SecA protein: Autoregulated initiator of secretory precursor protein translocation across theE. coli plasma membrane

Several classes ofsecA mutants have been isolated which reveal the essential role of this gene product forE. coli cell envelope protein secretion. SecA-dependent,in vitro protein translocation systems have been utilized to show that SecA is an essential, plasma membrane-associated, protein translocation factor, and that SecA's ATPase activity appears to play an essential but as yet undefined role in this process. Cell fractionation studies suggested that SecA protein is in a dynamic state within the cell, occurring in soluble, peripheral, and integral membraneous states. These data have been used to argue that SecA is likely to promote the initial insertion of secretory precursor proteins into the plasma membrane in a manner dependent on ATP hydrolysis. The protein secretion capability of the cell has been shown to translationally regulatesecA expression with SecA protein serving as an autogenous repressor, although the exact mechanism and purpose of this regulation need to be defined further.     

In vitro translocation of secretory proteins possessing no charges at the mature domain takes place efficiently in a protonmotive force-dependent manner.

The effect of charges existing on the mature domain of secretory proteins on the efficiency and protonmotive force dependence of translocation into everted membrane vesicles of Escherichia coli was studied. Model secretory proteins devoid of charges on the mature domain were constructed at the DNA level using proOmpF-Lpp as the starting protein. The chargeless presecretory proteins thus constructed were translocated and processed for the signal peptide much faster than proOmpF-Lpp and the rate of translocation was appreciably enhanced by imposition of the protonmotive force. Not only the membrane potential but also delta pH were effective in stimulating the rate of translocation of the chargeless proteins. The results indicate that the mature domain does not have to be charged for the secretory translocation and that the major requirement of the protonmotive force for the secretory translocation is not for the movement, including an electrophoretic one, of charged regions of the mature domain. All of the proOmpF-Lpp derivatives thus constructed were translocated efficiently into everted membrane vesicles in a SecA-dependent manner, irrespective of their size. The mature domain of the smallest one was 45 amino acid residues in length. Contrary to the views previously presented by other workers, these results suggest that there is no sharp boundary at the reported regions for the translocation of presecretory proteins across the cytoplasmic membrane or for the requirement of SecA.     

Protein translocationin vitro: Biochemical characterization of genetically defined translocation components

Recent years have seen the convergence of both genetic and biochemical approaches in the study of protein translocation inE. coli. The powerful combination of these approaches is exemplified in the use of anin vitro protein synthesis-protein translocaltion system to analyze the role of genetically defined components of the protein translocation machinery. We describe in this review recent results focusing on the function of thesecA, secB, andsecY gene products and the demonstration of their requirement forin vitro protein translocation. The SecA protein was recently shown to possess ATPase activity and was proposed to be a component of the translocation ATPase. We present a speculative working model whereby the translocator complex is composed of the integral membrane proteins SecY, SecD, SecE, and SecF, forming an aqueous channel in the cytoplasmic membrane, and the tightly associated peripheral membrane protein SecA functioning as the catalytic subunit of the translocator or protein-ATPase.     

The effects of partial uncoupling upon the kinetics of ATP synthesis by vesicles from Paracoccus denitrificans and by bovine heart submitochondrial particles. Implications for the mechanism of the proton-translocating ATP synthase

1. Reduction in the magnitude of the respiration-dependent protonmotive force (proton electrochemical gradient in mV) of vesicles from Paracoccus denitrificans, and of submitochondrial particles, has been found to be paralleled small increases in S50% values for both ADP and Pi. For example, reduction of the protonmotive force of P. denitrificans vesicles from 145 mV to 110 mV was accompanied by an increase of S50% (ADP) from 8 microM to 18 microM, and an increase of S50% (Pi) from 0.33 mM to 1.4 mM. This result was obtained with partial uncoupling quantities of both carbonyl-cyanide p-trifluoromethoxyphenylhydrazone and of the synergistic combination of nigericin plus valinomycin in the presence of K+. In view of the similar effects of these two different methods of uncoupling it is concluded that the changes in S50% were a consequence of the diminished protonmotive force acting on the ATP synthase rather than of a secondary, direct interaction of the uncouplers with the enzyme. Changes in S50% rather than Km are described because under several sets of conditions double-reciprocal plots were nonlinear. 2. For equivalent attenuations in the rate of ATP synthesis by submitochondrial particles, 2,4-dinitrophenol caused much larger increases in S50% (ATP) than did carbonylcyanide p-trifluoromethoxyphenylhydrazone. Therefore it is concluded that the effect of 2,4-dinitrophenol was primarily a consequence of its previously recognized direct interaction with the F1 segment of the mitochondrial ATPase. The concentration range of 2,4-dinitrophenol that raised S50% (ADP) is similar to that which weakens the binding of ADP to a particular type of site on the purified F1 sector of ATP synthase. This correlation is consistent with such a site having a catalytic role during ATP synthesis. 3. A titration of the rate of ATP synthesis by vesicles of P. denitrificans with increasing quantities of carbonylcyanide p-trifluoromethoxyphenylhydrazone showed that the initial titres of the uncoupler caused large decreases in the rate of ATP synthesis for relatively small attenuations in the protonmotive force. Thus the initial 20 mV drop in the protonmotive force was accompanied by a reduction of more than 65% in the rate of ATP synthesis. Over the lowest range of values of protonmotive force that drove detectable rates of ATP synthesis however, the dependence of the rate was a less steep function of the protonmotive force. A plot of the logarithm of the rate of ATP synthesis against protonmotive force reveals a biphasic relationship. There does not appear to be a 'threshold' value of the protonmotive force below which ATP synthesis is blocked by kinetic factors. 4. The relationships of the protonmotive force with S50% values and with the rate of ATP synthesis (at near saturating concentrations of ADP and Pi) are discussed in relation to possible mechanisms for the coupling of proton translocation to ATP synthesis.     

Internal ATP is the only energy requirement for the translocation of precursor proteins across chloroplastic membranes

The energy requirements for the import of nuclear-encoded proteins into isolated chloroplasts have been reinvestigated. We have shown that, in contrast to protein import into mitochondria, the translocation of the precursors to ferredoxin, plastocyanin (prPC) and the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (prSS) across all chloroplastic membranes is independent of a protonmotive force and requires only ATP. This extends previous works in which investigations were limited to prSS and demonstrates that our results are probably general to all chloroplastic protein precursors. Our results are particularly interesting for the import of prPC, since in addition to the two envelope membranes, this protein must traverse the energy-transducing thylakoid membranes en route to its proper location in the thylakoid lumen. This lack of involvement of a protonmotive force, specifically of a transmembrane electric potential, demonstrates that separate mechanisms operate during the import of proteins into chloroplasts and mitochondria. We also examined the question of whether ATP is utilized inside or outside of chloroplasts during protein import. Previous attempts to resolve this question have resulted in conflicting answers. We found, by two independent approaches, that ATP for protein import is utilized inside chloroplasts. The implications of these results on the possible mechanisms of protein import into chloroplasts are discussed.     

Two distinct ATP-binding domains are needed to promote protein export by Escherichia coli SecA ATPase

Six putative ATP-binding motifs of SecA protein were altered by oligonucleotide-directed mutagenesis to try to define the ATP-binding regions of this multifunctional protein. The effects of the mutations were analysed by genetic and biochemical assays. The results show that SecA contains two essential ATP-binding domains. One domain is responsible for high-affinity ATP binding and contains motifs AO and BO, located at amino acid residues 102-109 and 198-210, respectively. A second domain is responsible for low-affinity ATP binding and contains motifs A3 and a predicted B motif located at amino acid residues 503-511 and 631-653, respectively. The ATP-binding properties of both domains were essential for SecA-dependent translocation ATPase and in vitro protein translocation activities. The significance of these findings for the mechanism of SecA-dependent protein translocation is discussed.     

Energy use by biological protein transport pathways

The targeting of proteins into and across biological membranes to their correct cellular locations is mediated by a variety of transport pathways. These systems must couple the thermodynamically unfavorable processes of substrate translocation and integration with the expenditure of metabolic energy, using the free energy of ATP and GTP hydrolysis and/or a transmembrane protonmotive force. Several recent advances in our knowledge of the structure and function of these transport systems have provided insights into the mechanisms of energy transduction, force generation and energy use by different protein transport pathways.     

In vitro translocation of protein across Escherichia coli membrane vesicles requires both the proton motive force and ATP

The energy requirement for protein translocation across membrane was studied with inverted membrane vesicles from an Escherichia coli strain that lacks all components of F1F0-ATPase. An ompF-lpp chimeric protein was used as a model secretory protein. Translocation of the chimeric protein into membrane vesicles was totally inhibited in the presence of carbonyl cyanide m-chlorophenylhydrazone (CCCP) or valinomycin and nigericin and partially inhibited when either valinomycin or nigericin alone was added. Depletion of ATP with glucose and hexokinase resulted in the complete inhibition of the translocation process, and the inhibition was suppressed by the addition of ATP-generating systems such as phosphoenolpyruvate-pyruvate kinase or creatine phosphate-creatine kinase. These results indicate that both the proton motive force and ATP are required for the translocation process. The results further suggest that both the membrane potential and the chemical gradient of protons (delta pH), of which the proton motive force is composed, participate in the translocation process.     

Active transport of Ca2+ in bacteria: Bioenergetics and function

Summary The bioenergetics of Ca²⁺ transport in bacteria are discussed with special emphasis on the interrelationship between transport and other cellular functions such as substrate oxidation by the respiratory chain and oxidative phosphorylation. The unusual polarity of Ca²⁺ movement provides an exceptional tool to compare active transport and other ATP requiring or generating processes since this ion is actively taken up by everted vesicles in which the coupling-factor ATPase is exposed to the external medium. As inferred from studies with everted vesicles, the active extrusion of Ca²⁺ by whole cells can be accomplished by substrate driven respiration, hydrolysis of ATP or as in the case ofStreptococcus faecalis by a nonhydrolytic unknown process which involves ATP directly. Substrate oxidation and the hydrolysis of ATP result in the generation of a pH gradient which can energize the Ca²⁺ uptake directly (Ca²⁺/H⁺ antiport) or via a secondary Na⁺ gradient (Ca²⁺/Na⁺ antiport). In contrast to exponentially growing cells sporulating Bacilli accumulate Ca²⁺ during the synthesis of dipicolinic acid. Studies involving Ca²⁺ transport provided evidence in support of the hypothesis that the Mg²⁺ ATPase fromEscherichia coli not only provides the driving force for various cellular functions but exerts a regulatory role by controlling the permeability of the membrane to protons. The different specificity requirements of various naphthoquinone analogs in the restoration of transport or oxidative phosphorylation, after the natural menaquinone has been destroyed by irradiation, has indicated that a protonmotive force is sufficient to drive active transport. However, in addition to the driving force (protonmotive force) necessary to establish oxidative phosphorylation, a specific spatial orientation of the respiratory components, such as the naphthoquinones, is essential for the utilization of the proton gradient or membrane potential or both. Finally evidence suggesting that intracellular Ca²⁺ levels might play a fundamental role in bacterial homeostasis is discussed, in particular the role of Ca²⁺ in the process of chemiotaxis and in conferring bacteria heat stability. A vitamin K-dependent carboxylation reaction has been found inEscherichia coli which is similar to that reported in mammalian systems which results in γ carboxylation of glutamate residues. Although all of the proteins containing γ-carboxyglutamate described so far are involved in Ca²⁺ metabolism, the role of these proteins inEscherichia coli is unknown and remains to be elucidated. Dr. A. F. Brodie deceased on January 24, 1981.     

SecA protein: autoregulated ATPase catalysing preprotein insertion and translocation across the Escherichia coli inner membrane

Recent insight into the biochemical mechanism of protein translocation in Escherichia coli indicates that SecA ATPase is required both for the initial binding of preproteins to the inner membrane as well as subsequent translocation across this structure. SecA appears to promote these events by direct recognition of the preprotein or preprotein-SecB complex, binding to inner-membrane anionic phospholipids, insertion into the membrane biiayer and association with the preprotein translocator, SecY/SecE. ATP binding appears to control the affinity of SecA for the various components of the system and ATP hydrolysis promotes cycling between its different biochemical states. As a component likely to catalyse a rate-determining step in protein secretion, SecA synthesis is co-ordinated with the activity of the protein export pathway. This form of negative reguiation appears to rely on SecA protein binding to its mRNA and repressing translation if conditions of rapid protein secretion prevail within the cell. A precise biochemical scheme for SecA-dependent catalysis of protein export and the details of secA regulation appear to be close at hand. The evolutionary conservation of SecA protein among eubacteria as well as the general requirement for translocation ATPases in other protein secretion systems argues for a mechanistic commonality of all prokaryotic protein export pathways.     

Comparative analysis of twin-arginine (Tat)-dependent protein secretion of a heterologous model protein (GFP) in three different Gram-positive bacteria

In contrast to the general protein secretion (Sec) system, the twin-arginine translocation (Tat) export pathway allows the translocation of proteins across the bacterial plasma membrane in a fully folded conformation. Due to this feature, the Tat pathway provides an attractive alternative to the secretory production of heterologous proteins via the Sec system. In this study, the potential for Tat-dependent heterologous protein secretion was compared in the three Gram-positive bacteria Staphylococcus carnosus, Bacillus subtilis, and Corynebacterium glutamicum using green fluorescent protein (GFP) as a model protein. In all three microorganisms, fusion of a Tat signal peptide to GFP resulted in its Tat-dependent translocation across the corresponding cytoplasmic membranes. However, striking differences with respect to the final localization and folding status of the exported GFP were observed. In S. carnosus, GFP was trapped entirely in the cell wall and not released into the supernatant. In B. subtilis, GFP was secreted into the supernatant, however, in an inactive form. In contrast, C. glutamicum effectively secreted active GFP. Our results clearly demonstrate that a comparative evaluation of different Gram-positive host microorganisms is a crucial step on the way to an efficient Tat-mediated secretory production process for a desired heterologous target protein. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. This paper is dedicated to Hermann Sahm on the occasion of his 65th birthday.     

Biochemical investigations of ionic channels in excitable membranes

Summary Ion channels of excitable membranes are composed of a gating device and a selectivity filter. Two strategies are discussed in this review for the biochemical isolation and characterization of these two functional subunits of channels: Membrane molecules involved in ion translocation can be identified in vitro by their pharmacological properties, i.e. by binding assays with radioactive drugs known to selectively affect a special channel in vivo. More desirable is an assay of their true biological function, i.e. translocation of ions through a membrane. Ion flux measurements with natural and reconstituted membrane systems in vitro are recently available.This article summarizes our present knowledge of electrically excitable sodium and potassium channels of nerve membranes and of the chemically excitable sodium/potassium channel of cholinergic synapses, the acetylcholine receptor complex (AChR). Because of the availability of a great variety of drugs binding with high affinity to axonal sodium channels its investigation is more advanced than that of the axonal potassium channel. The lack of high affinity labels for the latter can be possibly overcome by photoaffinity labels which label components of the channel in situ. Initial success is reported with a photoafinity label derived from the potassium channel blocker TEA.Most advanced is the biochemical investigation of the acetylcholine receptor (AChR) which has been purified in milligram quantities. It represents a protein complex composed of different polypeptide chains with different functions regulating the sodium/potassium permeability of cholinergic postsynaptic membranes. Experiments are described to elucidate the quaternary structure, the site of binding of cholinergic ligands and neurotoxins and to prove dynamic conformation changes of the protein which may be the cause for permeability changes of the membrane. The gating device and the ion translocation system (selectivity filter, ionophor) appear to be present in the receptor complex though located possible in different subunits. This is evidenced by reconstitution of excitable membranes from purified AChR and exogenous lipids by a novel and reproducible method.An invited review article.     

Control of the protonmotive force in Rhodopseudomonas sphaeroides in the light and dark and its effect on the initiation of flagellar rotation

The addition of low concentrations of the uncoupler of CCP (0.01−0.1 μM) to actinically illuminated, photosynthetically grown Rhodopseudomonas sphaeroides did not inhibit motility. When CCCP addition was followed by a period of dark, anaerobic incubation the bacteria became nonmotile, and motility was not regained immediately on actinic re-illumination. The length of the delay before the onset of motility on re-illumination was proportional to the concentration of uncoupler added, until at higher concentrations (0.5−5 μM) maximum motility was not regained. Flagellar rotation depends on the protonmotive force, therefore the total pmf and the electrical and chemical components were measured under a variety of environmental conditions. The addition of the uncoupler to dark-incubated bacteria caused the collapse of the respiratory protonmotive force, but had no effect on the rapid reformation of the full protonmotive force on re-illumination. The time-course of protonmotive force generation was very similar to that measured in untreated bacteria and showed little change with increasing concentrations of uncoupler, although the size of the induced protonmotive force was eventually reduced. The ΔpH component of the protonmotive force developed more slowly than the Δψ component, but the time taken for the development of the ΔpH did not increase as the CCCP concentration increased. The delay in motility was longer under conditions where ΔpH was the sole or major component of the protonmotive force. ATP is required for taxis but not motility in bacteria. The addition of CCCP to dark-incubated bacteria caused a rapid fall in intracellular ATP which recovered rapidly on re-illumination. At high uncoupler concentrations the ATP content fell as the protonmotive force was reduced. However, the delay in resumption of motility was observed at CCCP concentrations which did not affect either the protonmotive force or the ATP concentration reached on illumination. There was no delay in recovery of motility when protonmotive force was increased but ATP levels reduced by the addition of the ATPase inhibitor venturicidin. In its proposed that initiation of flagellar rotation involves a protonmotive force dependent modification of the motor and that this modification acts as the on-off switch for the motor.     

Functional transitions of F0F1-ATPase mediated by the inhibitory peptide IF1 in yeast coupled submitochondrial particles.

The mechanism of inhibition of yeast F(0)F(1)-ATPase by its naturally occurring protein inhibitor (IF1) was investigated in submitochondrial particles by studying the IF1-mediated ATPase inhibition in the presence and absence of a protonmotive force. In the presence of protonmotive force, IF1 added during net NTP hydrolysis almost completely inhibited NTPase activity. At moderate IF1 concentration, subsequent uncoupler addition unexpectedly caused a burst of NTP hydrolysis. We propose that the protonmotive force induces the conversion of IF1-inhibited F(0)F(1)-ATPase into a new form having a lower affinity for IF1. This form remains inactive for ATP hydrolysis after IF1 release. Uncoupling simultaneously releases ATP hydrolysis and converts the latent form of IF1-free F(0)F(1)-ATPase back to the active form. The relationship between the different steps of the catalytic cycle, the mechanism of inhibition by IF1 and the interconversion process is discussed.     

Mechanistic and phenomenological features of proton pumps in the respiratory chain of mitochondria

Various direct, indirect (kinetic and thermodynamic), and combined mechanisms have been proposed to explain the conversion of redox energy into a transmembrane protonmotive force (p) by enzymatic complexes of respiratory chains. The conceptual evolution of these models is examined. The characteristics of thermodynamic coupling between redox transitions of electron carriers and scalar proton transfer in cytochromec oxidase and its possible involvement in proton pumping is discussed. Other aspects dealt with in this paper are: (i) variability of H⁺/e ^– stoichiometries, in cytochromec oxidase and cytochromec reductase and its mechanistic implications; (ii) possible models by which the reduction of dioxygen to water at the binuclear heme-copper center of protonmotive oxidases can be directly involved in proton pumping. Finally a unifying concept for proton pumping by the redox complexes of respiratory chain is presented.     

Bacterial protein translocation: kinetic and thermodynamic role of ATP and the protonmotive force.

The energetic mechanism of preprotein export in Escherichia coli has been a source of controversy for many years. In vitro studies of translocation reactions that use purified soluble and membrane components have not clarified the main features of this mechanism. Translocation occurs through consecutive steps which each have distinct energy requirements. Initiation of translocation requires ATP and the SecA protein. Most of the further steps can be driven by the protonmotive force (delta p).     

Evaluation of the annexins as potential mediators of membrane fusion in exocytosis

Membrane fusion is a central event in the process of exocytosis. It occurs between secretory vesicle membranes and the plasma membrane and also among secretory vesicle membranes themselves during compound exocytosis. In many cells the fusion event is regulated by calcium. Since the relevant membranes do not undergo fusion in vitro when highly purified, much attention has been paid to possible protein mediators of these calcium-dependent fusion events. The annexins comprise a group of calcium-dependent membrane-aggregating proteins, of which synexin is the prototype, which can initiate contacts between secretory vesicle membranes which will then fuse if the membranes are further perturbed by the addition of exogenous free fatty acids. This review discusses the secretory pathway and the evidence obtained fromin vitro studies that suggests the annexins may be mediators or regulators of membrane fusion in exocytosis.     

The Startling Properties of Fibroblast Growth Factor 2: How to Exit Mammalian Cells without a Signal Peptide at Hand

For a long time, protein transport into the extracellular space was believed to strictly depend on signal peptide-mediated translocation into the lumen of the endoplasmic reticulum. More recently, this view has been challenged, and the molecular mechanisms of unconventional secretory processes are beginning to emerge. Here, we focus on unconventional secretion of fibroblast growth factor 2 (FGF2), a secretory mechanism that is based upon direct protein translocation across plasma membranes. Through a combination of genome-wide RNAi screening approaches and biochemical reconstitution experiments, the basic machinery of FGF2 secretion was identified and validated. This includes the integral membrane protein ATP1A1, the phosphoinositide phosphatidylinositol-4,5-bisphosphate (PI(4,5)P₂), and Tec kinase, as well as membrane-proximal heparan sulfate proteoglycans on cell surfaces. Hallmarks of unconventional secretion of FGF2 are: (i) sequential molecular interactions with the inner leaflet along with Tec kinase-dependent tyrosine phosphorylation of FGF2, (ii) PI(4,5)P₂-dependent oligomerization and membrane pore formation, and (iii) extracellular trapping of FGF2 mediated by heparan sulfate proteoglycans on cell surfaces. Here, we discuss new developments regarding this process including the mechanism of FGF2 oligomerization during membrane pore formation, the functional role of ATP1A1 in FGF2 secretion, and the possibility that other proteins secreted by unconventional means make use of a similar mechanism to reach the extracellular space. Furthermore, given the prominent role of extracellular FGF2 in tumor-induced angiogenesis, we will discuss possibilities to develop highly specific inhibitors of FGF2 secretion, a novel approach that may yield lead compounds with a high potential to develop into anti-cancer drugs.     

Electrophysiological Studies in Xenopus Oocytes for the Opening of Escherichia coli SecA-Dependent Protein-Conducting Channels

Protein translocation in Escherichia coli requires protein-conducting channels in cytoplasmic membranes to allow precursor peptides to pass through with adenosine triphosphate (ATP) hydrolysis. Here, we report a novel, sensitive method that detects the opening of the SecA-dependent protein-conducting channels at the nanogram level. E. coli inverted membrane vesicles were injected into Xenopus oocytes, and ionic currents were recorded using the two-electrode voltage clamp. Currents were observed only in the presence of E. coli SecA in conjunction with E. coli membranes. Observed currents showed outward rectification in the presence of KCl as permeable ions and were significantly enhanced by coinjection with the precursor protein proOmpA or active LamB signal peptide. Channel activity was blockable with sodium azide or adenylyl 5′-(β,γ-methylene)-diphosphonate, a nonhydrolyzable ATP analogue, both of which are known to inhibit SecA protein activity. Endogenous oocyte precursor proteins also stimulated ion current activity and can be inhibited by puromycin. In the presence of puromycin, exogenous proOmpA or LamB signal peptides continued to enhance ionic currents. Thus, the requirement of signal peptides and ATP hydrolysis for the SecA-dependent currents resembles biochemical protein translocation assay with E. coli membrane vesicles, indicating that the Xenopus oocyte system provides a sensitive assay to study the role of Sec and precursor proteins in the formation of protein-conducting channels using electrophysiological methods.