Requirements for the Translocation of Elongation-arrested, Ribosome-associated OmpA across the Plasma Membrane ofEscherichia coli

Abstract

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, NH 2 -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, ribosomeassociated 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.Protein export across the plasma (cytoplasmic, inner) membrane of Escherichia coli is achieved by the concerted action of a distinct set of Sec proteins (summarized in Ref. 1). Most of them are integral membrane proteins of the plasma membrane. SecY and SecE most likely are core constituents of the translocation pore in the membrane; SecG appears to change its membrane topography during polypeptide translocation (2); the exact roles of SecD, SecF, and YajC are yet to be established. SecA on the other hand, is a peripheral membrane protein that binds to SecYE (3) probably serving as a membrane receptor for a preprotein-SecB complex. SecB functions as a chaperone (4) and as a targeting factor (5). Due to its ATPase activity, SecA inserts in, and deinserts from, the membrane in a cyclic manner (6, 7), which leads to the stepwise translocation of the precursor across the membrane bilayer (8, 9). In addition to ATP, the H ϩ -motive force (⌬H ϩ ) is utilized as an energy source for translocation.This model does not ascribe a function to the soluble form of SecA, which in E. coli partitions roughly equally between cytosol/ribosomes and the plasma membrane (10, 11). The occurrence of cytosolic complexes between precursor proteins and SecA (12, 13) suggests that SecA actually might interact with its protein substrate well before it has been targeted to the membrane. Besides the Sec proteins, E. coli possesses a signal recognition particle (SRP) 1 /SRP receptor system whose eukaryotic equivalents mediate the cotranslational targeting of nascent secretory and membrane proteins to the endoplasmic reticulum (14). Whereas more recent results (15-18) indicate that the bacterial SRP has a specialized role in the integrati...

Separate Analysis of Twin-arginine Translocation (Tat)-specific Membrane Binding and Translocation in Escherichia coli

“…The inserted DNA was verified by sequencing. Plasmids pMW18 encoding the fusion protein TorA-23K (18) and pDMB encoding pre-OmpA (19) have been described.…”

In Vitro Studies with Purified Components Reveal Signal Recognition Particle (SRP) and SecA/SecB as Constituents of Two Independent Protein-targeting Pathways ofEscherichia coli

“…Subcloning the mtlA gene under the control of the SP6 phage promoter was performed by ligating the StuI-SalI fragment of pCD7.5 into vector pSELECT (Promega, Madison, WI) cut with SmaI and SalI yielding plasmid pMtlII-9. Plasmid pDMB contains the ompA gene under the T7 phage promoter (Behrmann et al, 1998). Plasmid pJM8CS7, carrying the secY gene under the T7 phage promotor, was constructed by subcloning the KpnI-PstI fragment of pNO1573 (Akiyama and Ito, 1985) into pBluescript (Stratagene, La Jolla, CA), resulting in pJMsecY7.…”

“…Urea was added to 500 l of reaction mixture to a final concentration of 8 M, and the mixture was incubated for 30 min at 4°C. After 1:1 dilution with translocation buffer (Behrmann et al, 1998), it was buffer exchanged against translocation buffer and concentrated twofold (with respect to the starting material) by ultrafiltration using Centricon 3 microconcentrators (Amicon). The OmpA::tRNA-containing mixture (250 l) was incubated with 7.5 l INV (10 eq; 1 eq is the amount used per 25 l cell-free synthesis reaction) in the presence of 2.5 mM ATP, 8 mM creatine phosphate, 40 g/ml creatine phosphokinase, 2 mM DTT for 15 min at 37°C.…”

“…This technique was applied to generate a pOmpA derivative carrying a tRNA at its COOH terminus (pOmpA::tRNA). The fusion was synthesized in vitro by transcription/translation of the ompA gene in the presence of an oligonucleotide to generate 125-amino acid ribosome-associated nascent chains (Behrmann et al, 1998), which were subsequently released from the ribosomes with 8 M urea, leaving the peptidyl-tRNA bond intact. Upon translocation into INV, the bulky tRNA moiety should cause stalling of the translocating polypeptide, resulting in a blocked SecYEG translocation pore.…”

“…Ribosome-associated, nascent chains of pOmpA were synthesized with the ␤5-oligodeoxynucleotides as described earlier (Behrmann et al, 1998) in a 20-fold scaled-up reaction. Urea was added to 500 l of reaction mixture to a final concentration of 8 M, and the mixture was incubated for 30 min at 4°C.…”