Molecular Determinants for PspA-Mediated Repression of the AAA Transcriptional Activator PspF

Abstract

The Escherichia coli phage shock protein system (pspABCDE operon and pspG gene) is induced by numerous stresses related to the membrane integrity state. Transcription of the psp genes requires the RNA polymerase containing the 54 subunit and the AAA transcriptional activator PspF. PspF belongs to an atypical class of 54 AAA activators in that it lacks an N-terminal regulatory domain and is instead negatively regulated by another regulatory protein, PspA. PspA therefore represses its own expression. The PspA protein is distributed between the cytoplasm and the inner membrane fraction. In addition to its transcriptional inhibitory role, PspA assists maintenance of the proton motive force and protein export. Several lines of in vitro evidence indicate that PspA-PspF interactions inhibit the ATPase activity of PspF, resulting in the inhibition of PspF-dependent gene expression. In this study, we characterize sequences within PspA and PspF crucial for the negative effect of PspA upon PspF. Using a protein fragmentation approach, we show that the integrity of the three putative N-terminal ␣-helical domains of PspA is crucial for the role of PspA as a negative regulator of PspF. A bacterial two-hybrid system allowed us to provide clear evidence for an interaction in E. coli between PspA and PspF in vivo, which strongly suggests that PspA-directed inhibition of PspF occurs via an inhibitory complex. Finally, we identify a single PspF residue that is a binding determinant for PspA.

Possible function of VIPP1 in maintaining chloroplast membranes

“…The protein containing only HD1, HD2, and HD3 forms dimers. Apparently, HD4 is involved in forming a 36-mer ring [32,33]. Recently, two amphipathic helices, ahA and ahB, in PspA HD1 have been studied in detail.…”

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

“…In addition, PspA is known to interact with the regulatory protein PspF that can activate a 54 -dependent transcription of the psp genes under membrane stress conditions (45). To find out whether these interactions are important for the TatA-PspA contact, we carried out our analyses in E. coli strains that were deleted in the genes encoding PspB, PspC, or PspF (Fig.…”

Induction and Function of the Phage Shock Protein Extracytoplasmic Stress Response in Escherichia coli

“…PspA specifically and directly binds to the AAAϩ transcription activation domain of PspF, inhibiting pspA-E and pspG transcription (Ref. 9 and for review, see Ref. 1 and 2).…”