SecA: the ubiquitous component of preprotein translocase in prokaryotes

SecA is an obligatory component of the complex hetero-septameric translocase of prokaryotes. It is unique in that it exists as two forms within the holoenzyme; first, as a structural component of the preprotein channel and second, as an ATP-dependent membrane cycling factor facilitating the translocation of a broad class of proteins across the cytoplasmic membrane. While the translocase activity of SecA appears to be functionally conserved, it is not clear whether the mechanisms of regulation of the secA gene are similarly maintained. The recent characterization of an ATP-dependent RNA helicase activity of SecA offers a unique mechanism for SecA to communicate the secretion status of the cell to the appropriate regulatory circuits simply by the unwinding of an appropriate RNA target. Resolution of these two activities through combined biochemical, genetic, and biophysical studies should lead to a better understanding of the role of SecA in bacterial secretion.     

Dimeric SecA couples the preprotein translocation in an asymmetric manner

The Sec translocase mediates the post-translational translocation of a number of preproteins through the inner membrane in bacteria. In the initiatory translocation step, SecB targets the preprotein to the translocase by specific interaction with its receptor SecA. The latter is the ATPase of Sec translocase which mediates the post-translational translocation of preprotein through the protein-conducting channel SecYEG in the bacterial inner membrane. We examined the structures of Escherichia coli Sec intermediates in solution as visualized by negatively stained electron microscopy in order to probe the oligomeric states of SecA during this process. The symmetric interaction pattern between the SecA dimer and SecB becomes asymmetric in the presence of proOmpA, and one of the SecA protomers predominantly binds to SecB/proOmpA. Our results suggest that during preprotein translocation, the two SecA protomers are different in structure and may play different roles.     

Preprotein transfer to the Escherichia coli translocase requires the co-operative binding of SecB and the signal sequence to SecA

In Escherichia coli , precursor proteins are targeted to the membrane-bound translocase by the cytosolic chaperone SecB. SecB binds to the extreme carboxy-terminus of the SecA ATPase translocase subunit, and this interaction is promoted by preproteins. The mutant SecB proteins, L75Q and E77K, which interfere with preprotein translocation in vivo , are unable to stimulate in vitro translocation. Both mutants bind proOmpA but fail to support the SecA-dependent membrane binding of proOmpA because of a marked reduction in their binding affinities for SecA. The stimulatory effect of preproteins on the interaction between SecB and SecA exclusively involves the signal sequence domain of the preprotein, as it can be mimicked by a synthetic signal peptide and is not observed with a mutant preprotein (Δ8proOmpA) bearing a non-functional signal sequence. Δ8proOmpA is not translocated across wild-type membranes, but the translocation defect is suppressed in inner membrane vesicles derived from a prlA4 strain. SecB reduces the translocation of Δ8proOmpA into these vesicles and almost completely prevents translocation when, in addition, the SecB binding site on SecA is removed. These data demonstrate that efficient targeting of preproteins by SecB requires both a functional signal sequence and a SecB binding domain on SecA. It is concluded that the SecB–SecA interaction is needed to dissociate the mature preprotein domain from SecB and that binding of the signal sequence domain to SecA is required to ensure efficient transfer of the preprotein to the translocase.     

Delta mu H+ and ATP function at different steps of the catalytic cycle of preprotein translocase.

Preprotein translocation in E. coli requires ATP, the membrane electrochemical potential delta mu H+, and translocase, an enzyme with an ATPase domain (SecA) and the membrane-embedded SecY/E. Studies of translocase and proOmpA binds to the SecA domain. Second, SecA binds ATP. Third, ATP-binding energy permits translocation of approximately 20 residues of proOmpA. Fourth, ATP hydrolysis releases proOmpA. ProOmpA may then rebind to SecA and reenter this cycle, allowing progress through a series of transmembrane intermediates. In the absence of delta mu H+ or association with SecA, proOmpA passes backward through the membrane, but moves forward when either ATP and SecA or a membrane electrochemical potential is supplied. However, in the presence of delta mu H+ (fifth step), proOmpA rapidly completes translocation. delta mu H(+)-driven translocation is blocked by SecA plus nonhydrolyzable ATP analogs, indicating that delta mu H+ drives translocation when ATP and proOmpA are not bound to SecA.     

Sec-dependent membrane protein biogenesis: SecYEG, preprotein hydrophobicity and translocation kinetics control the stop-transfer function.

Preprotein translocase catalyzes membrane protein integration as well as complete translocation. Membrane proteins must interrupt their translocation and be laterally released from the translocase into the lipid bilayer. We have analyzed the translocation arrest and lateral release activities of Escherichia coli preprotein translocase with an in vitro reaction and the preprotein proOmpA carrying a synthetic stop-transfer sequence. Membrane protein integration is catalytic, occurs with kinetics similar to those of proOmpA itself and only requires the functions of SecYEG and SecA. Though a strongly hydrophobic segment will direct the protein to leave the translocase and enter the lipid bilayer, a protein with a segment of intermediate hydrophobicity partitions equally between the translocated and membrane-integrated states. Analysis of the effects of PMF, varied ATP concentrations or synthetic translocation arrest show that the stop-translocation efficiency of a mildly hydrophobic segment depends on the translocation kinetics. In contrast, the lateral partitioning from translocase to lipids depends solely on temperature and does not require SecA ATP hydrolysis or SecA membrane cycling. Thus translocation arrest is controlled by the SecYEG translocase activity while lateral release and membrane integration are directed by the hydrophobicity of the segment itself. Our results suggest that a greater hydrophobicity is required for efficient translocation arrest than for lateral release into the membrane.     

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.     

Nucleotide binding induces changes in the oligomeric state and conformation of Sec A in a lipid environment: a small-angle neutron-scattering study

In Escherichia coli, SecA is a large, multifunctional protein that is a vital component of the general protein secretion pathway. In its membrane-bound form it functions as the motor component of the protein translocase, perhaps through successive rounds of membrane insertion and ATP hydrolysis. To understand both the energy conversion process and translocase assembly, we have used contrast-matched, small-angle neutron-scattering (SANS) experiments to examine SecA in small unilamellar vesicles of E.coli phospholipids. In the absence of nucleotide, we observe a dimeric form of SecA with a radius of gyration comparable to that previously observed for SecA in solution. In contrast, the presence of either ADP or a non-hydrolyzable ATP analog induces conversion to a monomeric form. The larger radius of gyration for the ATP-bound relative to the ADP-bound form suggests the former has a more expanded global conformation. This is the first direct structural determination of SecA in a lipid bilayer. The SANS data indicate that nucleotide turnover can function as a switch of conformation of SecA in the membrane in a manner consistent with its proposed role in successive cycles of deep membrane penetration and release with concommitant preprotein insertion.     

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.     

The catalytic cycle of the escherichia coli SecA ATPase comprises two distinct preprotein translocation events.

SecA is the ATP-dependent force generator in the Escherichia coli precursor protein translocation cascade, and is bound at the membrane surface to the integral membrane domain of the preprotein translocase. Preproteins are thought to be translocated in a stepwise manner by nucleotide-dependent cycles of SecA membrane insertion and de-insertion, or as large polypeptide segments by the protonmotive force (Deltap) in the absence of SecA. To determine the step size of a complete ATP- and SecA-dependent catalytic cycle, translocation intermediates of the preprotein proOmpA were generated at limiting SecA translocation ATPase activity. Distinct intermediates were formed, spaced by intervals of approximately 5 kDa. Inhibition of the SecA ATPase by azide trapped SecA in a membrane-inserted state and shifted the step size to 2-2.5 kDa. The latter corresponds to the translocation elicited by binding of non-hydrolysable ATP analogues to SecA, or by the re-binding of partially translocated polypeptide chains by SecA. Therefore, a complete catalytic cycle of the preprotein translocase permits the stepwise translocation of 5 kDa polypeptide segments by two consecutive events, i.e. approximately 2.5 kDa upon binding of the polypeptide by SecA, and another 2.5 kDa upon binding of ATP to SecA.     

Bacterial preprotein translocase: mechanism and conformational dynamics of a processive enzyme

Preprotein translocase, the membrane transporter for secretory proteins, is a processive enzyme. It comprises the membrane proteins SecYEG(DFYajC) and the peripheral ATPase SecA, which acts as a motor subunit. Translocase subunits form dynamic complexes in the lipid bilayer and build an aqueous conduit through which preprotein substrates are transported at the expense of energy. Preproteins bind to translocase and trigger cycles of ATP binding and hydrolysis that drive a transition of SecA between two distinct conformational states. These changes are transmitted to SecG and lead to inversion of its membrane topology. SecA conformational changes promote directed migration of the polymeric substrate through the translocase, in steps of 20–30 aminoacyl residues. Translocase dissociates from the substrate only after the whole preprotein chain length has been transported to the trans side of the membrane, where it is fully released.     

Distinct catalytic roles of the SecYE, SecG and SecDFyajC subunits of preprotein translocase holoenzyme.

Escherichia coli preprotein translocase contains a membrane-embedded trimeric complex of SecY, SecE and SecG (SecYEG) and the peripheral SecA protein. SecYE is the conserved functional 'core' of the SecYEG complex. Although sufficient to provide sites for high-affinity binding and membrane insertion of SecA, and for its activation as a preprotein-dependent ATPase, SecYE has only very low capacity to support translocation. The proteins encoded by the secD operon--SecD, SecF and YajC--also form an integral membrane heterotrimeric complex (SecDFyajC). Physical and functional studies show that these two trimeric complexes are associated to form SecYEGDFyajC, the hexameric integral membrane domain of the preprotein translocase 'holoenzyme'. Either SecG or SecDFyajC can support the translocation activity of SecYE by facilitating the ATP-driven cycle of SecA membrane insertion and de-insertion at different stages of the translocation reaction. Our findings show that each of the prokaryote-specific subunits (SecA, SecG and SecDFyajC) function together to promote preprotein movement at the SecYE core of the translocase.     

Sec-dependent preprotein translocation in bacteria

Translocation of precursor proteins across the cytoplasmic membrane in bacteria is mediated by a multi-subunit protein complex termed translocase, which consists of the integral membrane heterotrimer SecYEG and the peripheral homodimeric ATPase SecA. Preproteins are bound by the cytosolic molecular chaperone SecB and targeted in a complex with SecA to the translocation site at the cytoplasmic membrane. This interaction with SecYEG allows the SecA/preprotein complex to insert into the membrane by binding of ATP to the high affinity nucleotide binding site of SecA. At that stage, presumably recognition and proofreading of the signal sequence occurs. Hydrolysis of ATP causes the release of the preprotein in the translocation channel and drives the withdrawal of SecA from the membrane-integrated state. Hydrolysis of ATP at the low-affinity nucleotide binding site of SecA converts the protein into a compact conformational state and releases it from the membrane. In the absence of the proton motive force, SecA is able to complete the translocation stepwise by multiple nucleotide modulated cycles. Received: 4 August 1995 / Accepted: 9 October 1995     

以SecA蛋白为“动力泵”的细菌Sec蛋白转运途径

细菌细胞中,三分之一的蛋白质是在合成后被转运到细胞质外才发挥功能的.其中大多数蛋白是通过Sec途径(即分泌途径secretion pathway)进行跨膜运动的.Sec转运酶是一个多组分的蛋白质复合体,膜蛋白三聚体SecYEG及水解ATP的动力蛋白SecA构成了Sec转运酶的核心.整合膜蛋白SecD,SecF和vajC形成了一个复合体亚单位,可与SecYEG相连并稳定SecA蛋白的膜结合形式.SecB是蛋白质转运中的伴侣分子,可以和很多蛋白质前体结合.SecM是由位于secA基因上游的secM基因编码的,可调节SecA蛋白的合成量,维持细胞在不同环境条件下的正常生长.新生肽链的信号肽被高度保守的SRP特异性识别.伴侣分子SecB通过与细胞膜上的SecA二聚体特异性结合将蛋白质前体引导至Sec转运途径,起始转运过程.结合蛋白质前体的SecA与组成转运通道的SecYEG复合体具有较高的亲和性.SecA经历插入和脱离细胞内膜SecYEG通道的循环,为转运提供所需的能量,每一次循环可推动20多个氨基酸的连续跨膜运动.     

SecA protein needs both acidic phospholipids and SecY/E protein for functional high-affinity binding to the Escherichia coli plasma membrane.

Translocation of preproteins across the Escherichia coli inner membrane requires acidic phospholipids. We have studied the translocation of the precursor protein proOmpA across inverted inner membrane vesicles prepared from cells depleted of phosphatidylglycerol and cardiolipin. These membranes support neither translocation nor the translocation ATPase activity of the SecA subunit of preprotein translocase. We now report that inner membrane vesicles which are depleted of acidic phospholipids are unable to bind SecA protein with high affinity. These membranes can be restored to translocation competence by fusion with liposomes containing phosphatidylglycerol, suggesting that the defect in SecA binding is a direct effect of phospholipid depletion rather than a general derangement of inner membrane structure. Reconstitution of SecY/E, the membrane-embedded domain of translocase, into proteoliposomes containing predominantly a single synthetic acidic lipid, dioleoylphosphatidylglycerol, allows efficient catalysis of preprotein translocation.     

YidC, the Escherichia coli homologue of mitochondrial Oxa1p, is a component of the Sec translocase

In Escherichia coli, both secretory and inner membrane proteins initially are targeted to the core SecYEG inner membrane translocase. Previous work has also identified the peripherally associated SecA protein as well as the SecD, SecF and YajC inner membrane proteins as components of the translocase. Here, we use a cross-linking approach to show that hydrophilic portions of a co-translationally targeted inner membrane protein (FtsQ) are close to SecA and SecY, suggesting that insertion takes place at the SecA/Y interface. The hydrophobic FtsQ signal anchor sequence contacts both lipids and a novel 60 kDa translocase-associated component that we identify as YidC. YidC is homologous to Saccharomyces cerevisiae Oxa1p, which has been shown to function in a novel export pathway at the mitochondrial inner membrane. We propose that YidC is involved in the insertion of hydrophobic sequences into the lipid bilayer after initial recognition by the SecAYEG translocase.     

以SecA为靶点的新型抗绿脓杆菌药物细胞水平筛选模型的建立和应用

Sec途径(即分泌途径secretion pathway)是蛋白质转运的主要途径.其中,最为关键的组分之一是SecAATP酶,是蛋白质转运途径中的"动力泵",通过ATP的水解循环驱使蛋白质前体穿过细菌内膜,在细菌中是不可缺少的.我们推测抑制SecAATP酶活性的化合物.必然会在一定程度上抑制蛋白质的转运和分泌.通过绿脓杆菌与大肠杆菌SecA蛋白的互补作用,利用本实验室构建的高效表达SecA蛋白的基因工程菌,建立了SecA蛋白ATP酶活性抑制剂的细胞水平筛选模型.利用所纯化的绿脓杆菌SecA蛋白的ATP酶活测定体系,验证了所建立的细胞水平筛选模型具有一定的特异性.研究结果表明其中两个酯相组分在细胞水平和蛋白水平均具有活性,值得进行深入的研究.     

Characterization of a mutant form of SecA that alleviates a SecY defect at low temperature and shows a synthetic defect with SecY alteration at high temperature

The secY205 mutant is cold-sensitive for protein export, with an in vitro defect in supporting ATP- and preprotein-dependent insertion of SecA into the membrane. We characterized SecA81 with a Gly516 to Asp substitution near the minor ATP-binding region, which suppresses the secY205 defect at low temperature and exhibits an allele-specific synthetic defect with the same SecY alteration at 42 degrees C. The overproduced SecA81 aggregated in vivo at temperatures above 37 degrees C. Purified SecA81 exhibited markedly enhanced intrinsic and membrane ATPase activities at 30 degrees C, while it was totally inactive at 42 degrees C. The trypsin digestion patterns indicated that SecA81 has some disorder in the central region of SecA, which encompasses residues 421-575. This conformational abnormality may result in unregulated ATPase at low temperature as well as the thermosensitivity of the mutant protein. In the presence of both proOmpA and the wild-type membrane vesicles, however, the thermosensitivity was alleviated, and SecA81 was able to catalyze significant levels of proOmpA-stimulated ATP hydrolysis as well as proOmpA translocation at 42 degrees C. While SecA81 was able to overcome the SecY205 defect at low temperature, the SecY205 membrane vesicles could not significantly support the translocation ATPase or the proOmpA translocation activity of SecA81 at 42 degrees C. The inactivated SecA81 molecules seemed to jam the translocase since it interfered with translocase functions at 42 degrees C. Based on these results, we propose that under preprotein-translocating conditions, the SecYEG channel can stabilize and activate SecA, and that this aspect is defective for the SecA81-SecY205 combination. The data also suggest that the conformation of the central region of SecA is important for the regulation of ATP hydrolysis and for the productive interaction of SecA with SecY.     

Indecisive M13 procoat protein mutants bind to SecA but do not activate the translocation ATPase

The M13 procoat protein serves as the paradigm for the Sec-independent membrane insertion pathway. This protein is inserted into the inner membrane of Escherichia coli with two hydrophobic regions and a central periplasmic loop region of 20 amino acid residues. Extension of the periplasmic loop region renders M13 procoat membrane insertion Sec-dependent. Loop regions with 118 or more residues required SecA and SecYEG and were efficiently translocated in vivo. Two mutants having loop regions of 80 and 100 residues, respectively, interacted with SecA but failed to activate the membrane translocation ATPase of SecA in vitro. Similarly, a procoat mutant with two additional glutamyl residues in the loop region showed binding to SecA but did not stimulate the ATPase. The three mutants were also defective for precursor-stimulated binding of SecA to the membrane surface. Remarkably, the mutant proteins act as competitive inhibitors of the Sec translocase. This suggests that the region to be translocated is sensed by SecA but the activation of the SecA translocation ATPase is only successful for substrates with a minimum length of the translocated region.     

Preprotein translocation by a hybrid translocase composed of Escherichia coli and Bacillus subtilis subunits

Bacterial protein translocation is mediated by translocase, a multisubunit membrane protein complex that consists of a peripheral ATPase SecA and a preprotein-conducting channel with SecY, SecE, and SecG as subunits. Like Escherichia coli SecG, the Bacillus subtilis homologue, YvaL, dramatically stimulated the ATP-dependent translocation of precursor PhoB (prePhoB) by the B. subtilis SecA-SecYE complex. To systematically determine the functional exchangeability of translocase subunits, all of the relevant combinations of the E. coli and B. subtilis secY, secE, and secG genes were expressed in E. coli. Hybrid SecYEG complexes were overexpressed at high levels. Since SecY could not be overproduced without SecE, these data indicate a stable interaction between the heterologous SecY and SecE subunits. E. coli SecA, but not B. subtilis SecA, supported efficient ATP-dependent translocation of the E. coli precursor OmpA (proOmpA) into inner membrane vesicles containing the hybrid SecYEG complexes, if E. coli SecY and either E. coli SecE or E. coli SecG were present. Translocation of B. subtilis prePhoB, on the other hand, showed a strict dependence on the translocase subunit composition and occurred efficiently only with the homologous translocase. In contrast to E. coli SecA, B. subtilis SecA binds the SecYEG complexes only with low affinity. These results suggest that each translocase subunit contributes in an exclusive manner to the specificity and functionality of the complex.     

Binding of SecA to the SecYEG complex accelerates the rate of nucleotide exchange on SecA

SecYEG translocase mediates the transport of preproteins across the inner membrane of Escherichia coli. SecA binds the membrane-embedded SecYEG protein-conducting channel with high affinity and then drives the stepwise translocation of preproteins across the membrane through multiple cycles of ATP binding and hydrolysis. We have investigated the kinetics of nucleotide binding to SecA while associated with the SecYEG complex. Lipid-bound SecA was separated from Se-cYEG-bound SecA by sedimentation of the proteoliposomes through a glycerol cushion, which maintains the SecA native state and effectively removes the lipid-bound SecA fraction. Nucleotide binding was assessed by means of fluorescence resonance energy transfer using fluorescent ATP analogues as acceptors of the intrinsic SecA tryptophan fluorescence in the presence of a tryptophanless variant of the SecYEG complex. Binding of SecA to the SecYEG complex elevated the rate of nucleotide exchange at SecA independently of the presence of preprotein. This defines a novel pretranslocation activated state of SecA that is primed for ATP hydrolysis upon preprotein interaction.