Alice Robson

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.

The oligomeric state and arrangement of the active bacterial translocon.

Protein secretion in bacteria is driven through the ubiquitous SecYEG complex by the ATPase SecA. The structure of SecYEG alone or as a complex with SecA in detergent reveal a monomeric heterotrimer enclosing a central protein channel, yet in membranes it is dimeric. We have addressed the functional significance of the oligomeric status of SecYEG in protein translocation using single molecule and ensemble methods. The results show that while monomers are sufficient for the SecA- and ATP-dependent association of SecYEG with pre-protein, active transport requires SecYEG dimers arranged in the back-to-back conformation. Molecular modeling of this dimeric structure, in conjunction with the new functional data, provides a rationale for the presence of both active and passive copies of SecYEG in the functional translocon.

Energy transduction in protein transport and the ATP hydrolytic cycle of SecA

The motor protein SecA drives the transport of polypeptides through the ubiquitous protein channel SecYEG. Changes in protein-nucleotide binding energy during the hydrolytic cycle of SecA must be harnessed to drive large conformational changes resulting in channel opening and vectorial substrate polypeptide transport. Here, we elucidate the ATP hydrolysis cycle of SecA from Escherichia coli by transient and steady-state methods. The basal ATPase activity of SecA is very slow with the release of ADP being some 600-fold slower than hydrolysis. Upon binding to SecYEG the release of ADP is stimulated but remains rate-limiting. ADP release is fastest in the fully coupled system when a substrate protein is being translocated; in this case hydrolysis and ADP release occur at approximately the same rate. The data imply that ADP dissociation from SecA is accompanied by a structural rearrangement that is strongly coupled to the protein interface and protein translocation through SecYEG.

Allosteric Regulation of SecA MAGNESIUM-MEDIATED CONTROL OF CONFORMATION AND ACTIVITY

In bacteria, the SecA protein associates with a ubiquitous protein channel SecYEG where it drives the post-translational secretion of pre-proteins across the plasma membrane. The high-resolution structures of both proteins have been determined in their resting states; however, the mechanism that couples ATP hydrolysis to active transport of substrate proteins through the membrane is not well understood. An analysis of the steady-state ATPase activity of the enzyme reveals that there is an allosteric binding site for magnesium distinct from that associated with hydrolysis of ATP. We have demonstrated that this regulation involves a large conformational change to the SecA dimer, which exerts a strong influence on the turnover and affinity for ATP, as well as the affinity for ADP. The strong inhibitory influence of magnesium on the ATPase activity can be countered by cardiolipin and conditions that promote protein translocation.

Mobility of the SecA 2-helix-finger is not essential for polypeptide translocation via the SecYEG complex.

Polypeptide translocation in bacteria, once underway, requires only one copy each of SecA and SecYEG and does not require the mobility of the SecA 2-helix-finger.

The Structure of the Sec complex and the problem of protein translocation

Proteins synthesized in the cytosol either remain there or are localized to a specific membrane and subsequently translocated to another cellular compartment. These extracytosolic proteins have to cross, or be inserted into, a phospholipid bilayer—a process governed by membrane‐bound protein transporters designed to recognize and receive appropriate polypeptides and thread them through the membrane. One such translocation complex, SecY/Sec61, is found in every cell, in either the plasma membrane of bacteria and archaea or the endoplasmic reticulum membrane of eukaryotes. Recent structural findings, combined with previous genetic and biochemical studies, have helped to describe how the passage of proteins through the membrane might occur, but several points of uncertainty remain.

The dynamic action of SecA during the initiation of protein translocation.

The motor ATPase SecA drives protein secretion through the bacterial Sec complex. The PPXD (pre-protein cross-linking domain) of the enzyme has been observed in different positions, effectively opening and closing a clamp for the polypeptide substrate. We set out to explore the implicated dynamic role of the PPXD in protein translocation by examining the effects of its immobilization, either in the position occupied in SecA alone with the clamp held open or when in complex with SecYEG with the clamp closed. We show that the conformational change from the former to the latter is necessary for high-affinity association with SecYEG and a corresponding activation of ATPase activity, presumably due to the PPXD contacting the NBDs (nucleotide-binding domains). In either state, the immobilization prevents pre-protein transport. However, when the PPXD was attached to an alternative position in the associated SecYEG complex, with the clamp closed, the transport capability was preserved. Therefore large-scale conformational changes of this domain are required for the initiation process, but not for translocation itself. The results allow us to refine a model for protein translocation, in which the mobility of the PPXD facilitates the transfer of pre-protein from SecA to SecYEG.

Epididymal specific, selenium-independent GPX5 protects cells from oxidative stress-induced lipid peroxidation and DNA mutation

STUDY QUESTION Can selenium (Se) independent, epididymal-specific glutathione peroxidase 5 (GPX5) protect CHO-K1 cells from oxidative damage and, more specifically, from lipid peroxidation and DNA mutation? SUMMARY ANSWER CHO-K1 cells expressing GPX5 have increased resistance to oxidative challenge and, more specifically, decreased levels of lipid peroxidation and decreased levels of the downstream DNA lesion 8-oxo-7,8-dihydroguanine (8-oxodG) compared with control cells. WHAT IS KNOWN ALREADY GPX5 associates with sperm during transit of the epididymis, and has been postulated to protect sperm from peroxide-mediated attack. However, its function as an active glutathione peroxidase has been questioned due to substitution of the classical selenocysteine residue at its active site. Indirect evidence for a functional role for GPX5 has been provided by in vivo studies, in particular from the GPX5 knockout mouse whereby offspring sired by GPX5(-/-) males have a higher rate of spontaneous abortion and developmental defects, attributed to increased oxidative injury (8-oxodG) to sperm DNA, but only when the GPX5(-/-) males are over 1 year of age. Interestingly, we have previously shown severely reduced levels of GPX5 in humans. STUDY DESIGN, SIZE, DURATION To look more directly at its role in protection against oxidative damage, we have used an in vitro system, generating a CHO-K1 mammalian cell line expressing recombinant rat GPX5. PARTICIPANTS/MATERIALS, SETTING, METHODS We have used the recombinant CHO-K1 cells to determine whether GPX5 is able to protect these cells from an administered oxidative challenge, using a range of approaches. We compared the viability of GPX5-expressing cells with control cells by both MTT and trypan blue exclusion assays. We next investigated whether GPX5 protects the cells specifically from lipid peroxidation, by using the fluorescent reporter molecule C11-BODIPY(581/591), and thus from downstream DNA mutation, by comparing levels of the DNA lesion 8-oxodG. We also investigated whether GPX5 can be transferred to rat sperm via epididymosomes. MAIN RESULTS AND THE ROLE OF CHANCE GPX5-expressing CHO-K1 cells had increased viability compared with control cells following oxidative challenge (P < 0.005). We also found that GPX5-expressing CHO-K1 cells had significantly lower levels of C11-BODIPY(581/591) oxidation, and hence lipid peroxidation, compared with control cells. Levels of 8-oxodG DNA damage were also markedly lower in the nuclei of GPX5-expressing cells than in control cells. Finally, we showed that GPX5 can be transferred to rat sperm via epididymosomes. LIMITATIONS, REASONS FOR CAUTION GPX5 is not active in glutathione peroxidase assays using H₂O₂ as the substrate. However, the related non-mammalian Se-independent GPXs show preference for electron donors other than glutathione, with a number utilizing thioredoxin as a reducing equivalent. Hence, the in vitro activity of GPX5 needs to be assessed using a range of alternative substrates and electron donors. GPX5 is secreted by the epididymis and associates with the sperm plasma membrane. We showed that this transfer can occur via epididymosomes; however, the mechanism for transfer and the identity of a potential binding partner in the sperm membrane needs to be determined. Finally, our study utilized an in vitro system that needs to be translated to human sperm. WIDER IMPLICATIONS OF THE FINDINGS Our study supports an important role for GPX5 as an antioxidant, possibly acting as a phospholipid hydroperoxidase and participating in the maintenance of cell and DNA integrity.

Synthetic peptides identify a second periplasmic site for the plug of the SecYEG protein translocation complex

A short helix in the centre of the SecY subunit serves as a ‘plug’ blocking the protein channel. This site must be vacated if the channel is to open and accommodate translocating protein. We have synthesised a peptide mimic of this plug, and show that it binds to E. coli SecYEG, identifying a distinct and peripheral binding site. We propose that during active translocation the plug moves to this second discrete site and chart its position. Deletion of the plug in SecY increases the stoichiometry of the peptide–SecYEG interaction by also exposing the location it occupies in the channel. Binding of the plug peptide to the channel is unaffected by SecA.

Unlocking the Bacterial SecY Translocon

Summary The Sec translocon performs protein secretion and membrane protein insertion at the plasma membrane of bacteria and archaea (SecYEG/β), and the endoplasmic reticular membrane of eukaryotes (Sec61). Despite numerous structures of the complex, the mechanism underlying translocation of pre-proteins, driven by the ATPase SecA in bacteria, remains unresolved. Here we present a series of biochemical and computational analyses exploring the consequences of signal sequence binding to SecYEG. The data demonstrate that a signal sequence-induced movement of transmembrane helix 7 unlocks the translocon and that this conformational change is communicated to the cytoplasmic faces of SecY and SecE, involved in SecA binding. Our findings progress the current understanding of the dynamic action of the translocon during the translocation initiation process. The results suggest that the converging effects of the signal sequence and SecA at the cytoplasmic face of SecYEG are decisive for the intercalation and translocation of pre-protein through the SecY channel.