Stabilization of SecA ATPase by the primary cytoplasmic salt of Escherichia coli

Abstract

Much is known about the structure, function, and stability of the SecA motor ATPase that powers the secretion of periplasmic proteins across the inner membrane of Escherichia coli. Most studies of SecA are carried out in buffered sodium or potassium chloride salt solutions. However, the principal intracellular salt of E. coli is potassium glutamate (KGlu), which is known to stabilize folded proteins and protein‐nucleic acid complexes. Here we report that KGlu stabilizes SecA, including its dimeric state, and increases its ATPase activity, suggesting that SecA is likely fully folded, stable, and active in vivo at 37°C. Furthermore, KGlu also stabilizes a precursor form of the secreted maltose‐binding protein.

Binding of SecA ATPase monomers and dimers to lipid vesicles

“…We showed earlier that KGlu stabilizes SecA against thermal unfolding based upon both tryptophan fluorescence and CD measurements [45]. Specifically, for KCl solutions the denaturation midpoints determined by Trp fluorescence and CD spectroscopy were 38.2 ± 0.4 °C and 43.6 ± 0.5 °C, respectively, independent of KCl concentration.…”

“…First, we wanted to obtain accurate quantitative data on the interactions of SecA with LUV membranes of different lipid compositions. Second, in light of the fact that potassium glutamate (KGlu)-the principal cytoplasmic salt [43,44] of E. coli-significantly enhances the thermal stability of SecA monomers and dimers in solution [45], we wished to establish how KGlu affects the partitioning of SecA into membranes. Third, we wished to explore whether SecA partitions into membranes as a monomer or as a dimer, specifically the 1M6N dimer.…”

“…It is better to think of partitioning between two phases: the aqueous phase and the bilayer phase [42]. We use that approach here to determine the free energy of transfer of SecA from water to bilayer, ΔG wb , under a wide range of lipid and salt conditions including potassium glutamate (KGlu), which is the principal cytoplasmic salt of E. coli that is known to stabilize monomeric and dimeric SecA [43][44][45]. We also examine the partitioning of two disulfide-stabilized dimers based upon the 1M6N dimer.…”

Unsaturated fatty acids augment protein transport via the SecA:SecYEG translocon

“…2B and C). Next to that, the threefold lower amplitude of the FRET signal observed in POPC : POPG further suggested that a fraction of SecA : SecYEG complexes did not completely translocate the proOmpA domain, likely due to the slower translocation kinetics accompanied by inactivation of the temperature‐labile SecA ATPase [16]. Together, the results of the functional assays reveal that UFAs within the physiologically fluid lipid membrane stimulate the efficiency of the SecA : SecYEG translocon and increase the rate of the polypeptide chain transport.…”

Unsaturated fatty acids augment protein transport via the SecA:SecYEG translocon

“…twofold higher rates of the intermediate formation compared to SecYEG in POPC:POPG liposomes (Figure 1E and 1F). The lower amplitude of the FRET signal observed in POPC:POPG further suggested that a fraction of SecA:SecYEG complexes did not completely translocate the proOmpA domain, likely due to the slower translocation kinetics accompanied by inactivation of the temperature-labile SecA ATPase [17]. Together, the results of the functional assays reveal that UFAs within the physiologically fluid lipid membrane stimulate the efficiency of the SecA:SecYEG translocon and increase the rate of the polypeptide chain transport.…”