From the Adolf Butenandt Institut für Physikalische Biochemie, Ludwig-Maximilians-Universität München, D-80336 München, Germany
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ABSTRACT |
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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.
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INTRODUCTION |
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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+-motive force
(µH+) 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 integration of hydrophobic membrane proteins, several reports had suggested a role of the bacterial SRP in the export of signal sequence-containing proteins (19-21).
To elucidate early events during translocation of bacterial secretory proteins in general, and the function of soluble SecA in particular, the translocation of ribosome-associated, nascent chains of OmpA was analyzed. The outer membrane protein OmpA was chosen because it is one of the most widely studied model precursors of E. coli. In vitro synthesis of ribosome-associated, nascent polypeptide chains requires a truncated mRNA lacking a stop codon so that ribosomes carrying nascent chains stall at the end of this mRNA fragment. Truncation of mRNA is most commonly achieved by the use of linearized DNA. This method has successfully been used with both eukaryotic and prokaryotic mRNAs translated in a wheat germ extract to demonstrate direct molecular interaction of the E. coli SRP subunit Ffh (P48) with signal sequences (22, 23). In our hands, however, linearized DNA turned out to be extremely unstable in cell-free transcription/translation systems made from E. coli, giving rise to a large population of undefined peptides. The use of a homologous translation system from E. coli, however, appeared to be imperative for the analysis of early translocation steps, since recent studies show that results obtained with wheat germ ribosomes might not necessarily reflect the authentic events occurring in E. coli (24).
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EXPERIMENTAL PROCEDURES |
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Subcloning and Transcription of ompA-- An EcoRI-PstI fragment of plasmid pRD87 (25) containing the E. coli ompA gene was subcloned into vector pGEM-3Z (Promega) to yield plasmid pDMB in which ompA is under the control of the T7 phage promoter. For in vitro transcription, pDMB was linearized with PstI. 20 mg of linearized DNA was transcribed in 100-µl reactions containing 40 mM Tris/HCl, pH 7.5; 10 mM NaCl; 6 mM MgCl2; 2 mM spermidine; 10 mM dithiothreitol; a 0.5 mM concentration each of ATP, GTP, CTP, and UTP; 0.1 mg/ml acetylated bovine serum albumin; 400 units/ml placental RNase inhibitor; and 600 units/ml T7 RNA polymerase. Incubation was for 60 min at 37 °C. The mRNA was precipitated with ethanol in the presence of 200 mM NH4CH3COO and redissolved in 50 µl of H2O.
Cell-free Protein Synthesis-- A nuclease-treated wheat germ extract was prepared, and the translation system was operated as described (26) with the following modifications. The final concentrations of K+, Mg2+, spermidine, and S-adenosylmethionine were 80 mM, 2.5 mM, 0.08 mM, and 5 µM, respectively; 25-µl reactions contained 4 µl of wheat germ extract, 12 units of placental RNase inhibitor, 2 µl of transcribed ompA RNA, and 10 µCi of [35S]methionine (>1000 Ci/mmol). Incubation was for 2 h at 25 °C.
The components of the reconstituted system (RCS), salt-washed ribosomes, initiation factors, and the undefined fraction of soluble translation factors were prepared as described (27). In vitro protein synthesis by use of the RCS was performed in 25-µl aliquots of translation buffer (40 mM triethanolamine/CH3COO, pH 7.5, 70 mM KCH3COO, 10 mM Mg(CH3COO)2, 0.8 mM spermidine) containing 3.2% polyethylene glycol 6000; 2.5 mM ATP; a 0.5 mM concentration each of GTP, CTP, and UTP; 0.01 N KOH; 12 mM phosphoenolpyruvate; 8 mM creatine phosphate; 40 µg/ml creatine phosphokinase; 2 mM dithiothreitol; a 40 µM concentration each of 19 amino acids; 10 µCi of [35S]methionine (>1000 Ci/mmol); 0.2 mg/ml E. coli tRNA; 6 units of T7 RNA polymerase; 8 units of placental RNase inhibitor; and 1 µg of pDMB as well as optimal amounts of ribosomes, initiation factor 2, and undefined fraction. Initiation factors 1 and 3 were omitted. Incubations were for 30 min at 37 °C.Isolation of Ribosome-associated, Nascent Chains
(RANCs)--
RANCs of pOmpA were synthesized by the RCS in the
presence of the oligodeoxynucleotides 2
(5'-AAGCCAACATACGGGTTAAC-3'),
5 (5'-TAAACGTTGGATTTAGTGTC-3'), and
8 (5'-GGAGCTGCCTCGCCCTGACC-3') (amounts are given in the legend to
Fig. 1). Aliquots of 25-100 µl were
centrifuged through 100 µl of 20% (w/v) sucrose prepared in
translation buffer in a Beckman Airfuge at 30 p.s.i. for 60 min at
4 °C. Pelleted RANCs were resuspended in the original volume of
translocation buffer (translation buffer containing all low molecular
weight substances of the RCS except for amino acids). Resuspension in
an initial volume of 20 µl was carried out by shaking three times for
3 min each in an Eppendorf test tube shaker in the cold with 5-min
intervals when samples were kept on ice. This was followed by repeated
pipetting steps. After removal of the liquid, the procedure was
repeated with another aliquot of 30 µl of buffer. Resuspended RANCs
could be stored at
80 °C for further use.
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Membrane Vesicles-- Gradient-purified inner membrane vesicles were obtained as described (28) and extracted with 6 M urea (29). For extraction with 1 M KCH3COO, 50-µl aliquots of inside-out, plasma membrane vesicles (INV) were incubated with 25 µl of 3 M KCH3COO for 15 min on ice and centrifuged through a cushion of 30% sucrose prepared in 50 mM triethanolamine/CH3COO, pH 7.5, 1 mM dithiothreitol for 10 min at 30 p.s.i. in a Beckman Airfuge. Low salt washing of INV (wINV) was performed as described (30).
Purification of Components-- The purification of SecA (29), SecB (13), and F1-ATPase (up to the gel filtration step; Ref. 30) followed previously reported protocols. His-tagged Ffh (P48) and 4.5 S RNA were prepared as detailed elsewhere.2 FtsY was purified according to Luirink et al. (21) except that the first MonoQ column was replaced by Q-Sepharose.
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RESULTS |
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Synthesis of Discrete Nascent Chains of pOmpA by Wheat Germ and E. coli Ribosomes--
In order to synthesize nascent chains of pOmpA
in vitro, we adopted a method described previously (31) to
create elongation-arrested chains by use of oligodeoxynucleotides
complementary to discrete sections of the mRNA under investigation.
The DNA-RNA hybrids thus formed in vitro are cleaved by
endogenous RNase H, leading to 5'-fragments of mRNA. Three
oligodeoxynucleotides were designed so that the resulting nascent
chains would be caused by translational stops after the second, the
fifth, and the eighth -strand of OmpA (32): pOmpA-
2, 65 amino
acids; pOmpA-
5, 125 amino acids; pOmpA-
8, 191 amino acids.
The Translocation of Nascent pOmpA Chains Is Mediated by SecA and
SecB--
Next, the translocation of RANCs of pOmpA into INV of
E. coli was examined (translocation was assayed via
resistance to proteinase K as illustrated in Fig.
2D, lanes 5-7, and
Fig. 5, lanes 18-20). Routinely, INV had been extracted
with 1 M potassium acetate (K-INV), which removes a
considerable amount of the INV-bound SecA (13). Only background levels
of translocation into K-INV were observed when pOmpA-5 was
synthesized by wheat germ ribosomes (Fig. 2A, compare
lanes 8 and 10). Translocation was completely
suppressed by removing additional SecA from the INV by extraction with
6 M urea (U-INV; Fig. 2A, lane 4).
Translocation into both types of membranes, however, was clearly
stimulated by the addition of pure SecA and SecB (Fig. 2A,
lanes 1-4 and 5-8).
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The Bacterial SRP Is Not Required for Translocation of Nascent
Chains of pOmpA--
The finding that RANCs of pOmpA could not be
translocated into U-INV by SecA/B alone when synthesized in the
E. coli cell-free system prompted the question of whether an
additional necessary component had been removed from INV by urea
treatment. We have recently found that urea treatment of INV also
interferes with the integration of membrane proteins, a property that
is partially restored by the addition of the purified components of the
bacterial SRP/SRP-receptor system but not by SecA and
SecB.2 In contrast, the failure of SecA/B to translocate
pOmpA-5 into U-INV (Fig. 3,
lanes 1-4) was not overcome by Ffh (P48), FtsY, and 4.5 S
RNA when added as purified components (lanes 5 and
6) at concentrations that had restored integration of a
membrane protein to U-INV.2 There was also no translocation
of pOmpA-
5 and full-length pOmpA when the SRP components were added
without SecA/B (lanes 7 and 8). However,
translocation of pOmpA-
5 into U-INV occurred if, in addition to
SecA/B, purified F1-ATPase was provided to restore the
µH+ (not shown).
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Cotranslational Binding of SecA/B to Nascent pOmpA Is Not Linked to
a Cotranslational Membrane Targeting--
In the previous experiments,
SecA and SecB had always been substituted together. The SecB-binding
site of OmpA has been localized to the first 229 amino acids of the
mature protein (34), which include the entire -stranded domain.
Because pOmpA-
5 contains only five of the eight
-strands, we
assumed that its membrane translocation might not to the same extent
depend on SecB as that of pOmpA-
8. In fact, Fig.
5 illustrates that the SecA-mediated translocation of pOmpA-
8 into K-INV (lanes 14 and
15) was further stimulated by SecB (lanes 16 and
17), whereas that of pOmpA-
5 was not (compare lanes
5 and 6 with lanes 7 and 8).
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DISCUSSION |
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Results Obtained with RANCs Depend on the Origin of
Ribosomes--
Noticeable differences between the plant and the
bacterial translation system were that the former (i) was less
stringently dependent on µH+ for translocation and
(ii) still allowed for considerable processing even if little
translocation was observed in the absence of SecA/B (cf.
Fig. 2A, lanes 5-8). In the E. coli
system, however, both processing and translocation were abolished under
these conditions (Fig. 5, compare lanes 1 and 2 to lanes 7 and 8). These differences underline
the necessity to use a homologous in vitro system in order
to examine the authentic events during the biogenesis and translocation
of bacterial nascent preproteins.
No Indication for a Ribosome-involving Membrane Targeting of RANCs in E. coli-- We have obtained no evidence for a targeting of ribosome/nascent chain complexes to the E. coli membrane vesicles. Thus, isolated RANCs of pOmpA did not bind to INV in a way that would have withstood reisolation of the membranes. Such binding of RANCs to membrane vesicles is characteristic for the SRP-mediated targeting of eukaryotic nascent chains to the endoplasmic reticulum and has frequently been demonstrated with both native (cf. Refs. 36 and 37) and reconstituted (38) microsomes. On the other hand, Josefsson and Randall (39) demonstrated that in E. coli, cleavage of the signal sequence occurs with unfinished polypeptide chains and concluded that translocation can initiate late during the synthesis of bacterial precursor proteins. We assume that in this case membrane targeting of nascent chains, which have already grown up to 80% of their final size, does not involve a ribosome/membrane junction but is rather achieved by a SecA/B-dependent insertion of the NH2-terminal portion of the proteins.
Previous reports had suggested a role of the bacterial SRP in the export of signal sequence-containing proteins (19-21) including that of OmpA (40). We have recently found that the RCS used here for in vitro transcription/translation contains sufficient amounts of the SRP components when combined with K-INV to allow for the integration of an SRP-dependent membrane protein.2 Under the same experimental conditions, targeting of nascent pOmpA to K-INV could not be detected, indicating that the bacterial SRP is not involved in such a targeting step. Consistently, the use of GMP-PNP and GDP-A Precursor Recognition Function for SecA-- The method used here to create RANCs yielded peptides of largely distinct lengths, which is a prerequisite to show that the SecB dependence of translocation correlates with the length of the nascent pOmpA chains. Using a similar approach of producing nascent chains of the maltose-binding protein, a critical length was defined below which tight binding of SecB was not observed (43). Most of the data collected so far, including the data presented here, are consistent with the view that SecB binds within the mature part of preproteins (reviewed in Ref. 44), although involvement of the signal sequence in the binding of SecB has also been postulated (45, 46).
We demonstrate here that SecA can tightly bind to nascent pOmpA and remain functional for a proficient interaction with the translocase in the plasma membrane. We therefore propose that in addition to its widely accepted function as a membrane-located receptor for precursor proteins (1), SecA also operates as recognition factor for nascent secretory proteins. This mode of action would then be represented by the fraction of non-membrane-associated SecA, completely compatible with the fact that a considerable amount of soluble SecA is recovered from the ribosomes (10, 47). Conceivably, this property of SecA has so far not received much attention because most of the functions of SecA were deduced from studies employing purified, denatured precursor proteins, a strategy that bypasses intermolecular contacts during the biogenesis of presecretory proteins. Interaction of SecA with a precursor protein was reported to involve specific features of the signal sequence such as the positively charged NH2 terminus (48). Is it also influenced by SecB? A molecular interaction between SecB and SecA was first deduced from the finding that SecB binds with high affinity to INV only in the presence of SecA (49). Later on, it was directly demonstrated to occur by the purification of an enzymatically active complex between SecA and SecB from E. coli (13). Moreover, SecA and SecB were both found attached to a completed precursor synthesized in vitro (13). Further experimentation is required to examine whether or not recognition of precursors by SecA is modulated by SecB. ![]() |
FOOTNOTES |
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* This work was supported by a grant from the Sonderforschungsbereich 184 and the Fonds der Chemischen Industrie.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Present address: Institut für Biochemie und
Molekularbiologie, Universität Freiburg, Hermann-Herderstr. 7, D-79104 Freiburg, Germany.
§ To whom correspondence should be addressed. Tel.: 761-203-5265; Fax: 761-203-5274; E-mail: mumatthi{at}ruf.uni-freiburg.de.
1
The abbreviations used are: SRP, signal
recognition particle; RCS, reconstituted system; RANC,
ribosome-associated, nascent chain; INV, inside-out, plasma membrane
vesicle(s); AMP-PNP, 5'-adenylyl ,
-imidodiphosphate; GMP-PNP,
5'-guanylyl
,
-imidodiphosphate; GDP-
S, guanosine
5'-O-2-(thio)diphosphate.
2 T. Hengelage, J. MacFarlane, H. K. Hoffschulte, and M. Müller, unpublished results.
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REFERENCES |
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