An essential component of a novel bacterial protein export system with homologues in plastids and mitochondria

Proteins are transported across the bacterial plasma membrane and the chloroplast thylakoid membrane by means of protein translocases that recognize N-terminal targeting signals in their cognate substrates. Transport of many of these proteins involves the well defined Sec apparatus that operates in both membranes. We describe here the identification of a novel component of a bacterial Sec-independent translocase. The system probably functions in a similar manner to a Sec-independent translocase in the thylakoid membrane, and substrates for both systems bear a characteristic twin-arginine motif in the targeting peptide. The translocase component is encoded in Escherichia coli by an unassigned reading frame, yigU, disruption of which blocks the export of at least five twin-Arg-containing precursor proteins that are predicted to bind redox cofactors, and hence fold, prior to translocation. The Sec pathway remains unaffected in the deletion strain. The gene has been designated tatC (for twin-arginine translocation), and we show that homologous genes are present in a range of bacteria, plastids, and mitochondria. These findings suggest a central role for TatC-type proteins in the translocation of tightly folded proteins across a spectrum of biological membranes.     

Alcohol intake of P rats is regulated by muscarinic receptors in the pedunculopontine nucleus and VTA

The CtpA protein in the cyanobacterium Synechocystis 6803 is a C-terminal processing protease that is essential for the assembly of the manganese cluster of the photosystem II complex. When fused to different chloroplast-targeting transit peptides, CtpA can be imported into isolated spinach chloroplasts and is subsequently translocated into the thylakoid lumen. Thylakoid transport is mediated by the cyanobacterial signal peptide which demonstrates that the protein transport machinery in thylakoid membranes is functionally conserved between chloroplasts and cyanobacteria. Transport of CtpA across spinach thylakoid membranes is affected by both nigericin and sodium azide indicating that the SecA protein and a transthylakoidal proton gradient are involved in this process. Saturation of the Sec-dependent thylakoid transport route by high concentrations of the precursor of the 33-kDa subunit of the oxygen-evolving system leads to a strongly reduced rate of thylakoid translocation of CtpA which demonstrates transport by the Sec pathway. However, thylakoid transport of CtpA is affected also by excess amounts of the 23-kDa subunit of the oxygen-evolving system, though to a lesser extent. This suggests that the cyanobacterial protein is capable of also interacing with components of the deltapH-dependent route and that transport of a protein across the thylakoid membrane may not always be restricted to a single pathway.     

Mutant ATP-binding RNA aptamers reveal the structural basis for ligand binding

A nuclear mutant of maize, tha1, which exhibited defects in the translocation of proteins across the thylakoid membrane, was described previously. A transposon insertion at the tha1 locus facilitated the cloning of portions of the tha1 gene. Strong sequence similarity with secA genes from bacteria, pea and spinach indicates that tha1 encodes a SecA homologue (cp-SecA). The tha1-ref allele is either null or nearly so, in that tha1 mRNA is undetectable in mutant leaves and cp-SecA accumulation is reduced > or = 40-fold. These results, in conjunction with the mutant phenotype described previously, demonstrate that cp-SecA functions in vivo to facilitate the translocation of OEC33, PSI-F and plastocyanin but does not function in the translocation of OEC23 and OEC16. Our results confirm predictions for cp-SecA function made from the results of in vitro experiments and establish several new functions for cp-SecA, including roles in the targeting of a chloroplast-encoded protein, cytochrome f, and in protein targeting in the etioplast, a nonphotosynthetic plastid type. Our finding that the accumulation of properly targeted plastocyanin and cytochrome f in tha1-ref thylakoid membranes is reduced only a few-fold despite the near or complete absence of cp-SecA suggests that cp-SecA facilitates but is not essential in vivo for their translocation across the membrane.     

The SecDFyajC domain of preprotein translocase controls preprotein movement by regulating SecA membrane cycling

Escherichia coli preprotein translocase comprises a membrane-embedded hexameric complex of SecY, SecE, SecG, SecD, SecF and YajC (SecYEGDFyajC) and the peripheral ATPase SecA. The energy of ATP binding and hydrolysis promotes cycles of membrane insertion and deinsertion of SecA and catalyzes the movement of the preprotein across the membrane. The proton motive force (PMF), though not essential, greatly accelerates late stages of translocation. We now report that the SecDFyajC domain of translocase slows the movement of preprotein in transit against both reverse and forward translocation and exerts this control through stabilization of the inserted form of SecA. This mechanism allows the accumulation of specific translocation intermediates which can then complete translocation under the driving force of the PMF. These findings establish a functional relationship between SecA membrane insertion and preprotein translocation and show that SecDFyajC controls SecA membrane cycling to regulate the movement of the translocating preprotein.     

Sec/SRP-independent insertion of two thylakoid membrane proteins bearing cleavable signal peptides

Two imported thylakoid membrane proteins, PSII-X and PSII-W, are synthesised with cleavable N-terminal signal peptides that closely resemble those of Sec-dependent lumenal proteins. In this report we have reconstituted the insertion of pre-PSII-X and pre-PSII-W into isolated thylakoids. We show that insertion does not require either nucleoside triphosphates or stromal extracts, both of which are required for Sec- and signal recognition particle (SRP)-dependent targeting mechanisms. Insertion is furthermore unaffected by protease treatments that destroy the known protein translocation apparatus in the thylakoid membrane. We conclude that these membrane proteins are inserted by an unusual Sec/SRP-independent mechanism that probably resembles that used by CFoII, and we discuss possible parallels with the biogenesis of phage M13 procoat.     

PrlA4 prevents the rejection of signal sequence defective preproteins by stabilizing the SecA-SecY interaction during the initiation of translocation

In Escherichia coli, precursor proteins are translocated across the cytoplasmic membrane by translocase. This multisubunit enzyme consists of a preprotein-binding and ATPase domain, SecA, and the SecYEG complex as the integral membrane domain. PrlA4 is a mutant of SecY that enables the translocation of preproteins with a defective, or missing, signal sequence. Inner membranes of the prlA4 strain efficiently translocate Delta8proOmpA, a proOmpA derivative with a non-functional signal sequence. Owing to the signal sequence mutation, Delta8proOmpA binds to the translocase with a lowered affinity and the recognition is not restored by the prlA4 SecY. At the ATP-dependent initiation of translocation, the binding affinity of SecA for SecYEG is lowered causing the premature loss of bound preproteins from the translocase. The prlA4 membranes, however, bind SecA with a much higher affinity than the wild-type, and during initiation, the SecA and preprotein remain bound at the translocation site allowing an improved efficiency of translocation. It is concluded that the prlA4 strain prevents the rejection of defective preproteins from the export pathway by stabilizing SecA at the SecYEG complex.     

Biogenesis of Tim23 and Tim17, integral components of the TIM machinery for matrix-targeted preproteins

We analysed the import pathway of Tim23 and of Tim17, components of the mitochondrial import machinery for matrix-targeted preproteins. Tim23 contains two independent import signals. One is located within the first 62 amino acid residues of the hydrophilic domain that, in the assembled protein, is exposed to the intermembrane space. This signal mediates translocation of Tim23 across the outer membrane independently of the membrane potential, DeltaPsi. A second import signal is located in the C-terminal membrane-integrated portion of Tim23. It mediates translocation across the outer membrane and insertion into the inner membrane in a strictly DeltaPsi-dependent fashion. Structurally, Tim17 is related to Tim23 but lacks a hydrophilic domain. It contains an import signal in the C-terminal half and its import requires DeltaPsi. The DeltaPsi-dependent import signals of Tim23 and Tim17 are located at corresponding sites in these two homologous proteins. They exhibit features reminiscent of the positively charged N-terminal presequences of matrix-targeted precursors. Import of Tim23 and its insertion into the inner membrane requires Tim22 but not functional Tim23. Thus, biogenesis of the Tim23.17 complex depends on the Tim22 complex, which is the translocase identified as mediating the import of carrier proteins.     

Multiple pathways for protein transport into or across the thylakoid membrane

Many thylakoid proteins are cytosolically synthesized and have to cross the two chloroplast envelope membranes as well as the thylakoid membrane en route to their functional locations. In order to investigate the localization pathways of these proteins, we over-expressed precursor proteins in Escherichia coli and used them in competition studies. Competition was conducted for import into the chloroplast and for transport into or across isolated thylakoids. We also developed a novel in organello method whereby competition for thylakoid transport occurred within intact chloroplasts. Import of all precursors into chloroplasts was similarly inhibited by saturating concentrations of the precursor to the OE23 protein. In contrast, competition for thylakoid transport revealed three distinct precursor specificity groups. Lumen-resident proteins OE23 and OE17 constitute one group, lumenal proteins plastocyanin and OE33 a second, and the membrane protein LHCP a third. The specificity determined by competition correlates with previously determined protein-specific energy requirements for thylakoid transport. Taken together, these results suggest that thylakoid precursor proteins are imported into chloroplasts on a common import apparatus, whereupon they enter one of several precursor-specific thylakoid transport pathways.     

In vivo cross-linking of the SecA and SecY subunits of the Escherichia coli preprotein translocase

Precursor protein translocation across the Escherichia coli inner membrane is mediated by the translocase, which is composed of a heterotrimeric integral membrane protein complex with SecY, SecE, and SecG as subunits and peripherally bound SecA. Cross-linking experiments were conducted to study which proteins are associated with SecA in vivo. Formaldehyde treatment of intact cells results in the specific cross-linking of SecA to SecY. Concurrently with the increased membrane association of SecA, an elevated amount of cross-linked product was obtained in cells harboring overproduced SecYEG complex. Cross-linked SecA copurified with hexahistidine-tagged SecY and not with SecE. The data indicate that SecA and SecY coexist as a stable complex in the cytoplasmic membrane in vivo.     

Syd, a SecY-interacting protein, excludes SecA from the SecYE complex with an altered SecY24 subunit

Syd is an Escherichia coli cytosolic protein that interacts with SecY. Overproduction of this protein causes a number of protein translocation-related phenotypes, including the strong toxicity against the secY24 mutant cells. Previously, this mutation was shown to impair the interaction between SecY and SecE, the two fundamental subunits of the membrane-embedded part of protein translocase. We have now studied in vitro the mechanisms of the Syd-directed inhibition of protein translocation. Pro-OmpA translocation into inverted membrane vesicles (IMVs) prepared from the secY24 mutant cells as well as the accompanied translocation ATPase activity of SecA were rapidly inhibited by purified Syd protein. In the course of protein translocation, high affinity binding of preprotein-bearing SecA to the translocase on the IMV is followed by ATP-driven insertion of the 30-kDa SecA segment into the membrane. Our experiments using 125I-labeled SecA and the secY24 mutant IMV showed that Syd abolished both the high affinity SecA binding and the SecA insertion. Syd was even able to release the inserted form of SecA that had been stabilized by a nonhydrolyzable ATP analog. Syd affected markedly the proteolytic digestion pattern of the IMV-integrated SecY24 protein, suggesting that Syd exerts its inhibitory effect by interacting directly with the SecY24 protein. In accordance with this notion, a SecY24 variant with a second site mutation (secY249) resisted the Syd action both in vivo and in vitro. Thus, Syd acts against the SecY24 form of translocase, in which SecY-SecE interaction has been compromised, to exclude the SecA motor protein from the SecYE channel complex.     

Components of the protein translocation machinery in the thermophilic cyanobacterium Phormidium laminosum

Cyanobacteria possess cytoplasmic membranes, which are common to all eubacteria, and an additional internal network of thylakoid membranes. We have investigated the distribution of the protein translocation machinery between the two membrane systems. We have demonstrated that leader peptidase activity exists in both membrane systems, suggesting that newly synthesized protein precursors are targeted directly rather than sorted after translocation. Southern blot analysis suggests that the leader peptidase is likely to be a single enzyme common to both membrane systems. We have also shown that cyanobacteria possess a single SecA homologue. These results indicate that the protein translocation machinery in the two membrane systems is likely to be the same and leaves open the question of what mechanism enables the cell to sort proteins between them.     

The isolated complex of the translocase of the outer membrane of mitochondria. Characterization of the cation-selective and voltage-gated preprotein-conducting pore

The complex of the translocase mitochondrial outer membrane (TOM), mediates recognition, unfolding, and translocation of preproteins. We have used a combination of biochemical and electrophysiological methods to study the properties of the preprotein-conducting pore of the purified TOM complex. The pore is cation-selective and voltage-gated. It shows three main conductance levels with characteristic slow and fast kinetics transitions to states of lower conductance following application of transmembrane voltages. These electrical properties distinguish it from the mitochondrial voltage-dependent anion channel (porin) and are identical to those of the previously described peptide-sensitive channel. Binding of antibodies to the C terminus of Tom40 on the intermembrane space side of the outer membrane modifies the channel properties and allows determination of the orientation of the channel within the lipid bilayer. Mitochondrial presequence peptides specifically interact with the pore and decrease the ion flow through the channel in a voltage-dependent manner. We propose that the presequence-induced closures of the pore are related to structural alterations of the TOM complex observed during the various stages of preprotein movement across the mitochondrial outer membrane.     

Carrier protein import into mitochondria mediated by the intermembrane proteins Tim10/Mrs11 and Tim12/Mrs5

Import of nuclear-encoded precursor proteins into mitochondria and their subsequent sorting into mitochondrial subcompartments is mediated by translocase enzymes in the mitochondrial outer and inner membranes. Precursor proteins carrying amino-terminal targeting signals are translocated into the matrix by the integral inner membrane proteins Tim23 and Tim17 in cooperation with Tim44 and mitochondrial Hsp70. We describe here the discovery of a new pathway for the transport of members of the mitochondrial carrier family and other inner membrane proteins that contain internal targeting signals. Two related proteins in the intermembrane space, Tim10/Mrs11 and Tim12/Mrs5, interact sequentially with these precursors and facilitate their translocation across the outer membrane, irrespective of the membrane potential. Tim10 and Tim12 are found in a complex with Tim22, which takes over the precursor and mediates its membrane-potential-dependent insertion into the inner membrane. This interaction of Tim10 and Tim12 with the precursors depends on the presence of divalent metal ions. Both proteins contain a zinc-finger-like motif with four cysteines and bind equimolar amounts of zinc ions.     

On the relationship between the mitochondrial inner membrane anion channel and the adenine nucleotide translocase

The mitochondrial inner membrane anion channel (IMAC) is a transport pathway which is believed to be involved in mitochondrial volume homeostasis. The protein, however, has not been identified. In this paper, we examine the relationship between IMAC and the adenine nucleotide translocator. Many inhibitors of the adenine nucleotide translocase are shown to block IMAC, including Cibacron blue 3GA, bromcresol green, alizarin red S, agaric acid, palmitoyl-CoA, and the fluorescein derivatives erythrosin B, erythrosin isothiocyanate, rose bengal, and eosin Y. The following evidence suggests that Cibacron blue, agaric acid, and palmitoyl-CoA inhibit by binding to a common site. 1) They all only partially block the transport of small anions such as Cl-, NO3-, and HCO3-, but completely block the transport of larger anions such as malonate. 2) They decrease the IC50 values of each other in a manner consistent with competitive binding. 3) N-Ethylmaleimide decreases their IC50 values by a similar extent. 4) Inhibition by all shows no dependence on matrix pH and only a small dependence on medium pH. It is suggested that these agents may selectively bind to an open state of IMAC and inhibit by decreasing its conductance. The physiological nucleotides CoA, NAD+, NADH, NADP+, NADH, and ATP do not inhibit; in fact, IMAC is shown to transport ATP. Despite these similarities between IMAC and the adenine nucleotide translocase, IMAC appears to be a separate entity, since some of the IC50 values differ by up to 8-fold, and carboxyatracyloside, the most selective inhibitor of the adenine nucleotide translocase, has no effect on IMAC. In addition, IMAC is also able to transport AMP, while the adenine nucleotide translocase does not.     

Old and new pathways of protein export in chloroplasts and bacteria

Targeting of chloroplast proteins to the thylakoid membrane is analogous to bacterial secretion, and much of what we know has been learned from secretory mechanisms in Escherichia coli. However, chloroplasts also use a delta pH-dependent pathway to target thylakoid proteins, at least some of which are folded before transport. Previously, this pathway seemed to have no cognate in bacteria, but recent results have shown that the HCF106 gene in maize encodes a component of this pathway and has bacterial homologues. This delta pH-dependent pathway might be an ancient conserved mechanism for protein translocation that evolved before the endosymbiotic origin of plastids and mitochondria.     

Detection of subtle abnormalities on chest radiographs after irreversible compression

In both oxidative phosphorylation and photo-phosphorylation, electron flow through a carrier is linked to the generation of ATP. The energy released by electron transport is converted to potential energy forming a proton gradient across the membranes in chloroplasts. The proton gradient can be measured by a pH microelectrode. In this report, pH changes produced by photo-induced proton transport through spinach chloroplast membranes were measured by a glass microelectrode. The effect of 5,5'-dithiobis(2-nitrobenzoate) (DTNB) on the kinetics of proton movement across the thylakoid membranes was studied. The results showed that the rate of proton uptake was reduced with increasing DTNB concentration. The rate of leakage of accumulated protons through thylakoid membranes also decreased. The results support the notion that cysteinyl residue is involved in proton translocation. The inhibition of proton transport would subsequently affect the chemical reactions of the Calvin Cycle that takes place in the stroma which is the soluble compartment surrounding the thylakoid membranes.     

Common principles of protein translocation across membranes

Most major systems that transport proteins across a membrane share the following features: an amino-terminal transient signal sequence on the transported protein, a targeting system on the cis side of the membrane, a hetero-oligomeric transmembrane channel that is gated both across and within the plane of the membrane, a peripherally attached protein translocation motor that is powered by the hydrolysis of nucleoside triphosphate, and a protein folding system on the trans side of the membrane. These transport systems are divided into two families: export systems that export proteins out of the cytosol, and import systems that transport proteins into cytosol-like compartments.     

A GTP-dependent "push" is generally required for efficient protein translocation across the mitochondrial inner membrane into the matrix

Mitochondrial biogenesis requires translocation of numerous preproteins across both outer and inner membranes into the matrix of the organelle. This translocation process requires a membrane potential (DeltaPsi) and ATP. We have recently demonstrated that the efficient import of a urea-denatured preprotein into the matrix requires GTP hydrolysis (Sepuri, N. B. V., Schülke, N., and Pain, D. (1998) J. Biol. Chem. 273, 1420-1424). We now demonstrate that GTP is generally required for efficient import of various preproteins, both native and urea-denatured. The GTP participation is localized to a particular stage in the protein import process. In the presence of DeltaPsi but no added nucleoside triphosphates, the transmembrane movement of preproteins proceeds only to a point early in their translocation across the inner membrane. The completion of translocation into the matrix is independent of DeltaPsi but is dependent on a GTP-mediated "push." This push is likely mediated by a membrane-bound GTPase on the cis side of the inner membrane. This conclusion is based on two observations: (i) GTP does not readily cross the inner membrane barrier and hence, primarily acts outside the inner membrane to stimulate import, and (ii) the GTP-dependent stage of import does not require soluble constituents of the intermembrane space and can be observed in isolated mitoplasts. Efficient import into the matrix, however, is achieved only through the coordinated action of a cis GTP-dependent push and a trans ATP-dependent "pull."