In vivo cross-linking of the SecA and SecY subunits of the Escherichia coli preprotein translocase

Precursor protein translocation across the Escherichia coli inner membrane is mediated by the translocase, which is composed of a heterotrimeric integral membrane protein complex with SecY, SecE, and SecG as subunits and peripherally bound SecA. Cross-linking experiments were conducted to study which proteins are associated with SecA in vivo. Formaldehyde treatment of intact cells results in the specific cross-linking of SecA to SecY, Concurrently with the increased membrane association of SecA, an elevated amount of cross-linked product was obtained in cells harboring overproduced SecYEG complex. Cross-linked SecA copurified with hexahistidine-tagged SecY and not with SecE, The data indicate that SecA and SecY coexist as a stable complex in the cytoplasmic membrane in vivo.</p

A Single Amino Acid Substitution in SecY Stabilizes the Interaction with SecA

The SecYEG complex constitutes a protein conducting channel across the bacterial cytoplasmic membrane. It binds the peripheral ATPase SecA to form the translocase. When isoleucine 278 in transmembrane segment 7 of the SecY subunit was replaced by a unique cysteine, SecYEG supported an increased preprotein translocation and SecA translocation ATPase activity, and allowed translocation of a preprotein with a defective signal sequence. SecY(I278C)EG binds SecA with a higher affinity than normal SecYEG, in particular in the presence of ATP. The increased translocation activity of SecY(I278C)EG was confirmed in a purified system consisting of SecYEG proteoliposomes, while immunoprecipitation in detergent solution reveal that translocase-preprotein complexes are more stable with SecY(I278C) than with normal SecY. These data imply an important role for SecY transmembrane segment 7 in SecA binding. As improved SecA binding to SecY was also observed with the prlA4 suppressor mutation, it may be a general mechanism underlying signal sequence suppression.

Temporal Expression of the Bacillus subtilis secA Gene, Encoding a Central Component of the Preprotein Translocase

In Bacillus subtilis, the secretion of extracellular proteins strongly increases upon transition from exponential growth to the stationary growth phase. It is not known whether the amounts of some or all components of the protein translocation apparatus are concomitantly increased in relation to the increased export activity. In this study, we analyzed the transcriptional organization and temporal expression of the secA gene, encoding a central component of the B. subtilis preprotein translocase. We found that secA and the downstream gene (prfB) constitute an operon that is transcribed from a vegetative (σA-dependent) promoter located upstream of secA. Furthermore, using different independent methods, we found that secA expression occurred mainly in the exponential growth phase, reaching a maximal value almost precisely at the transition from exponential growth to the stationary growth phase. Following to this maximum, the de novo transcription of secA sharply decreased to a low basal level. Since at the time of maximal secA transcription the secretion activity of B. subtilis strongly increases, our results clearly demonstrate that the expression of at least one of the central components of the B. subtilis protein export apparatus is adapted to the increased demand for protein secretion. Possible mechanistic consequences are discussed

Temporal Expression of the Bacillus subtilis secA Gene, Encoding a Central Component of the Preprotein Translocase

In Bacillus subtilis, the secretion of extracellular proteins strongly increases upon transition from exponential growth to the stationary growth phase. It is not known whether the amounts of some or all components of the protein translocation apparatus are concomitantly increased in relation to the increased export activity. In this study, we analyzed the transcriptional organization and temporal expression of the secA gene, encoding a central component of the B. subtilis preprotein translocase. We found that secA and the downstream gene (prfB) constitute an operon that is transcribed from a vegetative (ς(A)-dependent) promoter located upstream of secA. Furthermore, using different independent methods, we found that secA expression occurred mainly in the exponential growth phase, reaching a maximal value almost precisely at the transition from exponential growth to the stationary growth phase. Following to this maximum, the de novo transcription of secA sharply decreased to a low basal level. Since at the time of maximal secA transcription the secretion activity of B. subtilis strongly increases, our results clearly demonstrate that the expression of at least one of the central components of the B. subtilis protein export apparatus is adapted to the increased demand for protein secretion. Possible mechanistic consequences are discussed

Interaction between SecA and SecYEG in Micellar Solution and Formation of the Membrane-Inserted State

Preprotein translocation in Escherichia coli is mediated by the translocase with SecA as peripheral ATPase and SecY, SecE, and SecG as membrane domain. To facilitate large-scale purification of the SecYEG heterotrimer, SecY was fused at its amino terminus with a hexahistidine tag and co-overexpressed with SecE and SecG. The presence of the His tag allowed purification of homogeneously pure SecYEG complex by a single anion-exchange chromatographic step starting from octyl glucoside-solubilized inner membranes. Endogenous levels of SecD and SecF copurified with the SecYEG protein. Purified SecYEG complex retained a nativelike, α-helical conformation in octyl glucoside and in micellar solution binds SecA with high affinity. In the presence of the nonhydrolyzable nucleotide analogue adenosine 5'-(β,γ-imidotriphosphate), octyl glucoside-solubilized SecYEG is nearly as effective as the reconstituted enzyme in inducing the formation of a proteinase K-protected 30 kDa fragment of 125I-labeled SecA, while SecYEG is proteolyzed to fragments smaller than 6 kDa. These data demonstrate that the 30-kDa SecA fragment is not protected by the lipid phase nor by SecYEG but rather indicate that it represents a SecYEG- and nucleotide-induced stable conformational state of a SecA domain.

Cysteine-Directed Cross-Linking Demonstrates That Helix 3 of SecE Is Close to Helix 2 of SecY and Helix 3 of a Neighboring SecE

Preprotein translocation in Escherichia coli is mediated by translocase, a multimeric membrane protein complex with SecA as the peripheral ATPase and SecYEG as the translocation pore. Unique cysteines were introduced into transmembrane segment (TMS) 2 of SecY and TMS 3 of SecE to probe possible sites of interaction between the integral membrane subunits. The SecY and SecE single-Cys mutants were cloned individually and in pairs into a secYEG expression vector and functionally overexpressed. Oxidation of the single-Cys pairs revealed periodic contacts between SecY and SecE that are confined to a specific α-helical face of TMS 2 and 3, respectively. A Cys at the opposite α-helical face of TMS 3 of SecE was found to interact with a neighboring SecE molecule. Formation of this SecE dimer did not affect the high-affinity binding of SecA to SecYEG and ATP hydrolysis, but blocked preprotein translocation and thus uncouples the SecA ATPase activity from translocation. Conditions that prevent membrane deinsertion of SecA markedly stimulated the interhelical contact between the SecE molecules. The latter demonstrates a SecA-mediated modulation of the protein translocation channel that is sensed by SecE.

SecA is an intrinsic subunit of the Escherichia coli preprotein translocase and exposes its carboxyl terminus to the periplasm

SecA is the dissociable ATPase subunit of the Escherichia coli preprotein translocase, and cycles in a nucleotide-modulated manner between the cytosol and the membrane. Overproduction of the integral subunits of the translocase, the SecY, SecE and SecG polypeptides, results in an increased level of membrane-bound SecA. This fraction of Sec A is firmly associated with the membrane as it is resistant to extraction with the chaotropic agent urea, and appears to be anchored by SecYEG rather than by lipids. Topology analysis of this membrane-associated form of Sec A indicates that it exposes a carboxy-terminal domain to the periplasmic face of the membrane.