The bacterial ATPase SecA functions as a monomer in protein translocation

The ATPase SecA drives the post-translational translocation of proteins through the SecY channel in the bacterial inner membrane. SecA is a dimer that can dissociate into monomers under certain conditions. To address the functional importance of the monomeric state, we generated an Escherichia coli SecA mutant that is almost completely monomeric (>99%), consistent with predictions from the crystal structure of Bacillus subtilis SecA. In vitro, the monomeric derivative retained significant activity in various assays, and in vivo, it sustained 85% of the growth rate of wild type cells and reduced the accumulation of precursor proteins in the cytoplasm. Disulfide cross-linking in intact cells showed that mutant SecA is monomeric and that even its parental dimeric form is dissociated. Our results suggest that SecA functions as a monomer during protein translocation in vivo.     

Cross-linked SecA dimers are not functional in protein translocation

The ATPase SecA is involved in post-translational protein translocation through the SecY channel across the bacterial inner membrane. SecA is a dimer that can dissociate into monomers with translocation activity. Here, we have addressed whether dissociation of the SecA dimer is required for translocation. We show that a dimer in which the two subunits are cross-linked by disulfide bridges is inactive in protein translocation, translocation ATPase, and binding to a lipid bilayer. In contrast, upon reduction of the disulfide bridges, the resulting monomers regain these activities. These data support the notion that dissociation of SecA dimers into monomers occurs during protein translocation.     

Dissociation of the dimeric SecA ATPase during protein translocation across the bacterial membrane

The ATPase SecA mediates post-translational translocation of precursor proteins through the SecYEG channel of the bacterial inner membrane. We show that SecA, up to now considered to be a stable dimer, is actually in equilibrium with a small fraction of monomers. In the presence of membranes containing acidic phospholipids or in certain detergents, SecA completely dissociates into monomers. A synthetic signal peptide also affects dissociation into monomers. In addition, conversion into the monomeric state can be achieved by mutating a small number of residues in a dimeric and fully functional SecA fragment. This monomeric SecA fragment still maintains strong binding to SecYEG in the membrane as well as significant in vitro translocation activity. Together, the data suggest that the SecA dimer dissociates during protein translocation. Since SecA contains all characteristic motifs of a certain class of monomeric helicases, and since mutations in residues shared with the helicases abolish its translocation activity, SecA may function in a similar manner.     

Involvement of SecDF and YidC in the membrane insertion of M13 procoat mutants

The M13 phage Procoat protein is one of the best characterized substrates for the novel YidC pathway. It inserts into the membrane independent of the SecYEG complex but requires the 60 kDa YidC protein. Mutant Procoat proteins with alterations in the periplasmic region had been found to require SecYEG and YidC. In this report, we show that the membrane insertion of these mutants also strongly depends on SecDF that bridges SecYEG to YidC. In a cold-sensitive mutant of YidC, the Sec-dependent function of YidC is strongly impaired. We find that specifically the SecDF-dependent mutants are inhibited in the cold-sensitive YidC strain. Finally, we find that subtle changes in the periplasmic loop such as the number and location of negatively charged residues and the length of the periplasmic loop can make the Procoat strictly Sec-dependent. In addition, we successfully converted Sec-independent Pf3 coat into a Sec-dependent protein by changing the location of a negatively charged residue in the periplasmic tail. Protease mapping of Pf3 coat shows that the insertion-arrested proteins that accumulate in the YidC- or in the SecDF-deficient strains are not translocated. Taken together, the data suggest that the Sec-dependent mutants insert at the interface of YidC and the translocon with SecDF assisting in the translocation step in vivo.     

The purification of M13 procoat, a membrane protein precursor.

Many membrane proteins and most secreted proteins are initially made as precursors with an N-terminal leader sequence. We now report the isolation of M13 procoat, the precursor of the membrane-bound form of M13 coat protein. There are 40 000 copies of M13 procoat protein/cell during M13 amber 7 virus infection. Purified procoat is quantitatively cleaved by isolated leader peptidase to yield mature-length coat protein. Rabbit antibodies to M13 procoat will precipitate procoat but not coat, suggesting that the antibody molecules are specifically recognizing the leader sequence or the conformation which it induces in the whole procoat molecule.     

M13 procoat protein insertion into YidC and SecYEG proteoliposomes and liposomes

M13 procoat protein was one of the first model proteins used to study bacterial membrane protein insertion. It contains a signal peptide of 23 amino acid residues and is not membrane targeted by the signal recognition particle. The translocation of its periplasmic domain is independent of the preprotein translocase (SecAYEG) but requires electrochemical membrane potential and the membrane insertase YidC of Escherichia coli. We show here that YidC is sufficient for efficient membrane insertion of the purified M13 procoat protein into energized YidC proteoliposomes. When no membrane potential is applied, the insertion is substantially reduced. Only in the presence of YidC, membrane insertion occurs if bilayer integrity is preserved and membrane potential is stable for more than 20 min. A mutant of the M13 procoat protein, H5EE, with two additional negatively charged residues in the periplasmic domain inserted into YidC proteoliposomes and SecYEG proteoliposomes with equal efficiencies. We conclude that the protein can use both the YidC-only pathway and the Sec pathway. This poses the questions of how procoat H5EE is inserted in vivo and how insertion pathways are selected in the cell.     

SecA protein hydrolyzes ATP and is an essential component of the protein translocation ATPase of Escherichia coli.

Bacterial protein export requires two forms of energy input, ATP and the membrane electrochemical potential. Using an in vitro reaction reconstituted with purified soluble and peripheral membrane components, we can now directly measure the translocation-coupled hydrolysis of ATP. This translocation ATPase requires inner membrane vesicles, SecA protein and translocation-competent proOmpA. The stimulatory activity of membrane vesicles can be blocked by either antibody to the SecY protein or by preparing the membranes from a secY-thermosensitive strain which had been incubated at the non-permissive temperature in vivo. The SecA protein itself has more than one ATP binding site. 8-azido-ATP inactivates SecA for proOmpA translocation and for translocation ATPase, yet does not inhibit a low level of ATP hydrolysis inherent in the isolated SecA protein. These data show that the SecA protein has a central role in coupling the hydrolysis of ATP to the transfer of pre-secretory proteins across the membrane.     

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.     

Function of YidC for the insertion of M13 procoat protein in Escherichia coli: translocation of mutants that show differences in their membrane potential dependence and Sec requirement.

The membrane insertion of the Sec-independent M13 Procoat protein in bacteria requires the membrane electrochemical potential and the integral membrane protein YidC. We show here that YidC is involved in the translocation but not in the targeting of the Procoat protein, because we found the protein was partitioned into the membrane in the absence of YidC. YidC can function also to promote membrane insertion of Procoat mutants that insert independently of the membrane potential, proving that the effect of YidC depletion is not due to a dissipation of the membrane potential. We also found that YidC is absolutely required for Sec-dependent translocation of a long periplasmic loop of a mutant Procoat in which the periplasmic region has been extended from 20 to 194 residues. Furthermore, when Sec-dependent membrane proteins with large periplasmic domains were overproduced under YidC-limited conditions, we found that the exported proteins pro-OmpA and pre-peptidoglycan-associated lipoprotein accumulated in the cytoplasm. This suggests for Sec-dependent proteins that YidC functions at a late stage in membrane insertion, after the Sec translocase interacts with the translocating membrane protein. These studies are consistent with the understanding that YidC cooperates with the Sec translocase for membrane translocation and that YidC is required for clearing the protein-conducting channel.     

Arabidopsis dynamin-related protein 1A polymers bind, but do not tubulate, liposomes

The Arabidopsis dynamin-related protein 1A (AtDRP1A) is involved in endocytosis and cell plate maturation in Arabidopsis. Unlike dynamin, AtDRP1A does not have any recognized membrane binding or protein-protein interaction domains. We report that GTPase active AtDRP1A purified from Escherichia coli as a fusion to maltose binding protein forms homopolymers visible by negative staining electron microscopy. These polymers interact with protein-free liposomes whose lipid composition mimics that of the inner leaflet of the Arabidopsis plasma membrane, suggesting that lipid-binding may play a role in AtDRP1A function. However, AtDRP1A polymers do not appear to assemble and disassemble in a dynamic fashion and do not have the ability to tubulate liposomes in vitro, suggesting that additional factors or modifications are necessary for AtDRP1A’s in vivo function.     

Characterization of a Bacillus subtilis SecA mutant protein deficient in translocation ATPase and release from the membrane

SecA is the precursor protein binding subunit of the bacterial precursor protein translocase, which consists of the SecY/E protein as integral membrane domain. SecA is an ATPase, and couples the hydrolysis of ATP to the release of bound precursor proteins to allow their proton-motive-force-driven translocation across the cytoplasmic membrane. A putative ATP-binding motif can be predicted from the amino acid sequence of SecA with homology to the consensus Walker A-type motif. The role of this domain is not known. A lysine residue at position 106 at the end of the glycine-rich loop in the A motif of the Bacillus subtilis SecA was replaced by an asparagine through site-directed mutagenesis (K106N SecA). A similar replacement was introduced at an adjacent lysine residue at position 101 (K101N SecA). Wild-type and mutant SecA proteins were expressed to a high level and purified to homogeneity. The catalytic efficacy (k_cat/k_m) of the K106N SecA for lipid-stimulated ATP hydrolysis was only 1% of that of the wild-type and K101N SecA. K106N SecA retained the ability to bind ATP, but its ATPase activity was not stimulated by precursor proteins. Mutant and wild-type SecA bind with similar affinity to Escherichia coli inner membrane vesicles and insert into a phospholipid mono-layer, in contrast to the wild type, membrane insertion of the K106N SecA was not prevented by ATP. K106N SecA blocks the ATP and proton-motive-force-dependent chase of a translocation intermediate to fully translocated proOmpA. It is concluded that the GKT motif in the amino-terminal domain of SecA is part of the catalytic ATP-binding site. This site may be involved in the ATP-driven protein recycling function of SecA which allows the release of SecA from its association with precursor proteins, and the phospholipid bilayer.     

Efficient translocation of positively charged residues of M13 procoat protein across the membrane excludes electrophoresis as the primary force for membrane insertion.

The coat protein of bacteriophage M13 is inserted into the Escherichia coli plasma membrane as a precursor protein, termed procoat, with a typical leader peptide of 23 amino acid residues. Its membrane insertion requires the electrochemical potential but not the cellular components SecA and SecY. Since the electrochemical gradients result in the periplasmic side of the membrane being positively charged, the membrane potential could contribute to the transfer of the negatively charged central region of procoat across the membrane. Here we demonstrate that the central domain following the leader peptide can be translocated across the membrane even when the net charge of the region is changed from -3 to +3. This rules out an electrophoresis-like insertion mechanism for procoat. We also show that the sec independence of procoat insertion is linked to the presence of the second apolar domain. The deletion of most of the second apolar domain from a procoat fusion protein results in sec dependent membrane insertion of the hybrid protein. Moreover, like other proteins that require the sec genes, translocation of this sec dependent procoat protein is inhibited when positively charged residues are introduced after the leader peptide. Loop models involving one or two hydrophobic regions are presented that account for the differences in tolerance of positively charged residues.     

Fluorescence resonance energy transfer shows a close helix-helix distance in the transmembrane M13 procoat protein

During the membrane insertion process the major coat protein of bacteriophage M13 assumes a conformation in which two transmembrane helices corresponding to the leader sequence and the anchor region in the mature part of the protein coming into close contact with each other. Previous studies on the molecular mechanism of membrane insertion of M13 procoat protein have shown that this interaction between the two helices might drive the actual translocation process. We investigated the intramolecular distance between the two helices of the transmembrane procoat protein by measuring fluorescence resonance energy transfer (FRET) between the donor (Tyr) placed in one helix and the acceptor (Trp) placed in the other helix. Various mutant procoat proteins with differently positioned donor-acceptor pairs were generated, purified, and reconstituted into artificial lipid bilayers. The results obtained from the FRET measurements, combined with molecular modeling, show that the transmembrane helices are in close contact on the order of 1-1.5 nm. The present approach might be of general interest for determining the topology and the folding of membrane proteins.     

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.     

Helicase Motif III in SecA is essential for coupling preprotein binding to translocation ATPase

The SecA ATPase is a protein translocase motor and a superfamily 2 (SF2) RNA helicase. The ATPase catalytic core ('DEAD motor') contains the seven conserved SF2 motifs. Here, we demonstrate that Motif III is essential for SecA-mediated protein translocation and viability. SecA Motif III mutants can bind ligands (nucleotide, the SecYEG translocase 'channel', signal and mature preprotein domains), can catalyse basal and SecYEG-stimulated ATP hydrolysis and can be activated for catalysis. However, Motif III mutation specifically blocks the preprotein-stimulated 'translocation ATPase' at a step of the reaction pathway that lies downstream of ligand binding. A functional Motif III is required for optimal ligand-driven conformational changes and kinetic parameters that underlie optimal preprotein-modulated nucleotide cycling at the SecA DEAD motor. We propose that helicase Motif III couples preprotein binding to the SecA translocation ATPase and that catalytic activation of SF2 enzymes through Motif-III-mediated action is essential for both polypeptide and nucleic-acid substrates.     

Initial steps in protein membrane insertion. Bacteriophage M13 procoat protein binds to the membrane surface by electrostatic interaction.

Bacteriophage M13 procoat protein is synthesized on free polysomes prior to its assembly into the inner membrane of Escherichia coli. As an initial step of the membrane insertion pathway, the precursor protein interacts with the cytoplasmic face of the inner membrane. We have used oligonucleotide-directed mutagenesis to study the regions of the procoat protein involved in membrane binding. We find that there is an absolute requirement for positively charged amino acids at both ends of the protein. Replacing these with negatively charged residues resulted in an accumulation of the precursor in the cytoplasm. We propose that the positively charged amino acids are directly involved in membrane binding, possibly directly to the negatively charged phospholipid head groups. This was tested in vitro with artificial liposomes. Whereas wild-type procoat interacted with these liposomes, we found that procoat mutants with negatively charged amino acids at both ends did not bind. Therefore, we conclude that newly synthesized M13 procoat protein binds electrostatically to the negatively charged inner membrane of E. coli.     

The long alpha-helix of SecA is important for the ATPase coupling of translocation

SecA contains two ATPase folds (NBF1 and NBF2) and other interaction/regulatory domains, all of which are connected by a long helical scaffold domain (HSD) running along the molecule. Here we identified a functionally important and spatially adjacent pair of SecA residues, Arg-642 on HSD and Glu-400 on NBF1. A charge-reversing substitution at either position as well as disulfide tethering of these positions inactivated the translocation activity. Interestingly, however, the translocation-inactive SecA variants fully retained the ability to up-regulate the ATPase in response to a preprotein and the SecYEG translocon. The translocation defect was suppressible by second site alterations at the hinge-forming boundary of NBF2 and HSD. Based on these results, we propose that the motor function of SecA is realized by ligand-activated ATPase engine and its HSD-mediated conversion into the mechanical work of preprotein translocation.     

SecY and SecA interact to allow SecA insertion and protein translocation across the Escherichia coli plasma membrane.

SecA, the preprotein-driving ATPase in Escherichia coli, was shown previously to insert deeply into the plasma membrane in the presence of ATP and a preprotein; this movement of SecA was proposed to be mechanistically coupled with preprotein translocation. We now address the role played by SecY, the central subunit of the membrane-embedded heterotrimeric complex, in the SecA insertion reaction. We identified a secY mutation (secY205), affecting the most carboxyterminal cytoplasmic domain, that did not allow ATP and preprotein-dependent productive SecA insertion, while allowing idling insertion without the preprotein. Thus, the secY205 mutation might affect the SecYEG 'channel' structure in accepting the preprotein-SecA complex or its opening by the complex. We isolated secA mutations that allele-specifically suppressed the secY205 translocation defect in vivo. One mutant protein, SecA36, with an amino acid alteration near the high-affinity ATP-binding site, was purified and suppressed the in vitro translocation defect of the inverted membrane vesicles carrying the SecY205 protein. The SecA36 protein could also insert into the mutant membrane vesicles in vitro. These results provide genetic evidence that SecA and SecY specifically interact, and show that SecY plays an essential role in insertion of SecA in response to a preprotein and ATP and suggest that SecA drives protein translocation by inserting into the membrane in vivo.     

Both hydrophobic domains of M13 procoat are required to initiate membrane insertion.

M13 procoat protein has two hydrophobic domains, one in the leader peptide and one which anchors the mature coat protein in the membrane. Disruption of the membrane anchor region by insertion of arginyl residues does not yield periplasmic coat protein. Instead, the rate of membrane assembly is slowed greater than 100-fold (t1/2 less than 5 s for wild-type, t1/2 greater than 10 min for mutant). The hydrophobic region of mature coat protein not only functions as a membrane anchor, but has an important role in the membrane assembly process per se.