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The soluble cytoplasmic ATPase motor protein SecA powers protein transport across the Escherichia coli inner membrane via the SecYEG translocon. Although dimeric in solution, SecA associates monomerically with SecYEG during secretion according to several crystallographic and cryo-EM structural studies. The steps SecA follows from its dimeric cytoplasmic state to its active SecYEG monomeric state are largely unknown. We have previously shown that dimeric SecA in solution dissociates into monomers upon electrostatic binding to negatively charged lipid vesicles formed from E. coli lipids. Here we address the question of the disposition of SecA on the membrane prior to binding to membrane embedded SecYEG. We mutated to cysteine, one at a time, 25 surface-exposed residues of a Cys-free SecA. To each of these we covalently linked the polarity-sensitive fluorophore NBD whose intensity and fluorescence wavelength-shift change upon vesicle binding report on the the local membrane polarity. We established from these measurements the disposition of SecA bound to the membrane in the absence of SecYEG. Our results confirmed that SecA is anchored in the membrane interface primarily by the positive charges of the N terminus domain. But we found that a region of the nucleotide binding domain II is also important for binding. Both domains are rich in positively charged residues, consistent with electrostatic interactions playing the major role in membrane binding. Selective replacement of positively charged residues in these domains with alanine resulted in weaker binding to the membrane, which allowed us to quantitate the relative importance of the domains in stabilizing SecA on membranes. Fluorescence quenchers inside the vesicles had little effect on NBD fluorescence, indicating that SecA does not penetrate significantly across the membrane. Overall, the topology of SecA on the membrane is consistent with the conformation of SecA observed in crystallographic and cryo-EM structures of SecA-SecYEG complexes, suggesting that SecA can switch between the membrane-associated and the translocon-associated states without significant changes in conformation. ispartof: JOURNAL OF MOLECULAR BIOLOGY vol:434 issue:12 ispartof: location:Netherlands status: published

Under acid stress, Escherichia coli induce expression of CadA (lysine decarboxylase) and CadB (lysine/cadaverine antiporter) in a lysine-rich environment. The ToxR-like transcriptional activator CadC controls expression of the cadBA operon. Using a novel signal peptidase I (SPase I) cleavage assay, we show that CadC is a type II single-span membrane protein (S-SMP) with a cytoplasmic DNA-binding domain and a periplasmic sensor domain. We further show that, as long assumed, dimerization of the sensor domain is required for activating the cadBA operon. We prove this using a chimera in which the periplasmic domain of RodZ-a type II membrane protein involved in the maintenance of the rod shape of E. coli-replaces the CadC sensor domain. Because the RodZ periplasmic domain cannot dimerize, the chimera cannot activate the operon. However, replacement of the transmembrane (TM) domain of the chimera with the glycophorin A TM domain causes intramembrane dimerization and consequently operon activation. Using a low-expression protocol that eliminates extraneous TM helix dimerization signals arising from protein over-expression, we enhanced dramatically the dynamic range of the β-galactosidase assay for cadBA activation. Consequently, the strength of the intramembrane dimerization of the glycophorin A domain could be compared quantitatively with the strength of the much stronger periplasmic dimerization of CadC. For the signal peptidase assay, we inserted an SPase I cleavage site (AAA or AQA) at the periplasmic end of the TM helix. Cleavage occurred with high efficiency for all TM and periplasmic domains tested, thus eliminating the need for the cumbersome spheroplast-proteinase K method for topology determinations.

The transmembrane (TM) helices of most type II single-span membrane proteins (S-SMPs) of Escherichia coli occur near the N-terminus, where the cell's targeting mechanisms can readily identify it as it emerges from the ribosome. However, the TM helices of a few S-SMPs, such as RodZ, occur a hundred or more residues downstream from the N-terminus, which raises fundamental questions about targeting and assembly. Because of RodZ's novelty and potential usefulness for understanding TM helix insertion in vivo, we examined its membrane targeting and assembly. We used RodZ constructs containing immunotags before the TM domain to assess membrane insertion using proteinase K digestion. We confirmed the N(in)-C(out) (type II) topology of RodZ and established the absence of a targeting signal other than the TM domain. RodZ was not inserted into the membrane under SecA depletion conditions or in the presence of sodium azide, which is known to inhibit SecA. Insertion failed when the TM proton gradient was abolished with Carbonyl cyanide m-chlorophenyl hydrazone. Insertion also failed when RodZ was expressed in SecE-depleted E. coli, indicating that the SecYEG translocon is required for RodZ assembly. Protease accessibility assays of RodZ in other E. coli depletion strains revealed that insertion is independent of SecB, YidC, and SecD/F. Insertion was found to be only weakly dependent on the signal recognition particle pathway: insertion was weakly dependent on the Ffh but independent of FtsY. We conclude that membrane insertion of RodZ requires only the SecYEG translocon, the SecA ATPase motor, and the TM proton motive force.