Pathway specificity for a delta pH-dependent precursor thylakoid lumen protein is governed by a ''Sec-avoidance'' motif in the transfer peptide and a ''Sec-incompatible'' mature protein.

Cleavable N-terminal targeting signals direct the translocation of lumenal proteins across the chloroplast thylakoid membrane by either a Sec-type or delta pH-driven protein translocase. The targeting signals specify choice of translocation pathway, yet all resemble typical bacterial 'signal' peptides in possessing a charged N-terminus (N-domain), hydrophobic core region (H-domain) and more polar C-terminal region (C-domain). We have previously shown that a twin-arginine motif in the N-domain is essential for targeting by the delta pH-dependent pathway, but it has remained unclear why targeting signals for this system (transfer peptides) are not recognized by the Sec apparatus. We show here that the conserved charge distribution around the H-domain in the 23K transfer peptide (twin-Arg in the N-domain, Lys in the C-domain) constitutes a 'Sec-avoidance' signal. The C-domain Lys, while not important for delta pH-dependent targeting, is the only barrier to Sec-dependent translocation; its removal generates an apparently perfect signal peptide. Conversely, insertion of twin-Arg into the N-domain of a Sec substrate has little effect, as has insertion of a C-domain Lys, but the combined substitutions almost totally block transport. We also show that the 23K mature protein is incapable of being targeted by the Sec pathway, and it is proposed that the role of the Sec-avoidance motif in the transfer peptide is to prevent futile interactions with the Sec apparatus.     

Bacterial proteins carrying twin-R signal peptides are specifically targeted by the delta pH-dependent transport machinery of the thylakoid membrane system

Glucose-fructose oxidoreductase (GFOR), a periplasmic protein of Zymomonas mobilis, is synthesized as a precursor polypeptide with a twin-R signal peptide for Sec-independent protein export in bacteria. In higher plant chloroplasts, twin-R signal peptides are specific targeting signals for the Sec-independent delta pH pathway of the thylakoid membrane system. In agreement with the assumed common phylogenetic origin of the two protein transport mechanisms, GFOR can be efficiently translocated by the delta pH-dependent pathway when analyzed with isolated thylakoid membranes. Transport is sensitive to the ionophore nigericin and competes with specific substrates for the delta pH-dependent transport route. In contrast, neither sodium azide nor enzymatic destruction of the nucleoside triphosphates in the assays affects thylakoid transport of GFOR indicating that the Sec apparatus is not involved in this process. Mutagenesis of the twin-R motif in the GFOR signal peptide prevents membrane translocation of the protein emphasizing the importance of these residues for the transport process.     

Competition between Sec- and TAT-dependent protein translocation in Escherichia coli.

Recently, a new protein translocation pathway, the twin-arginine translocation (TAT) pathway, has been identified in both bacteria and chloroplasts. To study the possible competition between the TAT- and the well-characterized Sec translocon-dependent pathways in Escherichia coli, we have fused the TorA TAT-targeting signal peptide to the Sec-dependent inner membrane protein leader peptidase (Lep). We find that the soluble, periplasmic P2 domain from Lep is re-routed by the TorA signal peptide into the TAT pathway. In contrast, the full-length TorA-Lep fusion protein is not re-routed into the TAT pathway, suggesting that Sec-targeting signals in Lep can override TAT-targeting information in the TorA signal peptide. We also show that the TorA signal peptide can be converted into a Sec-targeting signal peptide by increasing the hydrophobicity of its h-region. Thus, beyond the twin-arginine motif, the overall hydrophobicity of the signal peptide plays an important role in TAT versus Sec targeting. This is consistent with statistical data showing that TAT-targeting signal peptides in general have less hydrophobic h-regions than Sec-targeting signal peptides.     

The thylakoid delta pH-dependent pathway machinery facilitates RR-independent N-tail protein integration

The thylakoidal DeltapH-dependent and bacterial twin arginine transport systems are structurally and functionally related protein export machineries. These recently discovered systems have been shown to transport folded proteins but are not known to assemble integral membrane proteins. We determined the translocation pathway of a thylakoidal FtsH homologue, plastid fusion/protein translocation factor, which is synthesized with a chloroplast-targeting peptide, a hydrophobic signal peptide, and a hydrophobic membrane anchor. The twin arginine motif in its signal peptide and its sole integration requirement of a DeltapH suggested that plastid fusion/protein translocation factor employs the DeltapH pathway. Surprisingly, changing the twin arginine to twin lysine or deleting the signal peptide did not abrogate integration capability or characteristics. Nevertheless, three criteria argue that all three forms require the DeltapH pathway for integration. First, integration was competed by an authentic DeltapH pathway precursor. Second, antibodies to DeltapH pathway component Hcf106 specifically inhibited integration. Finally, chloroplasts from the hcf106 null mutant were unable to integrate Pftf into their thylakoids. Thus, DeltapH pathway machinery facilitates both signal peptide-directed and N-tail-mediated membrane integration and does not strictly require the twin arginine motif.     

Post-translational protein translocation into thylakoids by the Sec and DeltapH-dependent pathways.

Two distinct protein translocation pathways that employ hydrophobic signal peptides function in the plant thylakoid membrane. These two systems are precursor specific and distinguished by their energy and component requirements. Recent studies have shown that one pathway is homologous to the bacterial general export system called Sec. The other one, called the DeltapH-dependent pathway, was originally considered to be unique to plant thylakoids. However, it is now known that homologous transport systems are widely present in prokaryotes and even present in archaea. Here we review these protein transport pathways and discuss their capabilities and mechanisms of operation.     

Tat-dependent protein targeting in prokaryotes and chloroplasts

The twin-arginine translocation (Tat) system operates in the chloroplast thylakoid and the plasma membranes of a wide range of bacteria. It recognizes substrates bearing cleavable signal peptides in which a twin-arginine motif almost invariably plays a key role in recognition by the translocation machinery. These signal peptides are surprisingly similar to those used to specify transport by Sec-type systems, but the Tat pathway differs in fundamental respects from Sec-type and other protein translocases. Its key attribute is its ability to translocate large, fully folded (even oligomeric) proteins across tightly sealed membranes. To date, three key tat genes have been characterised and the first details of the Tat system are beginning to emerge. In this article we review the salient features of Tat systems, with an emphasis on the targeting signals involved, the substrate specificities of Tat systems, our current knowledge of Tat complex structures and the known mechanistic features. Although the article is focused primarily on bacterial systems, we incorporate relevant aspects of plant thylakoid Tat work and we discuss how the plant and bacterial systems may differ in some respects.     

Transport of proteins into chloroplasts. Delineation of envelope "transit" and thylakoid "transfer" signals within the pre-sequences of three imported thylakoid lumen proteins.

The targeting of cytosolically synthesized proteins into the thylakoid lumen is mediated by an aminoterminal pre-sequence consisting of an "envelope transit" and a "thylakoid transfer" signal in tandem. We have investigated the structural characteristics of several thylakoid transfer signals by determining the intermediate sites at which the stromal processing peptidase cleaves to remove the transit sequences. Using this approach we have found that the thylakoid transfer signals of Silene pratensis plastocyanin, 23-kDa oxygen-evolving complex protein from wheat, and 33-kDa oxygen-evolving complex protein from wheat, are 25, 39, and 48 residues in length, respectively. All of the transfer signals contain hydrophobic core sequences and a "-3,-1" motif reminiscent of those found in signal sequences, but the amino-terminal regions of the transfer signals of the 23- and 33-kDa proteins are both longer and more highly charged. The net charge of each amino-terminal region of the transfer sequences is +1, including the amino-terminal amino group. In each case, the stromal processing peptidase cleaves immediately after a positively charged residue, but otherwise the cleavage sites exhibit no common elements of either primary or secondary structure.     

Phylogenetic transfer of organelle genes to the nucleus can lead to new mechanisms of protein integration into membranes

Subunits CFo-I and CFo-II of ATP synthase in chloroplast thylakoid membranes are two structurally and functionally closely related proteins of bitopic membrane topology which evolved from a common ancestral gene. In higher plants, CFo-I still originates in plastid chromosomes (gene: atpF), while the gene for CFo-II (atpG) was phylogenetically transferred to the nucleus. This gene transfer was accompanied by the reorganization of the topogenic signals and the mechanism of membrane insertion. CFo-I is capable of integrating correctly as the mature protein into the thylakoid membrane, whereas membrane insertion of CFo-II strictly depends on a hydrophobic targeting signal in the transit peptide. This requirement is caused by three negatively charged residues at the N-terminus of mature CFo-II which are lacking from CFo-I and which have apparently been added to the protein only after gene transfer has occurred. Accordingly, the CFo-II transit peptide is structurally and functionally equivalent to typical bipartite transit peptides, capable of also translocating hydrophilic lumenal proteins across the thylakoid membrane. In this case, transport takes place by the Sec-dependent pathway, despite the fact that membrane integration of CFo-II is a Sec-independent, and presumably spontaneous, process.     

The Tat protein export pathway

The Tat (twin-arginine translocation) system is a bacterial protein export pathway with the remarkable ability to transport folded proteins across the cytoplasmic membrane. Preproteins are directed to the Tat pathway by signal peptides that bear a characteristic sequence motif, which includes consecutive arginine residues. Here, we review recent progress on the characterization of the Tat system and critically discuss the structure and operation of this major new bacterial protein export pathway.     

Mutations in a signal sequence for the thylakoid membrane identify multiple protein transport pathways and nuclear suppressors

The apparatus that permits protein translocation across the internal thylakoid membranes of chloroplasts is completely unknown, even though these membranes have been the subject of extensive biochemical analysis. We have used a genetic approach to characterize the translocation of Chlamydomonas cytochrome f, a chloroplast-encoded protein that spans the thylakoid once. Mutations in the hydrophobic core of the cytochrome f signal sequence inhibit the accumulation of cytochrome f, lead to an accumulation of precursor, and impair the ability of Chlamydomonas cells to grow photosynthetically. One hydrophobic core mutant also reduces the accumulation of other thylakoid membrane proteins, but not those that translocate completely across the membrane. These results suggest that the signal sequence of cytochrome f is required and is involved in one of multiple insertion pathways. Suppressors of two signal peptide mutations describe at least two nuclear genes whose products likely describe the translocation apparatus, and selected second-site chloroplast suppressors further define regions of the cytochrome f signal peptide.     

Unusual signal peptide directs penicillin amidase from Escherichia coli to the Tat translocation machinery

The recently described Tat protein translocation system in Escherichia coli recognizes its protein substrates by the consensus twin arginine (SRRXFLK) motif in the signal peptide. The signal sequence of E. coli pre-pro-penicillin amidase bears two arginine residues separated by one aspargine and does not resemble the Tat-targeting motif but can nevertheless target the precursor to the Tat pathway. Mutational studies have shown that the hydrophobic core region acts in synergism with the positive charged N-terminal part of the signal peptide as a Tat recognition signal and contributes to the efficient Tat targeting of the pre-pro-penicillin amidase.     

Role of vesicle-inducing protein in plastids 1 in cpTat transport at the thylakoid

VIPP1 has been shown to be required for the proper formation of thylakoid membranes. However, studies on VIPP1 itself, as well as on PspA, its bacterial homolog, suggests that this protein may be involved in a number of additional functions, including protein translocation. The role of VIPP1 in protein translocation in the chloroplast has not been investigated. To this end, we conducted in vitro thylakoid protein transport assays to look at the effect of VIPP1 on the cpTat pathway, which is one of three translocation pathways found in both the chloroplast and its bacterial progenitor. We found that VIPP1 does indeed enhance protein transport through the cpTat pathway by up to 100%. The VIPP1 effect on cpTat activity occurs without interacting with the substrates or components of the translocon, and does not alter the energy potentials driving this translocation pathway. Instead, VIPP1 greatly enhances the amount of substrate bound productively to the thylakoids. Moreover, the presence of increasing VIPP1 concentrations in the reactions resulted in greater interactions between thylakoid membranes. Taken together, these results demonstrate a stimulatory role for VIPP1 in cpTat transport by enhancement of substrate binding, probably to the membrane lipid regions of the thylakoid. We propose a model in which VIPP1 facilitates reorganization of the thylakoid structure to increase substrate access to productive binding regions of the membrane as an early step in the cpTat pathway.     

Thylakoid targeting of Tat passenger proteins shows no delta pH dependence in vivo

The Tat pathway is a major route for protein export in prokaryotes and for protein targeting to thylakoids in chloroplasts. Based on in vitro studies, protein translocation through this pathway is thought to be strictly dependent on a transmembrane delta pH. In this paper, we assess the delta pH sensitivity of the Tat pathway in vivo. Using Chlamydomonas reinhardtii, we observed changes in the efficiency of thylakoid targeting in vivo by mutating the Tat signal of the Rieske protein. We then employed two endogenous pH probes located on the lumen side of the thylakoid membranes to estimate spectroscopically the delta pH in vivo. Using experimental conditions in which the trans-thylakoid delta pH was almost zero, we found no evidence for a delta pH dependence of the Tat pathway in vivo. We confirmed this observation in higher plants using attached barley leaves. We conclude that the Tat pathway does not require a delta pH under physiological conditions, but becomes delta pH sensitive when probed in vitro/in organello because of the loss of some critical intracellular factors.     

The Rieske protein from Paracoccus denitrificans is inserted into the cytoplasmic membrane by the twin-arginine translocase

The Rieske [2Fe-2S] protein (ISP) is an essential subunit of cytochrome bc(1) complexes in mitochondrial and bacterial respiratory chains. Based on the presence of two consecutive arginines, it was argued that the ISP of Paracoccus denitrificans, a Gram-negative soil bacterium, is inserted into the cytoplasmic membrane via the twin-arginine translocation (Tat) pathway. Here, we provide experimental evidence that membrane integration of the bacterial ISP indeed relies on the Tat translocon. We show that targeting of the ISP depends on the twin-arginine motif. A strict requirement is established particularly for the second arginine residue (R16); conservative replacement of the first arginine (R15K) still permits substantial ISP transport. Comparative sequence analysis reveals characteristics common to Tat signal peptides in several bacterial ISPs; however, there are distinctive features relating to the fact that the presumed ISP Tat signal simultaneously serves as a membrane anchor. These differences include an elevated hydrophobicity of the h-region compared with generic Tat signals and the absence of an otherwise well-conserved '+5'-consensus motif lysine residue. Substitution of the +5 lysine (Y20K) compromises ISP export and/or cytochrome bc(1) stability to some extent and points to a specific role for this deviation from the canonical Tat motif. EPR spectroscopy confirms cytosolic insertion of the [2Fe-2S] cofactor. Mutation of an essential cofactor binding residue (C152S) decreases the ISP membrane levels, possibly indicating that cofactor insertion is a prerequisite for efficient translocation along the Tat pathway.     

A universal system for the transport of redox proteins: early roots and latest developments

The transport of proteins binding redox cofactors across a biological membrane is complicated by the fact that insertion of the redox cofactor is often a cytoplasmic process. These cytoplasmically assembled redox proteins must thus be transported in partially or completely folded form. The need for a special transport system for redox proteins was first recognized for periplasmic hydrogenases in gram-negative bacteria. These enzymes, which catalyze the reaction H2 <--> 2H+ + 2e, are composed of a large and a small subunit. Only the small subunit has an unusually long signal sequence of 30-50 amino acid residues, characterized by a conserved motif (S/T)-R-R-x-F-L-K at the N-terminus. This sequence directs export of the large and small subunit complex to the periplasm. Sequencing of microbial genes and genomes has shown that signal sequences with this conserved motif, now referred to as twin-arginine leaders, occur ubiquitously and export different classes of redox proteins, containing iron sulfur clusters, molybdopterin cofactors, polynuclear copper sites or flavin adenine dinucleotide. Mutations in an Escherichia coli operon referred to as mtt (membrane targeting and translocation) or tat (twin arginine translocation) are pleiotropic, i.e. these prevent the expression of a variety of periplasmic oxido-reductases in functional form. The Mtt or Tat pathway is distinct from the well-known Sec pathway and occurs ubiquitously in prokaryotes. The fact that its component proteins share sequence homology with proteins of the delta pH pathway for protein transport associated with chloroplast thylakoid assembly, illustrates the universal nature of this novel protein translocation system.     

Structural determinants of SecB recognition by SecA in bacterial protein translocation

SecB is a bacterial chaperone involved in directing pre-protein to the translocation pathway by its specific interaction with the peripheral membrane ATPase SecA. The SecB-binding site on SecA is located at its C terminus and consists of a stretch of highly conserved residues. The crystal structure of SecB in complex with the C-terminal 27 amino acids of SecA from Haemophilus influenzae shows that the SecA peptide is structured as a CCCH zinc-binding motif. One SecB tetramer is bound by two SecA peptides, and the interface involves primarily salt bridges and hydrogen bonding interactions. The structure explains the importance of the zinc-binding motif and conserved residues at the C terminus of SecA in its high-affinity binding with SecB. It also suggests a model of SecB-SecA interaction and its implication for the mechanism of pre-protein transfer in bacterial protein translocation.     

Proteolytic processing of <Emphasis Type="Italic">Escherichia coli</Emphasis> twin-arginine signal peptides by LepB

The twin-arginine translocation (Tat) apparatus is a protein targeting system found in the cytoplasmic membranes of many prokaryotes. Substrate proteins of the Tat pathway are synthesised with signal peptides bearing SRRxFLK ‘twin-arginine’ amino acid motifs. All Tat signal peptides have a common tripartite structure comprising a polar N-terminal region, followed by a hydrophobic region of variable length and a polar C-terminal region. In Escherichia coli, Tat signal peptides are proteolytically cleaved after translocation. The signal peptide C-terminal regions contain conserved AxA motifs, which are possible recognition sequences for leader peptidase I (LepB). In this work, the role of LepB in Tat signal peptide processing was addressed directly. Deliberate repression of lepB expression prevented processing of all Tat substrates tested, including SufI, AmiC, and a TorA-23K reporter protein. In addition, electron microscopy revealed gross defects in cell architecture and membrane integrity following depletion of cellular LepB protein levels.     

Functional independence of the protein translocation machineries in mitochondrial outer and inner membranes: passage of preproteins through the intermembrane space.

The protein translocation machineries of the outer and inner mitochondrial membranes usually act in concert during translocation of matrix and inner membrane proteins. We considered whether the two machineries can function independently of each other in a sequential reaction. Fusion proteins (pF-CCHL) were constructed which contained dual targeting information, one for the intermembrane space present in cytochrome c heme lyase (CCHL) and the other for the matrix space contained in the signal sequence of the precursor of F1-ATPase beta-subunit (pF1 beta). In the absence of a membrane potential, delta psi, the fusion proteins moved into the intermembrane space using the CCHL pathway. In contrast, in the presence of delta psi they followed the pF1 beta pathway and eventually were translocated into the matrix. The fusion protein pF51-CCHL containing 51 amino acids of pF1 beta, once transported into the intermembrane space in the absence of a membrane potential, could be further chased into the matrix upon re-establishing delta psi. The sequential and independent movement of the fusion protein across the two membranes demonstrates that the translocation machineries act as distinct entities. Our results support a model in which the two translocation machineries can function independently of each other, but generally interact in a dynamic fashion to achieve simultaneous translocation across both membranes. In addition, the results provide information about the targeting sequences within CCHL. The protein does not contain a signal for retention in the intermembrane space; rather, it lacks matrix targeting information, and therefore is unable to undergo delta psi-dependent interaction with the protein translocation apparatus in the inner membrane.     

Effects of total hydrophobicity and length of the hydrophobic domain of a signal peptide on in vitro translocation efficiency.

The hydrophobic domain of the signal peptide of OmpF-Lpp, a model secretory protein, was systematically engineered so as to be composed of different lengths of polyleucine residues or polymers with alternate leucine and alanine residues, and the effects of the length and nature of the hydrophobic stretch on the rate of in vitro translocation were studied using everted membrane vesicles of Escherichia coli. The translocation reaction exhibited high substrate specificity as to the number of hydrophobic residues. The results suggest that the hydrophobic domain is recognized specifically by a component(s) of the secretory machinery rather than nonspecifically by the hydrophobic region of the membrane. The in vitro translocation thus demonstrated required SecA and ATP and was markedly enhanced upon imposition of the proton motive force, as in the case of secretory proteins possessing a natural signal peptide. The highest translocation rate was obtained with the octamer in the case of polyleucine-containing signal peptides, whereas it was the decamer in the case of ones containing both leucine and alanine. These results suggest that the total hydrophobicity of the hydrophobic region of the signal peptides is an important determinant of the substrate specificity.     

A subset of bacterial inner membrane proteins integrated by the twin-arginine translocase

A group of bacterial exported proteins are synthesized with N-terminal signal peptides containing a SRRxFLK 'twin-arginine' amino acid motif. Proteins bearing twin-arginine signal peptides are targeted post-translationally to the twin-arginine translocation (Tat) system which transports folded substrates across the inner membrane. In Escherichia coli, most integral inner membrane proteins are assembled by a co-translational process directed by SRP/FtsY, the SecYEG translocase, and YidC. In this work we define a novel class of integral membrane proteins assembled by a Tat-dependent mechanism. We show that at least five E. coli Tat substrate proteins contain hydrophobic C-terminal transmembrane helices (or 'C-tails'). Fusions between the identified transmembrane C-tails and the exclusively Tat-dependent reporter proteins TorA and SufI render the resultant chimeras membrane-bound. Export-linked signal peptide processing and membrane integration of the chimeras is shown to be both Tat-dependent and YidC-independent. It is proposed that the mechanism of membrane integration of proteins by the Tat system is fundamentally distinct from that employed for other bacterial inner membrane proteins.