Franck Duong

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.

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

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

Biogenesis of the Gram-Negative Bacterial Envelope

We thank Pamela Silver, Charles Barlowe, Carol Kumamoto, and (especially) Tony Pugsley for critical comments. Work in our laboratory has been supported by NIH grant GM23377. J. E. received fellowship support from the Human Frontier Science Program Organization and F. D. from the Association pour la Recherche sur le Cancer and the Institut de la Sante et de la Recherche Medicale.

The SecYEG preprotein translocation channel is a conformationally dynamic and dimeric structure

Escherichia coli preprotein translocase comprises a membrane‐embedded trimeric complex of SecY, SecE and SecG. Previous studies have shown that this complex forms ring‐like assemblies, which are thought to represent the preprotein translocation channel across the membrane. We have analyzed the functional state and the quaternary structure of the SecYEG translocase by employing cross‐linking and blue native gel electrophoresis. The results show that the SecYEG monomer is a highly dynamic structure, spontaneously and reversibly associating into dimers. SecG‐dependent tetramers and higher order SecYEG multimers can also exist in the membrane, but these structures form at high SecYEG concentration or upon overproduction of the complex only. The translocation process does not affect the oligomeric state of the translocase and arrested preproteins can be trapped with SecYEG or SecYE dimers. Dissociation of the dimer into a monomer by detergent induces release of the trapped preprotein. These results provide direct evidence that preproteins cross the bacterial membrane, associated with a translocation channel formed by a dimer of SecYEG.

Sequence of a cluster of genes controlling synthesis and secretion of alkaline protease in Pseudomonas aeruginosa: relationships to other secretory pathways

A genetic locus implicated in the synthesis and secretion of alkaline protease (APR) in Pseudomonas aeruginosa has been previously described [Guzzo et al., J. Bacteriol. 172 (1990) 942-948]. The nucleotide sequence of the DNA fragment encoding these functions was determined and revealed the existence of five open reading frames: aprA, the structural gene encoding APR; aprI, which encodes a protease inhibitor; and aprD, aprE, aprF whose products are involved in protease secretion. The AprD, AprE and AprF proteins share significant homology with proteins implicated in secretion of Erwinia chrysanthemi proteases and Escherichia coli alpha-haemolysin. These results provide further evidence for the existence of a specialized secretory system widespread among Gram- bacteria.

Nanodiscs unravel the interaction between the SecYEG channel and its cytosolic partner SecA

The translocon is a membrane‐embedded protein assembly that catalyzes protein movement across membranes. The core translocon, the SecYEG complex, forms oligomers, but the protein‐conducting channel is at the center of the monomer. Defining the properties of the SecYEG protomer is thus crucial to understand the underlying function of oligomerization. We report here the reconstitution of a single SecYEG complex into nano‐scale lipid bilayers, termed Nanodiscs. These water‐soluble particles allow one to probe the interactions of the SecYEG complex with its cytosolic partner, the SecA dimer, in a membrane‐like environment. The results show that the SecYEG complex triggers dissociation of the SecA dimer, associates only with the SecA monomer and suffices to (pre)‐activate the SecA ATPase. Acidic lipids surrounding the SecYEG complex also contribute to the binding affinity and activation of SecA, whereas mutations in the largest cytosolic loop of the SecY subunit, known to abolish the translocation reaction, disrupt both the binding and activation of SecA. Altogether, the results define the fundamental contribution of the SecYEG protomer in the translocation subreactions and illustrate the power of nanoscale lipid bilayers in analyzing the dynamics occurring at the membrane.

Binding, activation and dissociation of the dimeric SecA ATPase at the dimeric SecYEG translocase

The bacterial preprotein translocase is comprised of a membrane‐embedded oligomeric SecYEG structure and a cytosolic dimeric SecA ATPase. The associations within SecYEG oligomers and SecA dimers, as well as between these two domains are dynamic and reversible. Here, it is shown that a covalently linked SecYEG dimer forms a functional translocase and a high affinity binding site for monomeric and dimeric SecA in solution. The interaction between these two domains stimulates the SecA ATPase, and nucleotides modulate the affinity and ratio of SecA monomers and dimers bound to the linked SecYEG complex. During the translocation reaction, the SecA monomer remains in stable association with a SecYEG protomer and the translocating preprotein. The nucleotides and translocation‐dependent changes of SecA–SecYEG associations and the SecA dimeric state may reflect important facets of the preprotein translocation reaction.

Projection structure and oligomeric properties of a bacterial core protein translocase

The major route for protein export or membrane integration in bacteria occurs via the Sec‐dependent transport apparatus. The core complex in the inner membrane, consisting of SecYEG, forms a protein‐conducting channel, while the ATPase SecA drives translocation of substrate across the membrane. The SecYEG complex from Escherichia coli was overexpressed, purified and crystallized in two dimensions. A 9 Å projection structure was calculated using electron cryo‐microscopy. The structure exhibits P121 symmetry, having two asymmetric units inverted with respect to one another in the unit cell. The map shows elements of secondary structure that appear to be transmembrane helices. The crystallized form of SecYEG is too small to comprise the translocation channel and does not contain a large pore seen in other studies. In detergent solution, the SecYEG complex displays an equilibrium between monomeric and tetrameric forms. Our results therefore indicate that, unlike other known channels, the SecYEG complex can exist as both an assembled channel and an unassembled smaller unit, suggesting that transitions between the two states occur during a functional cycle.

Investigating the SecY plug movement at the SecYEG translocation channel

Protein translocation occurs across the energy‐conserving bacterial membrane at the SecYEG channel. The crystal structure of the channel has revealed a possible mechanism for gating and opening. This study evaluates the plug hypothesis using cysteine crosslink experiments in combination with various allelic forms of the Sec complex. The results demonstrate that the SecY plug domain moves away from the center of the channel toward SecE during polypeptide translocation, and further show that the translocation‐enhancing prlA3 mutation and SecG subunit change the properties of channel gating. Locking the plug in the open state preactivates the Sec complex, and a super‐active translocase can be created when combined with the prlA4 mutation located in the pore of the channel. Dimerization of the Sec complex, which is essential for translocase activity, relocates the plug toward the open position. We propose that oligomerization may result in SecYEG cooperative interactions important to prime the translocon function.

The action of cardiolipin on the bacterial translocon

Cardiolipin is an ever-present component of the energy-conserving inner membranes of bacteria and mitochondria. Its modulation of the structure and dynamism of the bilayer impacts on the activity of their resident proteins, as a number of studies have shown. Here we analyze the consequences cardiolipin has on the conformation, activity, and localization of the protein translocation machinery. Cardiolipin tightly associates with the SecYEG protein channel complex, whereupon it stabilizes the dimer, creates a high-affinity binding surface for the SecA ATPase, and stimulates ATP hydrolysis. In addition to the effects on the structure and function, the subcellular distribution of the complex is modified by the cardiolipin content of the membrane. Together, the results provide rare and comprehensive insights into the action of a phospholipid on an essential transport complex, which appears to be relevant to a broad range of energy-dependent reactions occurring at membranes.