Organization of the AAA(+) adaptor protein PspA is an oligomeric ring

The 25.3 kDa "adaptor" protein, PspA (phage shock protein A), is found in the cytoplasm and in association with the inner membrane of certain bacteria. PspA plays critical roles in negatively regulating the phage shock response and maintaining membrane integrity, especially during the export of proteins such as virulence factors. Homologues of PspA function exist for thylakoid biogenesis. Here we report the first three-dimensional reconstruction of a PspA assembly from Escherichia coli, visualized by electron microscopy and single particle analysis to a resolution of 30 Angstroms. The assembly forms a 9-fold rotationally symmetric ring with an outer diameter of 200 Angstroms, an inner diameter of 95 Angstroms, and a height of approximately 85 Angstroms. The molecular mass of the complex was calculated to be 1023 kDa by size exclusion chromatography, suggesting that each of the nine domains is likely to be composed of four PspA subunits. The functional implications of this PspA structure are discussed in terms of its interaction with the protein export machinery of the bacterial cell and its AAA(+) protein partner, PspF.     

The Tat System for Membrane Translocation of Folded Proteins Recruits the Membrane-stabilizing Psp Machinery in Escherichia coli

Tat systems transport folded proteins across energized membranes of bacteria, archaea, and plant plastids. In Escherichia coli, TatBC complexes recognize the transported proteins, and TatA complexes are recruited to facilitate transport. We achieved an abstraction of TatA from membranes without use of detergents and observed a co-purification of PspA, a membrane-stress response protein. The N-terminal transmembrane domain of TatA was required for the interaction. Electron microscopy displayed TatA complexes in direct contact with PspA. PspB and PspC were important for the TatA-PspA contact. The activator protein PspF was not involved in the PspA-TatA interaction, demonstrating that basal levels of PspA already interact with TatA. Elevated TatA levels caused membrane stress that induced a strictly PspBC- and PspF-dependent up-regulation of PspA. TatA complexes were found to destabilize membranes under these conditions. At native TatA levels, PspA deficiency clearly affected anaerobic TMAO respiratory growth, suggesting that energetic costs for transport of large Tat substrates such as TMAO reductase can become growth limiting in the absence of PspA. The physiological role of PspA recruitment to TatA may therefore be the control of membrane stress at active translocons.