoms of life (16). The crystal structure of Methanocaldococcus jannaschii (17) demonstrates that the largest subunit, SecY, consists of an N- and C-terminal domain that comprise TMs3 1–5 and 6–10, respectively. These two domains are organized as a clamshell-like structure that encompass an hourglass-shaped central pore. This putative pore is closed at the periplasmic face of the membrane by a short transmembrane helix, TM2a, which has been proposed to function as a plug domain. The clamshell-like structure of SecY is embraced by SecE that in its minimal form consists of a surface-localized amphiphatic helix and a highly tilted transmembrane segment that localizes to the “back” of the SecY protein. It has been proposed that the “front” of SecY creates a lateral opening of the central pore to the membrane between TM2b and TM7 and that this gate is used to release signal sequences and transmembrane segments from the translocase (17). Cryoelectron microscopy of the E. coli SecYEG complex bound to a translating ribosome (18) suggests that the ribosome-bound SecYEG is organized as a dimer with a front-to-front organization (18). It was proposed that individual pores of the dimer have distinct functions in protein translocation, i.e. vectorial protein translocation and lateral release of TMs into the membrane (19). Freeze-fracture rotational shadowing electron microscopy has provided evidence for oligomeric forms of SecYEG, and suggest that SecA recruits SecYEG monomers to form a dimeric complex (20). Within this dimeric SecYEG complex, only a single pore seems sufficient for the translocation of preproteins (10).The mechanism by which the translocase coordinates protein translocation is only poorly understood. SecA has been proposed to insert the signal sequence into the SecYEG pore where it may latch between TM2b and TM7 of the SecY lateral gate. This would result in a widening of the central pore constriction and a subsequent displacement of the periplasmic plug domain. Next, adjoining polypeptide segments of the preprotein may enter the opened aqueous pore, but it is not clear if under those conditions the lateral gate remains open or is closed. Despite this vast amount of experimental data available on the function of the SecYEG complex, the exact role of the putative lateral gate remains unknown. Thus far, the only study on its dynamics and role during translocation concerns a molecular dynamics simulation (21) that does not take SecA or ribosome binding into account. Here we have investigated the function and the dynamics of the proposed lateral gate located between TM2b and TM7 in protein translocation. The data demonstrates that the lateral gate needs to open to allow for SecA-mediated preprotein translocation.     

Structural and Functional Modularity of the Orange Carotenoid Protein: Distinct Roles for the N- and C-Terminal Domains in Cyanobacterial Photoprotection

The orange carotenoid protein (OCP) serves as a sensor of light intensity and an effector of phycobilisome (PB)–associated photoprotection in cyanobacteria. Structurally, the OCP is composed of two distinct domains spanned by a single carotenoid chromophore. Functionally, in response to high light, the OCP converts from a dark-stable orange form, OCP^O, to an active red form, OCP^R. The C-terminal domain of the OCP has been implicated in the dynamic response to light intensity and plays a role in switching off the OCP’s photoprotective response through its interaction with the fluorescence recovery protein. The function of the N-terminal domain, which is uniquely found in cyanobacteria, is unclear. To investigate its function, we isolated the N-terminal domain in vitro using limited proteolysis of native OCP. The N-terminal domain retains the carotenoid chromophore; this red carotenoid protein (RCP) has constitutive PB fluorescence quenching activity comparable in magnitude to that of active, full-length OCP^R. A comparison of the spectroscopic properties of the RCP with OCP^R indicates that critical protein–chromophore interactions within the C-terminal domain are weakened in the OCP^R form. These results suggest that the C-terminal domain dynamically regulates the photoprotective activity of an otherwise constitutively active carotenoid binding N-terminal domain.     

Transport of cytochrome c derivatives by the bacterial Tat protein translocation system

An experimental system developed previously for the heterologous expression of c-type cytochromes in Escherichia coli Q1has been adapted to monitor protein transfer across the bacteria's cytoplasmic membrane. Apocytochrome, lacking the haem cofactor and probably in an unfolded state, was readily transferred across the cytoplasmic membrane when fused to a Sec-specific signal peptide. Furthermore, cytochrome fused to a signal peptide regarded as specific for the twin arginine transport (Tat) system was translocated in an unfolded state by the Sec apparatus. After maturation and folding in the cytoplasm, Tat-mediated transfer of holocytochrome to the periplasm occurred. We conclude that, in addition to the nature of the specific signal peptide, the folding state of a particular protein also governs its acceptance by a given transport system.