Escherichia coli SecA helicase activity is not required in vivo for efficient protein translocation or autogenous regulation

SecA is an essential ATP-driven motor protein that binds to preproteins and the translocon to promote protein translocation across the eubacterial plasma membrane. Escherichia coli SecA contains seven conserved motifs characteristic of superfamily II of DNA and RNA helicases, and it has been shown previously to possess RNA helicase activity. SecA has also been shown to be an autogenous repressor that binds to its translation initiation region on secM-secA mRNA, thereby blocking and dissociating 30 S ribosomal subunits. Here we show that SecA is an ATP-dependent helicase that unwinds a mimic of the repressor helix of secM-secA mRNA. Mutational analysis of the seven conserved helicase motifs in SecA allowed us to identify mutants that uncouple SecA-dependent protein translocation activity from its helicase activity. Helicase-defective secA mutants displayed normal protein translocation activity and autogenous repression of secA in vivo. Our studies indicate that SecA helicase activity is nonessential and does not appear to be necessary for efficient protein secretion and secA autoregulation.     

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.     

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.     

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.     

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.     

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.     

SecA is an intrinsic subunit of the Escherichia coli preprotein translocase and exposes its carboxyl terminus to the periplasm

SecA is the dissociable ATPase subunit of the Escherichia coli preprotein translocase, and cycles in a nucleotide-modulated manner between the cytosol and the membrane. Overproduction of the integral subunits of the translocase, the SecY, SecE and SecG polypeptides, results in an increased level of membrane-bound SecA. This fraction of SecA is firmly associated with the membrane as it is resistant to extraction with the chaotropic agent urea, and appears to be anchored by SecYEG rather than by lipids. Topology analysis of this membrane-associated form of SecA indicates that it exposes a carboxy-terminal domain to the periplasmic face of the membrane.     

SecB, a molecular chaperone with two faces

SecB is a molecular chaperone unique to the phylum Proteobacteria, which includes the majority of known Gram-negative bacteria of medical, industrial and agricultural significance. SecB is involved in the translocation of secretory proteins across the cytoplasmic membrane. The crystal structure of the Haemophilus influenzae SecB provides new insights into how SecB simultaneously recognizes its two ligands: unfolded preproteins and SecA, the ATPase subunit of the translocase. SecB uses its entire molecular surface for these two functions, but for preprotein release and its own membrane release, SecB relies on the catalytic activity of SecA. This defines SecB as a translocation-specific molecular chaperone.     

绿脓杆菌SecA ATPase抑制剂筛选模型的建立和应用

Sec途径(分泌途径,Secretion pathway)是蛋白质转运的主要途径。其中,SecA ATPase是蛋白质转运途径中的"动力泵",它通过ATP的水解循环驱使蛋白质前体穿过细菌内膜。SecA蛋白在细菌中是独有且不可缺少的。克隆和高效表达绿脓杆菌PasecAN75蛋白(绿脓杆菌SecA蛋白N端645个氨基酸残基组成的片段,大小约75 kD)并优化其ATPase酶活测定体系,在此基础上建立了更为灵敏的SecA蛋白ATPase活性抑制剂的筛选模型。运用该模型从化合物库的3220个样品中筛选得到可抑制绿脓杆菌SecA ATP酶的活性阳性化合物4个,从7196个微生物发酵液中得到66个阳性样品,筛选阳性率为0.67%(以抑制率大于30%为筛选阳性标准)。而后通过已建立的细胞水平筛选模型对其抗菌活性进行验证。研究结果表明3个化合物样品和6个发酵液样品在酶水平和细胞水平对绿脓杆菌SecA ATPase均有较好的抑制作用,值得进一步研究。     

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.     

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.     

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.     

The bacterial Sec-translocase: structure and mechanism

Most bacterial secretory proteins pass across the cytoplasmic membrane via the translocase, which consists of a protein-conducting channel SecYEG and an ATP-dependent motor protein SecA. The ancillary SecDF membrane protein complex promotes the final stages of translocation. Recent years have seen a major advance in our understanding of the structural and biochemical basis of protein translocation, and this has led to a detailed model of the translocation mechanism.     

A single amino acid substitution in SecY stabilizes the interaction with SecA.

The SecYEG complex constitutes a protein conducting channel across the bacterial cytoplasmic membrane. It binds the peripheral ATPase SecA to form the translocase. When isoleucine 278 in transmembrane segment 7 of the SecY subunit was replaced by a unique cysteine, SecYEG supported an increased preprotein translocation and SecA translocation ATPase activity, and allowed translocation of a preprotein with a defective signal sequence. SecY(I278C)EG binds SecA with a higher affinity than normal SecYEG, in particular in the presence of ATP. The increased translocation activity of SecY(I278C)EG was confirmed in a purified system consisting of SecYEG proteoliposomes, while immunoprecipitation in detergent solution reveal that translocase-preprotein complexes are more stable with SecY(I278C) than with normal SecY. These data imply an important role for SecY transmembrane segment 7 in SecA binding. As improved SecA binding to SecY was also observed with the prlA4 suppressor mutation, it may be a general mechanism underlying signal sequence suppression.     

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.     

A molecular switch in SecA protein couples ATP hydrolysis to protein translocation

SecA, the dimeric ATPase subunit of bacterial protein translocase, catalyses translocation during ATP-driven membrane cycling at SecYEG. We now show that the SecA protomer comprises two structural modules: the ATPase N-domain, containing the nucleotide binding sites NBD1 and NBD2, and the regulatory C-domain. The C-domain binds to the N-domain in each protomer and to the C-domain of another protomer to form SecA dimers. NBD1 is sufficient for single rounds of SecA ATP hydrolysis. Multiple ATP turnovers at NBD1 require both the NBD2 site acting in cis and a conserved C-domain sequence operating in trans. This intramolecular regulator of ATP hydrolysis (IRA) mediates N-/C-domain binding and acts as a molecular switch: it suppresses ATP hydrolysis in cytoplasmic SecA while it releases hydrolysis in SecY-bound SecA during translocation. We propose that the IRA switch couples ATP binding and hydrolysis to SecA membrane insertion/deinsertion and substrate translocation by controlling nucleotide-regulated relative motions between the N-domain and the C-domain. The IRA switch is a novel essential component of the protein translocation catalytic pathway.     

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.     

Kinetic analysis of the translocation of fluorescent precursor proteins into Escherichia coli membrane vesicles

Protein secretion in Escherichia coli is mediated by translocase, a multi-subunit membrane protein complex with SecA as ATP-driven motor protein and the SecYEG complex as translocation pore. A fluorescent assay was developed to facilitate kinetic studies of protein translocation. Single cysteine mutants of proOmpA were site-specific labeled with fluorescent dyes, and the SecA and ATP-dependent translocation into inner membrane vesicles and SecYEG proteoliposomes was monitored by means of protease accessibility and in gel fluorescent imaging. The translocation of fluorescently labeled proOmpA was largely independent on the position and the size of the fluorescent label (up to a size of 13-16 A). A fluorophore at the +4 position blocked translocation, but inhibition was completely relieved in the PrlA4 mutant. The kinetics of translocation of the fluorescently labeled proOmpA could be directly monitored by means of fluorescence quenching. Inner membrane vesicles containing wild-type SecYEG were found to translocate proOmpA with a turnover of 4.5 molecules proOmpA/SecYEG complex/min and an apparent K(m) of 180 nm, whereas the PrlA4 mutant showed an almost 10-fold increase in turnover rate and a 3-fold increase of the apparent K(m) for proOmpA 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.     

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.