Effects of Nonlamellar-prone Lipids on the ATPase Activity of SecA Bound to Model Membranes

Self Cite

We examined the effects of synthetic signal peptides from the wild-type, export-defective mutant and its revertant species of ribose-binding protein on the phase properties of lipid bilayers. The lateral segregation of phosphatidylglycerol (PG) in the lipid bilayer was detected through quenching between NBD-PGs upon the reconstitution of signal peptide into the liposome made with the Escherichia coli inner membrane composition. The tendency of lipid segregation was highly dependent on the export competency of signal peptides in vivo, with a decreasing order of wild-type, revertant, and mutant species. The colocalizations of pyrene-PG with BODIPY-PG were also induced by the signal peptides, confirming the phase separation of the acidic phospholipid. The wildtype and revertant signal peptides predominantly formed ␣-helical conformations with the presence of acidic phospholipid as determined by circular dichroism spectroscopy. In addition, they restricted the motion of lipid acyl chains as monitored by fluorescence anisotropy of DPH, suggesting a deep penetration of signal peptide into the lipid bilayer. However, the ␣-helical content of mutant signal peptide was only about half that of the wild-type or revertant peptide with a significantly smaller degree of penetration into the bilayer. An association of the defective signal peptides into the membrane was affected by salt extraction, whereas the functional ones were not. The aforementioned results indicate that the functionality of signal peptide is accomplished through its topologies in the membrane and also by its ability to induce lateral segregation of acidic phospholipid. We propose that the clustering of acidic phospholipid by the functional signal peptide is responsible for the formation of non-bilayer membrane structure, thereby promoting an efficient translocation of secretory proteins.

supporting

confidence: 70%

The Phase Property of Membrane Phospholipids Is Affected by the Functionality of Signal Peptides from the Escherichia coli Ribose-binding Protein

Ahn

Kim

et al. 2002

Self Cite

“…When it is retracted from SecYEG, SecA could interact directly with the phospholipid bilayer, which would then have the opportunity to drive conformationally specific changes in SecA that could contribute to a carefully controlled progression of the overall conformational reaction cycle. Such interactions could account for the acceleration in the rate of preprotein translocation in the presence of bilayer destabilizing lipids (26), which increase the solvent exposure of the hydrocarbon moieties of the phospholipids (29).…”

Section: Discussionmentioning

confidence: 99%

“…The ability of SecA to interact with the hydrocarbon region of phospholipids in bilayer membranes is also supported by several experiments conducted using vesicles containing acidic phospholipids (26, 28 -30). Bilayer destabilizing lipids increase hydrocarbon exposure (29) and accelerate the rate of both preprotein translocation (26) and a conformational change that can be induced in SecA by interaction with vesicles (30). Moreover, SecA can be labeled by lipids containing photoactivatable groups in their hydrocarbon moieties (31,32) when such probes are incorporated into pure lipid vesicles, although experiments of this kind also indicate that SecA becomes shielded from such interactions when it binds to SecYEG (14,32).…”

mentioning

confidence: 99%

Phospholipid-induced Monomerization and Signal-peptide-induced Oligomerization of SecA

Benach¹,

Chou²,

Fak³

et al. 2003

100

114

The SecA ATPase drives the processive translocation of the N terminus of secreted proteins through the cytoplasmic membrane in eubacteria via cycles of binding and release from the SecYEG translocon coupled to ATP turnover. SecA forms a physiological dimer with a dissociation constant that has previously been shown to vary with temperature and ionic strength. We now present data showing that the oligomeric state of SecA in solution is altered by ligands that it interacts with during protein translocation. Analytical ultracentrifugation, chemical cross-linking, and fluorescence anisotropy measurements show that the physiological dimer of SecA is monomerized by long-chain phospholipid analogues. Addition of wild-type but not mutant signal sequence peptide to these SecA monomers redimerizes the protein. Physiological dimers of SecA do not change their oligomeric state when they bind signal sequence peptide in the compact, low temperature conformational state but polymerize when they bind the peptide in the domain-dissociated, high-temperature conformational state that interacts with SecYEG. This last result shows that, at least under some conditions, signal peptide interactions drive formation of new intermolecular contacts distinct from those stabilizing the physiological dimer. The observations that signal peptides promote conformationally specific oligomerization of SecA while phospholipids promote subunit dissociation suggest that the oligomeric state of SecA could change dynamically during the protein translocation reaction. Cycles of SecA subunit recruitment and dissociation could potentially be employed to achieve processivity in polypeptide transport.

“…SecA is water-soluble, but due to the nature of its activity has an intimate association with the lipid bilayer. It has been reported that acidic lipids and those that do not form lamellae increase SecA ATPase activity (28,29). Moreover, reports show that acidic phospholipids are required for protein translocation (39).…”

Section: Molecular Mass (Kda)mentioning

confidence: 99%

“…The basal ATPase activity of SecA is partially increased by the presence of acidic phospholipids and then fully stimulated by addition of vesicles containing SecYEG and precursor protein (28,29). The mechanism for this regulation is not clear.…”

mentioning

confidence: 99%

Allosteric Regulation of SecA

Gold

Robson

Clarke

et al. 2007

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.