In vitro studies with purified components reveal signal recognition particle (SRP) and SecA/SecB as constituents of two independent protein-targeting pathways of Escherichia coli.

The molecular requirements for the translocation of secretory proteins across, and the integration of membrane proteins into, the plasma membrane of Escherichia coli were compared. This was achieved in a novel cell-free system from E. coli which, by extensive subfractionation, was simultaneously rendered deficient in SecA/SecB and the signal recognition particle (SRP) components, Ffh (P48), 4. 5S RNA, and FtsY. The integration of two membrane proteins into inside-out plasma membrane vesicles of E. coli required all three SRP components and could not be driven by SecA, SecB, and DeltamicroH+. In contrast, these were the only components required for the translocation of secretory proteins into membrane vesicles, a process in which the SRP components were completely inactive. Our results, while confirming previous in vivo studies, provide the first in vitro evidence for the dependence of the integration of polytopic inner membrane proteins on SRP in E. coli. Furthermore, they suggest that SRP and SecA/SecB have different substrate specificities resulting in two separate targeting mechanisms for membrane and secretory proteins in E. coli. Both targeting pathways intersect at the translocation pore because they are equally affected by a blocked translocation channel.

Requirements for the translocation of elongation-arrested, ribosome-associated OmpA across the plasma membrane of Escherichia coli.

An oligodeoxynucleotide-dependent method to generate nascent polypeptide chains was adopted for use in a cell-free translation system prepared from Escherichia coli. In this way, NH2-terminal pOmpA fragments of distinct sizes were synthesized. Because most of these pOmpA fragments could be covalently linked to puromycin, precipitated with cetyltrimethylammonium bromide, and were enriched by sedimentation, they represent a population of elongation-arrested, ribosome-associated nascent chains. Translocation of these nascent pOmpA chains into inside-out membrane vesicles of E. coli required SecA and (depending on size) SecB. Whereas their translocation was strictly dependent on the H+-motive force of the vesicles, no indication for the involvement of the bacterial signal recognition particle was obtained. SecA and SecB, although required for translocation, did not mediate binding of the ribosome-associated pOmpA to membrane vesicles. However, SecA and SecB cotranslationally associated with nascent pOmpA, since they could be co-isolated with the ribosome-associated nascent chains and as such catalyzed translocation subsequent to the release of the ribosome. These results indicate that in E. coli, SecA also functionally interacts with preproteins before they are targeted to the translocase of the plasma membrane.

Comparative characterization of SecA from the alpha-subclass purple bacterium Rhodobacter capsulatus and Escherichia coli reveals differences in membrane and precursor specificity.

We have cloned the secA gene of the alpha-subclass purple bacterium Rhodobacter capsulatus, a close relative to the mitochondrial ancestor, and purified the protein after expression in Escherichia coli. R. capsulatus SecA contains 904 amino acids with 53% identity to E. coli and 54% identity to Caulobacter crescentus SecA. In contrast to the nearly equal partitioning of E. coli SecA between the cytosol and plasma membrane, R. capsulatus SecA is recovered predominantly from the membrane fraction. A SecA-deficient, cell-free synthesis-translocation system prepared from R. capsulatus is used to demonstrate translocation activity of the purified R. capsulatus SecA. This translocation activity is then compared to that of the E. coli counterpart by using various precursor proteins and inside-out membrane vesicles prepared from both bacteria. We find a preference of the R. capsulatus SecA for the homologous membrane vesicles whereas E. coli SecA is active with either type of membrane. Furthermore, the two SecA proteins clearly select between distinct precursor proteins. In addition, we show here for the first time that a bacterial c-type cytochrome utilizes the canonical, Sec-dependent export pathway.

Identification of a soluble SecA/SecB complex by means of a subfractionated cell-free export system.

We have reconstituted the cell-free synthesis of the Escherichia coli precursor protein LamB from partially purified subfractions of an E. coli cell extract. PreLamB synthesized in this manner is translocated into salt-extracted plasma membrane vesicles only in the presence of SecA/SecB- or SecB-containing preparations of the E. coli cytosol. The most active preparations obtained upon purification were those containing a soluble SecA/SecB complex. Complex formation between SecA and SecB was verified by co-sedimentation and co-immunoprecipitation. When preLamB was synthesized in the presence of this material, a considerable amount of precursor was recovered from a soluble ternary complex consisting of preLamB, SecA, and SecB. Our results suggest that a soluble SecA/SecB complex participates in the export of preLamB and that this complex is functionally equivalent to a previously described 12 S (7 S) export factor (Müller, M., and Blobel, G. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 7737-7741; Watanabe, M., and Blobel, G. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 2728-2732).

Precursor-specific requirements for SecA, SecB, and delta muH+ during protein export of Escherichia coli.

We compare translocation into inside-out plasma membrane vesicles (INV) of the in vitro synthesized outer membrane proteins LamB and OmpA and the periplasmic protein Skp of Escherichia coli and demonstrate a precursor-specific dependence on the export factors SecA, SecB, and the proton-motive force (delta mu H+). A partial reduction in soluble SecA caused a 50% decrease in translocation of preLamB. In contrast, removal of INV-bound SecA by urea extraction was required to see a decrease in translocation of preOmpA and preSkp, with 8% of preSkp still being translocated into urea-treated INV. Translocation of the three precursors into INV showed a corresponding differential sensitivity toward dissipation of delta mu H+ following removal of the F1-ATPase from the INV. While depletion of both F1 and SecA or simply lowering of the reaction temperature resulted in an inhibition of complete transmembrane translocation, it interfered less severely with signal sequence cleavage, indicating the formation of translocation intermediates under these conditions. The relative amounts of intermediate obtained were also different for the three preproteins correlating a low requirement for SecA and delta mu H+ with a facilitated initiation of translocation. Whereas preSkp was translocated independently of SecB, preLamB was not even targeted to the INV in its absence. Functional targeting of preOmpA required the presence of SecB during incubation of the precursor with INV and not during its synthesis. SecB, exogenously added during the period of synthesis, did not prevent the formation of translocation-incompetent preLamB. The latter results are consistent with an important targeting function of SecB, which so far has mostly been described as a molecular chaperone. The findings are discussed with respect to current models of bacterial protein export usually derived from the analysis of a single precursor.

Biochemical analysis of the biogenesis and function of the Escherichia coli export factor SecY.

SecY is an integral plasma-membrane protein of Escherichia coli which is essential for the export of periplasmic and outer-membrane proteins containing cleavable signal sequences. We have synthesized SecY in vitro using an E. coli transcription/translation system. In the absence of membranes, SecY remained largely soluble but cosedimented on sucrose gradients with the membrane fraction when inside-out plasma-membrane vesicles (INV) had been added cotranslationally. Membrane association of SecY was unaffected if the endogenous SecY of the INV had been inactivated by either antibodies, a mutation or trypsin treatment. In contrast, inactivation of the INV SecY interfered with membrane targeting and, consequently, the processing of precursors to beta-lactamase and lambda receptor. When SecY-deprived INV were, however, first functionally reconstituted with in-vitro-synthesized SecY, targeting and translocation of the lambda receptor were partially restored. Thus, the assembly of SecY into INV in vitro leads to an active enzyme. In addition, we show that the prlA4 allele of the secY gene suppresses signal-sequence mutations of the lambda receptor in vitro. Collectively, our results demonstrate that SecY, while functioning as a membrane-located receptor for precursors of exported proteins, appears to be virtually independent of pre-existing SecY for its own membrane integration.

A protein with sequence identity to Skp (FirA) supports protein translocation into plasma membrane vesicles of Escherichia coli.

We have purified to homogeneity a 15 kDa-protein from a ribosomal salt extract of Escherichia coli that compensates in vitro a defect of SecA but not of SecB. Removal of this protein from a cell-free transcription/translation system impairs translocation into plasma membrane vesicles of the precursors of LamB and to a lesser degree also of OmpA. These results suggest a role of the 15 kDa-protein in bacterial protein export. The NH2-terminal 35 amino acids were found to be identical to those of the skp (firA) gene product, to which several putative functions have previously been attributed.

Determinants of membrane-targeting and transmembrane translocation during bacterial protein export.

We have separately analyzed membrane-targeting and membrane translocation of an exported bacterial protein. The precursor of the outer membrane protein LamB of Escherichia coli was synthesized in vitro and translocated into inverted plasma membrane vesicles under co- and post-translational conditions. The translation/translocation products of LamB were subsequently resolved into soluble and membrane-associated material. Dissipation of the H(+)-motive force, depletion of ATP and treatment of membranes with N-ethylmaleimide each inhibited processing and translocation of preLamB without preventing its binding to the membranes. Hence, all three conditions block transmembrane passage rather than membrane-targeting. The latter was abolished by pretreatment of salt-extracted membrane vesicles with trypsin. It was also drastically reduced when preLamB was synthesized in cell extracts derived from either a secA amber or a secB null mutant. Membrane-targeting of preLamB therefore requires soluble SecA and SecB as well as a protease-sensitive membrane receptor. The finding that SecA is involved in targeting whereas ATP is required for the transmembrane passage suggests that SecA, which harbors an ATPase activity [Lill et al. (1989), EMBO J., 8, 961-966], might have a dual function in bacterial protein export.

In vitro studies on the translocation of acid phosphatase into the endoplasmic reticulum of the yeast Saccharomyces cerevisiae.

We demonstrate here the in vitro translocation of yeast acid phosphatase into rough endoplasmic reticulum. The precursor of the repressible acid phosphatase from Saccharomyces cerevisiae encoded by the PHO5 gene, was synthesized in a yeast lysate programmed with in vitro transcribed PHO5 mRNA. In the presence of yeast rough microsomes up to 16% of the acid phosphatase synthesized was found to be translocated into the microsomes, as judged by proteinase resistance, and fully core-glycosylated. The translocation efficiency however, decreased to 3% if yeast rough microsomes were added after synthesis of acid phosphatase had been terminated. When a wheat-germ extract was used for in vitro synthesis, the precursor of acid phosphatase was translocated into canine pancreatic rough microsomes and thereby core-glycosylated in a signal-recognition-particle-dependent manner. Replacing canine with yeast rough microsomes in the wheat-germ translation system, however, resulted in a significant decrease in the ability to translocate and glycosylate the precursor. Translocation and glycosylation were partially restored by a high-salt extract prepared from yeast ribosomes. The results presented here suggest that yeast-specific factors are needed to translocate and glycosylate acid phosphatase efficiently in vitro.

In vitro membrane assembly of a polytopic, transmembrane protein results in an enzymatically active conformation.

In vitro integration of the polytopic, transmembrane lactose permease into membrane vesicles from Escherichia coli is demonstrated. To this end the enzyme was synthesized in a homologous, cell-free transcription-translation system. In this system, synthesis occurred in an essentially membrane-free environment leading to the formation of lactose permease aggregates, which were resistant to protease digestion and detergent solubilization. However, if inverted membrane vesicles from E. coli were included in the synthesis reaction, most de novo-synthesized lactose permease could be recovered from a membrane-containing subfraction (enriched in leader [signal] peptidase activity). This membrane association of lactose permease was Na2CO3 resistant, detergent sensitive, and yielded a distinct pattern of proteolytic cleavage peptides. Moreover, membrane vesicles when present cotranslationally during synthesis of lactose permease, acquired the capability to accumulate lactose, strongly suggesting a correct in vitro assembly of the enzyme. Because of the extensive aggregation of lactose permease synthesized in the absence of membranes, only low amounts originating from the soluble enzyme pool integrated posttranslationally into the membrane vesicles. Unlike the translocation of the outer membrane protein LamB into membrane vesicles, integration of lactose permease was found to be independent of the H+-motive force.

DCCD inhibits protein translocation into plasma membrane vesicles from Escherichia coli at two different steps.

In vitro translocation of periplasmic and outer membrane proteins into inverted plasma membrane vesicles from Escherichia coli was completely prevented by the H+-ATPase inhibitor N,N'-dicyclohexylcarbodiimide (DCCD). DCCD was inhibitory to both co- and post-translational translocations, suggesting an involvement of the H+-translocating F1F0-ATPase in either mode of transport. This was verified by (i) the dependence of efficient co-translational translocation upon a low salt, i.e. F1-containing extract from membrane vesicles; (ii) the co-purification of the translocation activity present in this extract and F1-ATPase; (iii) the inability of either vesicles or their low-salt extract, derived from F1F0-ATPase-lacking mutant strains, to support translocation; and (iv) the greatly diminished extent of ATP-dependent, post-translational translocation into F1-deprived vesicles. Membranes devoid of F1 did show, however, residual translocation activity that was also found to be inhibitable by DCCD. These results suggest a dual target for DCCD in bacterial protein export, one being the H+-ATPase and the other an as yet unidentified translocation factor.