Component specificity for the thylakoidal Sec and Delta pH-dependent protein transport pathways.

Prokaryotes and prokaryote-derived thylakoid membranes of chloroplasts share multiple, evolutionarily conserved pathways for protein export. These include the Sec, signal recognition particle (SRP), and Delta pH/Tat systems. Little is known regarding the thylakoid membrane components involved in these pathways. We isolated a cDNA clone to a novel component of the Delta pH pathway, Tha4, and prepared antibodies against pea Tha4, against maize Hcf106, a protein implicated in Delta pH pathway transport by genetic studies, and against cpSecY, the thylakoid homologue of the bacterial SecY translocon protein. These components were localized to the nonappressed thylakoid membranes. Tha4 and Hcf106 were present in approximately 10-fold excess over active translocation sites. Antibodies to either Tha4 or Hcf106 inhibited translocation of four known Delta pH pathway substrate proteins, but not of Sec pathway or SRP pathway substrates. This suggests that Tha4 and Hcf106 operate either in series or as subunits of a heteromultimeric complex. cpSecY antibodies inhibited translocation of Sec pathway substrates but not of Delta pH or SRP pathway substrates. These studies provide the first biochemical evidence that Tha4 and Hcf106 are specific components of the Delta pH pathway and provide one line of evidence that cpSecY is used specifically by the Sec pathway.     

FtsH2 and FtsH5: two homologous subunits use different integration mechanisms leading to the same thylakoid multimeric complex

The Arabidopsis thylakoid FtsH protease complex is composed of FtsH1/FtsH5 (type A) and FtsH2/FtsH8 (type B) subunits. Type A and type B subunits display a high degree of sequence identity throughout their mature domains, but no similarity in their amino-terminal targeting peptide regions. In chloroplast import assays, FtsH2 and FtsH5 were imported and subsequently integrated into thylakoids by a two-step processing mechanism that resulted in an amino-proximal lumenal domain, a single transmembrane anchor, and a carboxyl proximal stromal domain. FtsH2 integration into washed thylakoids was entirely dependent on the proton gradient, whereas FtsH5 integration was dependent on NTPs, suggesting their integration by Tat and Sec pathways, respectively. This finding was corroborated by in organello competition and by antibody inhibition experiments. A series of constructs were made in order to understand the molecular basis for different integration pathways. The amino proximal domains through the transmembrane anchors were sufficient for proper integration as demonstrated with carboxyl-truncated versions of FtsH2 and FtsH5. The mature FtsH2 protein was found to be incompatible with the Sec machinery as determined with targeting peptide-swapping experiments. Incompatibility does not appear to be determined by any specific element in the FtsH2 domain as no single domain was incompatible with Sec transport. This suggests an incompatible structure that requires the intact FtsH2. That the highly homologous type A and type B subunits of the same multimeric complex use different integration pathways is a striking example of the notion that membrane insertion pathways have evolved to accommodate structural features of their respective substrates.     

Protein transport in Archaea: Sec and twin arginine translocation pathways

The transport of proteins into and across hydrophobic membranes is an essential cellular process. The majority of proteins that are translocated in an unfolded conformation traverse the membrane by way of the universally conserved Sec pathway, whereas the twin arginine translocation pathway is responsible for the transport of folded proteins across the membrane. Structural, biochemical and genetic analyses of these processes in Archaea have revealed unique archaeal features, and have also provided a better understanding of these pathways in organisms of all domains. Further study of these pathways in Archaea might elucidate fundamental principles involved in each type of transport and could help determine their relative costs and benefits as well as evolutionary adaptations in protein secretion strategies.     

Co-operation between different targeting pathways during integration of a membrane protein

Membrane protein assembly is a fundamental process in all cells. The membrane-bound Rieske iron-sulfur protein is an essential component of the cytochrome bc₁ and cytochrome b₆f complexes, and it is exported across the energy-coupling membranes of bacteria and plants in a folded conformation by the twin arginine protein transport pathway (Tat) transport pathway. Although the Rieske protein in most organisms is a monotopic membrane protein, in actinobacteria, it is a polytopic protein with three transmembrane domains. In this work, we show that the Rieske protein of Streptomyces coelicolor requires both the Sec and the Tat pathways for its assembly. Genetic and biochemical approaches revealed that the initial two transmembrane domains were integrated into the membrane in a Sec-dependent manner, whereas integration of the third transmembrane domain, and thus the correct orientation of the iron-sulfur domain, required the activity of the Tat translocase. This work reveals an unprecedented co-operation between the mechanistically distinct Sec and Tat systems in the assembly of a single integral membrane protein.     

Translocation of a phycoerythrin alpha subunit across five biological membranes

Cryptophytes, unicellular algae, evolved by secondary endosymbiosis and contain plastids surrounded by four membranes. In contrast to cyanobacteria and red algae, their phycobiliproteins do not assemble into phycobilisomes and are located within the thylakoid lumen instead of the stroma. We identified two gene families encoding phycoerythrin alpha and light-harvesting complex proteins from an expressed sequence tag library of the cryptophyte Guillardia theta. The proteins bear a bipartite topogenic signal responsible for the transport of nuclear encoded proteins via the ER into the plastid. Analysis of the phycoerythrin alpha sequences revealed that more than half of them carry an additional, third topogenic signal comprising a twin arginine motif, which is indicative of Tat (twin arginine transport)-specific targeting signals. We performed import studies with several derivatives of one member using a diatom transformation system, as well as intact chloroplasts and thylakoid vesicles isolated from pea. We demonstrated the different targeting properties of each individual part of the tripartite leader and show that phycoerythrin alpha is transported across the thylakoid membrane into the thylakoid lumen and protease-protected. Furthermore, we showed that thylakoid transport of phycoerythrin alpha takes place by the Tat pathway even if the 36 amino acid long bipartite topogenic signal precedes the actual twin arginine signal. This is the first experimental evidence of a protein being targeted across five biological membranes.     

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.     

Conservative sorting in the muroplasts of Cyanophora paradoxa: a reevaluation based on the completed genome sequence

After primary endosymbiosis, massive gene transfer occurred from the genome of the cyanobacterial endosymbiont to the nucleus of the protist host cell. In parallel, a specific protein import apparatus arose for reimport of many, but not all products of the genes moved to the nuclear genome. Presequences evolved to allow recognition of plastid proteins at the envelope and their translocation to the stroma. However, plastids (and cyanobacteria) also comprise five other subcompartments. Protein sorting to the cyanobacterial thylakoid membrane, the thylakoid lumen, the inner envelope membrane, the periplasmic space, and the outer envelope membrane is achieved by prokaryotic protein translocases recognizing, e.g., signal sequences. The “conservative sorting” hypothesis postulates that these translocases remained functional in endosymbiotic organelles and obtained their passengers not only from imported proteins but also from proteins synthesized in organello. For proteins synthesized in the cytosol, a collaboration of the general import apparatus and the former prokaryotic translocase is necessary which is often reflected by the use of bipartite presequences, e.g., stroma targeting peptide and signal peptide. For plants, this concept has been experimentally proven and verified. The muroplasts from Cyanophora paradoxa, that have several features more in common with cyanobacteria than with plastids, were analyzed with the availability of the recently completed nuclear genome sequence. Interesting findings include the absence of the post-translational signal recognition particle pathway, dual Sec translocases in thylakoid and inner envelope membranes that are produced from a single set of genes, and a co-translational signal recognition pathway operating without a 4.5S RNA component.     

Precursors bind to specific sites on thylakoid membranes prior to transport on the delta pH protein translocation system

The Delta pH pathway is one of two systems for protein transport to the thylakoid lumen. It is a novel transport system that requires only the thylakoidal DeltapH to power translocation. Several substrates of the Delta pH pathway, including the intermediate precursor form of OE17 (iOE17) and the truncated precursor form of OE17 (tOE17), were shown to bind to the membrane in the absence of the DeltapH and be transported into the lumen when the DeltapH was restored. Binding occurred without energy or soluble factors, and efficient transport from the bound state ( approximately 80-90%) required only the DeltapH. Binding is due to protein-protein interactions because protease pretreatment of thylakoids destroyed their binding capability. Precursors are bound to a specific site on the Delta pH pathway because binding was competed by saturating amounts of Delta pH pathway precursor proteins, but not by a Sec pathway precursor protein. These results suggested that precursor tOE17 binds to components of the Delta pathway translocation machinery. Hcf106 and Tha4 are two components of the Delta pH pathway machinery. Antibodies to Hcf106 or Tha4, when prebound to thylakoids, specifically inhibited precursor transport on the Delta pH pathway. However, only Hcf106 antibodies reduced the level of precursor binding. These results suggest that Hcf106 functions in early steps of the transport process.     

Chloroplast Oxa1p homolog albino3 is required for post-translational integration of the light harvesting chlorophyll-binding protein into thylakoid membranes

Multiple sorting pathways operate in chloroplasts to localize proteins to the thylakoid membrane. The signal recognition particle (SRP) pathway in chloroplasts employs the function of a signal recognition particle (cpSRP) to target light harvesting chlorophyll-binding protein (LHCP) to the thylakoid membrane. In assays that reconstitute stroma-dependent LHCP integration in vitro, the stroma is replaceable by the addition of GTP, cpSRP, and an SRP receptor homolog, cpFtsY. Still lacking is an understanding of events that take place at the thylakoid membrane including the identification of membrane proteins that may function at the level of cpFtsY binding or LHCP integration. The identification of Oxa1p in mitochondria, an inner membrane translocase component homologous to predicted proteins in bacteria and to the albino3 (ALB3) protein in thylakoids, led us to investigate the potential role of ALB3 in LHCP integration. Antibody raised against a 50-amino acid region of ALB3 (ALB3-50aa) identified a single 45-kDa thylakoid protein. Treatment of thylakoids with antibody to ALB3-50aa inhibited LHCP integration, whereas the same antibody treatment performed in the presence of antigen reversed the inhibition. In contrast, transport by the thylakoid Sec or Delta pH pathways was unaffected. These data support a model whereby a distinct translocase containing ALB3 is used to integrate LHCP into thylakoid membranes.     

Double mutation cpSRP43--/cpSRP54-- is necessary to abolish the cpSRP pathway required for thylakoid targeting of the light-harvesting chlorophyll proteins

Biochemical and genetic studies have established that the light-harvesting chlorophyll proteins (LHCPs) of the photosystems use the cpSRP (chloroplast signal recognition particle) pathway for their targeting to thylakoids. Previous analyses of single cpSRP mutants, chaos and ffc, deficient in cpSRP43 and cpSRP54, respectively, have revealed that half of the LHCPs are still integrated into the thylakoid membranes. Surprisingly, the effects of both mutations are additive in the double mutant ffc/chaos described here. This mutant has pale yellow leaves at all stages of growth and drastically reduced levels of all the LHCPs except Lhcb 4. Although the chloroplasts have a normal shape, the thylakoid structure is affected by the mutation, probably as a consequence of reduction of all the LHCPs. ELIPs (early light-inducible proteins), nuclear-encoded proteins related to the LHCP family and inducible by light stress, were also drastically reduced in the double mutant. However, proteins targeted by other chloroplastic targeting pathways (DeltapH, Sec and spontaneous pathways) accumulated to similar levels in the wild-type and the double mutant. Therefore, the near total loss of LHCPs and ELIPs in the double mutant suggests that cpSRP is the predominant, if not exclusive, targeting pathway for these proteins. Phenotypic analysis of the double mutant, compared to the single mutants, suggests that the cpSRP subunits cpSRP43 and cpSRP54 contribute to antenna targeting in an independent but additive way.     

The Twin-Arginine Signal Peptide of Bacillus subtilis YwbN Can Direct either Tat- or Sec-Dependent Secretion of Different Cargo Proteins: Secretion of Active Subtilisin via the B. subtilis Tat Pathway

Proteins that are produced for commercial purposes in Bacillus subtilis are commonly secreted via the Sec pathway. Despite its high secretion capacity, the secretion of heterologous proteins via the Sec pathway is often unsuccessful. Alternative secretion routes, like the Tat pathway, are therefore of interest. Two parallel Tat pathways with distinct specificities have previously been discovered in B. subtilis. To explore the application potential of these Tat pathways, several commercially relevant or heterologous model proteins were fused to the signal peptides of the known B. subtilis Tat substrates YwbN and PhoD. Remarkably, the YwbN signal peptide directed secretion of active subtilisin, a typical Sec substrate, via the B. subtilis TatAyCy route. In contrast, the same signal peptide directed Tat-independent secretion of the Bacillus licheniformis α-amylase (AmyL). Moreover, the YwbN signal peptide directed secretion of SufI, an Escherichia coli Tat substrate, in a Tat-independent manner, most likely via Sec. Our results suggest that cytoplasmic protein folding prior to translocation is probably a major determinant of Tat-dependent protein secretion in B. subtilis, as is the case with E. coli. We conclude that future applications for the Tat system of B. subtilis will most likely involve commercially interesting proteins that are Sec incompatible.     

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.     

Sec63p and Kar2p are required for the translocation of SRP-dependent precursors into the yeast endoplasmic reticulum in vivo

The translocation of secretory polypeptides into the endoplasmic reticulum (ER) occurs at the translocon, a pore-forming structure that orchestrates the transport and maturation of polypeptides at the ER membrane. In yeast, targeting of secretory precursors to the translocon can occur by two distinct pathways that are distinguished by their dependence upon the signal recognition particle (SRP). The SRP-dependent pathway requires SRP and its membrane-bound receptor, whereas the SRP-independent pathway requires a separate receptor complex consisting of Sec62p, Sec63p, Sec71p, Sec72p plus lumenal Kar2p/BiP. Here we demonstrate that Sec63p and Kar2p are also required for the SRP-dependent targeting pathway in vivo. Furthermore, we demonstrate multiple roles for Sec63p, at least one of which is exclusive to the SRP-independent pathway.     

Identification of a Role for an Azide-Sensitive Factor in the Thylakoid Transport of the 17-Kilodalton Subunit of the Photosynthetic Oxygen-Evolving Complex

We have examined the transport of the precursor of the 17-kD subunit of the photosynthetic O₂-evolving complex (OE17) in intact chloroplasts in the presence of inhibitors that block two protein-translocation pathways in the thylakoid membrane. This precursor uses the transmembrane pH gradient-dependent pathway into the thylakoid lumen, and its transport across the thylakoid membrane is thought to be independent of ATP and the chloroplast SecA homolog, cpSecA. We unexpectedly found that azide, widely considered to be an inhibitor of cpSecA, had a profound effect on the targeting of the photosynthetic OE17 to the thylakoid lumen. By itself, azide caused a significant fraction of mature OE17 to accumulate in the stroma of intact chloroplasts. When added in conjunction with the protonophore nigericin, azide caused the maturation of a fraction of the stromal intermediate form of OE17, and this mature protein was found only in the stroma. Our data suggest that OE17 may use the sec-dependent pathway, especially when the transmembrane pH gradient-dependent pathway is inhibited. Under certain conditions, OE17 may be inserted across the thylakoid membrane far enough to allow removal of the transit peptide, but then may slip back out of the translocation machinery into the stromal compartment.     

Targeting and translocation of two lipoproteins in Escherichia coli via the SRP/Sec/YidC pathway

In Escherichia coli, two main protein targeting pathways to the inner membrane exist: the SecB pathway for the essentially posttranslational targeting of secretory proteins and the SRP pathway for cotranslational targeting of inner membrane proteins (IMPs). At the inner membrane both pathways converge at the Sec translocase, which is capable of both linear transport into the periplasm and lateral transport into the lipid bilayer. The Sec-associated YidC appears to assist the lateral transport of IMPs from the Sec translocase into the lipid bilayer. It should be noted that targeting and translocation of only a handful of secretory proteins and IMPs have been studied. These model proteins do not include lipoproteins. Here, we have studied the targeting and translocation of two secretory lipoproteins, the murein lipoprotein and the bacteriocin release protein, using a combined in vivo and in vitro approach. The data indicate that both murein lipoprotein and bacteriocin release protein require the SRP pathway for efficient targeting to the Sec translocase. Furthermore, we show that YidC plays an important role in the targeting/translocation of both lipoproteins.     

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.     

Different lumen-targeting pathways for nuclear-encoded versus cyanobacterial/plastid-encoded Hcf136 proteins

Lumenal proteins are transported across the thylakoid membrane by two very different pathways: Sec-dependent or twin-arginine translocase (Tat)-dependent, where the substrate protein can be transported in a folded state. We present the first evidence that a given protein can be targeted by different pathways in different organisms. Arabidopsis Hcf136 is targeted exclusively by the Tat pathway in pea chloroplasts and no Sec-dependent transport is evident even when the twin-arginine is replaced by twin-lysine. However, twin-arginine motifs are absent from the presequences of Hcf136 proteins encoded by plastid or cyanobacterial genomes, strongly implying translocation by another pathway (presumably Sec). We suggest that the Hcf136 protein was transferred to the Tat pathway when the gene became incorporated into the nuclear genome, possibly due to the tighter folding associated with the more involved, post-translational targeting pathway.     

Multiple pathways used for the targeting of thylakoid proteins in chloroplasts

The assembly of the chloroplast thylakoid membrane requires the import of numerous proteins from the cytosol and their targeting into or across the thylakoid membrane. It is now clear that multiple pathways are involved in the thylakoid-targeting stages, depending on the type of protein substrate. Two very different pathways are used by thylakoid lumen proteins; one is the Sec pathway which has been well-characterised in bacteria, and which involves the threading of the substrate through a narrow channel. In contrast, the more recently characterised twin-arginine translocation (Tat) system is able to translocate fully folded proteins across this membrane. Recent advances on bacterial Tat systems shed further light on the structure and function of this system. Membrane proteins, on the other hand, use two further pathways. One is the signal recognition particle-dependent pathway, involving a complex interplay between many different factors, whereas other proteins insert without the assistance of any known apparatus. This article reviews advances in the study of these pathways and considers the rationale behind the surprising complexity.     

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.     

Export pathway selectivity of Escherichia coli twin arginine translocation signal peptides

The Escherichia coli genome encodes at least 29 putative signal peptides containing a twin arginine motif characteristic of proteins exported via the twin arginine translocation (Tat) pathway. Fusions of the putative Tat signal peptides plus six to eight amino acids of the mature proteins to three reporter proteins (short-lived green fluorescent protein, maltose-binding protein (MBP), and alkaline phosphatase) and also data from the cell localization of epitope-tagged full-length proteins were employed to determine the ability of the 29 signal peptides to direct export through the Tat pathway, through the general secretory pathway (Sec), or through both. 27/29 putative signal peptides could export one or more reporter proteins through Tat. Of these, 11 signal peptides displayed Tat specificity in that they could not direct the export of Sec-only reporter proteins. The rest (16/27) were promiscuous and were capable of directing export of the appropriate reporter either via Tat (green fluorescent protein, MBP) or via Sec (PhoA, MBP). Mutations that conferred a >or=+1 charge to the N terminus of the mature protein abolished or drastically reduced routing through the Sec pathway without affecting the ability to export via the Tat pathway. These experiments demonstrate that the charge of the mature protein N terminus affects export promiscuity, independent of the effect of the folding state of the mature protein.