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J Biol Chem, Vol. 273, Issue 29, 18003-18006, July 17, 1998
,, and
From the Department of Biological Sciences,
University of Warwick, Coventry CV4 7AL, United Kingdom, the
§ School of Biological Sciences, University of East Anglia,
Norwich NR4 7TJ, United Kingdom, and the ¶ Nitrogen Fixation
Laboratory, John Innes Centre, Colney Lane,
Norwich NR4 7UH, United Kingdom
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ABSTRACT |
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Top
Abstract
Introduction
Procedures
Results & Discussion
References
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Proteins are transported across the bacterial plasma membrane and the chloroplast thylakoid membrane by means of protein translocases that recognize N-terminal targeting signals in their cognate substrates. Transport of many of these proteins involves the well defined Sec apparatus that operates in both membranes. We describe here the identification of a novel component of a bacterial Sec-independent translocase. The system probably functions in a similar manner to a Sec-independent translocase in the thylakoid membrane, and substrates for both systems bear a characteristic twin-arginine motif in the targeting peptide. The translocase component is encoded in Escherichia coli by an unassigned reading frame, yigU, disruption of which blocks the export of at least five twin-Arg-containing precursor proteins that are predicted to bind redox cofactors, and hence fold, prior to translocation. The Sec pathway remains unaffected in the deletion strain. The gene has been designated tatC (for twin-arginine translocation), and we show that homologous genes are present in a range of bacteria, plastids, and mitochondria. These findings suggest a central role for TatC-type proteins in the translocation of tightly folded proteins across a spectrum of biological membranes.
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INTRODUCTION |
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Top
Abstract
Introduction
Procedures
Results & Discussion
References
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Numerous proteins are transported into the bacterial periplasmic space by means of N-terminal extensions, termed signal peptides, that direct their translocation across the plasma membrane by the Sec apparatus (reviewed in Ref. 1). The translocation of substrates by this system involves the participation of both cytoplasmic and membrane-bound components; the soluble components, SecB and SecA, serve to prevent folding of the protein until it is directed to the membrane-bound translocase, a complex of SecYEG together with several less well defined ancillary proteins. Translocation of a partially unfolded substrate protein through the SecYEG complex is then driven by the ATPase function of the SecA protein.
The Sec apparatus recognizes signal peptides that contain three
characteristic domains: an N-terminal charged domain (usually basic), a
hydrophobic core domain and a more polar C-terminal domain (reviewed in
Ref. 2). Similar signals have been shown to target proteins across the
chloroplast thylakoid membrane (3), and it is now clear that a
prokaryotic-like Sec system operates in this membrane, presumably
inherited from the cyanobacterial-type progenitor of the chloroplast
(4, 5). However, biochemical studies of thylakoid protein transport
(reviewed in Ref. 6) have pointed to the existence of a parallel
pathway that requires neither soluble factors nor ATP but that is
instead completely reliant on the thylakoidal 
pH (7-10).
Remarkably, the substrates on this pathway are synthesized with
signal-type peptides (transfer peptides) that nevertheless direct
translocation only by the pH-dependent pathway (11, 12).
The dominant factor in this sorting process is the presence of a
twin-arginine motif immediately upstream of the hydrophobic domain that
is essential for translocation by the pH-dependent
system (13). The structure of this Sec-independent system has been
unclear for some time, but a recent study on a maize mutant has
resulted in the cloning of a gene, hcf106, encoding the
first component (14). The Hcf106 protein is localized in the thylakoid
membrane and appears to comprise a single transmembrane span with the
bulk of the protein exposed to the stromal phase.
There is now clear evidence for the existence of a similar system in prokaryotes. It has been pointed out (15) that a subset of exported proteins are synthesized with twin-Arg-containing presequences, and this applies particularly to proteins that bind any of a range of complex redox cofactors, such as iron-sulfur clusters or molybdopterin cofactors. These cofactors are apparently inserted in the cytoplasm (15), which may well require the folding of substantial sections of the protein and hence preclude translocation by the Sec machinery. Consistent with this idea, one such protein has been found to be exported in a Sec-independent manner in Escherichia coli (16). Finally, homologues of Hcf106 are encoded by previously unassigned open reading frames in the majority of eubacterial and archaeal genomes, and recent studies on E. coli have shown that these homologues are indeed involved in Sec-independent protein export. However, the precise role of these proteins has been the subject of some confusion. Weiner et al. (17) isolated an E. coli mutant defective in the export of Me2SO reductase (a predicted substrate for this pathway) in which the mutation was found to lie in a previously unassigned gene, designated mttA (for membrane targeting and translocation). The product of this gene appeared to be homologous to Hcf106, and the gene was proposed to form an operon with two further genes, mttB and mttC. However, it now transpires that the operon structure is more complex than was at first apparent; we have recently shown that this operon comprises four distinct genes because of the presence of a stop codon in the gene identified as mttA. The first gene in this operon is homologous to hcf106, whereas the gene affected in the Weiner et al. (17) study lies in a separate gene unrelated to hcf106 (18). Disruption of the authentic hcf106 homologue was found to adversely affect the export of several cofactor-containing proteins, and a complete block in the export of four proteins was observed in a double mutant in which a second, unlinked hcf106 homologue was also disrupted. The four genes in the above operon were designated tatABCD (for twin-arginine translocation pathway), and the unlinked hcf106 homologue was designated tatE.
The components of the Sec-independent translocase analyzed to date (the Hcf106 homologues TatA and TatE) together with the gene product mutated in the Weiner et al. (17) study (TatB according to the above nomenclature) play important roles in the translocation process, but we have now addressed the question of whether additional components are involved. tatA and tatB form a transcriptional unit with two other unassigned reading frames including one that we have provisionally designated tatC (originally designated yigU). Because tatC homologues are present only in those prokaryotic genomes with genes for Hcf106-like proteins and are in many cases linked to such genes, we considered TatC a potential additional component of the Sec-independent protein export system. In this report we show that TatC plays a particularly crucial role in the translocation mechanism, and we show that homologues are present in a wide range of bacteria, plastids, and mitochondria.
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EXPERIMENTAL PROCEDURES |
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Top
Abstract
Introduction
Procedures
Results & Discussion
References
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Mutant Construction-- A 590-base pair fragment covering the upstream region and the first three codons of tatC was amplified by PCR1 using primers TATC1 (5'-GCGCTCTAGAGGCGGATACGAATCAGGAACAGGC-3') and TATC2 (5'-GCGCGGATCCTACAGACATGTTTACGGTTTATCACTC-3') with chromosomal DNA as template. The product was digested with XbaI and BamHI and cloned into the polylinker of pBluescript (Stratagene) to give plasmid pFAT21. A 592-base pair fragment covering the last four codons of tatC and downstream DNA was amplified using primers TATC3 (5'-CGCATCGATACTGAAGAATAAATTCAACCGCCCGTC-3') and TATC4 (5'-GCGCGGTACCTTCATCGCAAACCCAACCGGTAATGCC-3'), digested with ClaI and KpnI and cloned into pFAT21 to give plasmid pFAT23. The deletion construct, pFAT23, would therefore encode a protein of 20 amino acids, of which the three N- and three C-terminal residues are derived from TatC, and the remainder of the residues specified by pBluescript polylinker DNA. The DNA covering the in-frame deletion of tatC was excised by digestion with XbaI and KpnI and cloned into the polylinker of pMAK705 (19) to give the construct pFAT24. The mutant allele of tatC was transferred to the chromosome of strain MC4100 (20) as described (19). The mutant strain, B1LK0, obtained from this procedure was verified by PCR using primers TATC1 and TATC4, and the chromosomal PCR product was sequenced to ensure that no mismatched bases had been introduced during the procedure.
Construction of the TorA signal sequence-23K fusion was as follows. A 176-base pair fragment of chromosomal DNA was amplified with primers TorASS1 (5'-GCGGAATTCAAGAAGGAAGAAAAATAATG-3') and TorASS2 (5'-GCGGAATTCGGTACCGTCAGTCGCCGCTTG-3'). This covered DNA from 17 bases upstream of the TorA start codon to the sixth codon of the mature TorA sequence. The product was digested with EcoRI and cloned into the polylinker of pBluescript (Stratagene). A clone with the insert in the correct orientation was determined by digestion with KpnI and designated pMW11. The gene encoding the mature region of the spinach 23-kDa oxygen evolving complex protein (23K) was excised from plasmid pOEC23mp (provided by R. B. Klösgen) by digestion with AviII and SalI, end filled with Klenow DNA polymerase, and cloned into EcoRV-digested pMW11 to give plasmid pMW18. The DNA covering the TorA-23K fusion was excised by digestion with SacI and SalI and cloned into pDHB5700 (kindly provided by J. Beckwith) to give pMW23. All clones constructed from PCR-amplified DNA were sequenced to ensure that no mismatches had been introduced during amplification.Protein Methods--
Cells were cultured anaerobically in the
medium (CR) of Cohen and Rickenberg (21) supplemented with glycerol
together with the electron acceptor appropriate to the reductase to be
analyzed or fumarate for experiments with hydrogenases. Cells were
fractionated, and the fractions were analyzed by rocket
immunoelectrophoresis and activity staining as described previously
(22-25). For pulse-chase experiments, E. coli MC4100 cells
and the mutant strain were grown overnight in LB medium and then
diluted 1:75 in CR minimal medium supplemented with ammonium
molybdate/potassium selenite (1 µM each), thiamine
(0.001%), MgCl2 (1 mM), glucose (0.4%), and
sodium nitrate (40 mM). The culture was grown anaerobically
at 37 °C until midlog phase (A600 = 0.4).
Cells were then harvested and resuspended in CR medium lacking peptone
and casamino acids but supplemented with a methionine-free amino acid
mixture (0.2 mg/ml each amino acid). After growth for 1 h,
expression of TorA-23K was induced by the addition of
isopropyl-1-thio-
-D-galactopyranoside (0.04 mM) for 30 min. 5-ml aliquots of the culture were then
incubated with 30 µCi of [35S]methionine for 1 min,
after which cold methionine was added to a concentration of 0.5 mg/ml.
Where appropriate, spheroplasts were formed by collection at 14,000 rpm
for 2 min, resuspension in ice-cold buffer (40% w/v sucrose, 33 mM Tris, pH 8.0), and incubation with lysozyme (5 µg/ml
in 1 mM EDTA) for 15 min on ice. Aliquots of the
spheroplasts were incubated on ice for 1 h in either the presence
or the absence of 0.3 mg/ml proteinase K. At the end of this period,
phenylmethylsulfonyl fluoride was added (final concentration, 0.33 mg/ml), and the samples were precipitated with trichloroacetic acid
(5%). The precipitate was pelleted, resuspended in 10 mM
Tris/2% SDS, and immunoprecipitated with antiserum to OmpA (kindly
provided by G. von Heijne) or 23K.
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RESULTS AND DISCUSSION |
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Top
Abstract
Introduction
Procedures
Results & Discussion
References
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The Export of Five Different Cofactor-containing Proteins Is
Blocked in a tatC Mutant--
To test the role of the
tatC gene product we constructed a strain in which the
tatC gene was inactivated by an in-frame deletion as
described under "Experimental Procedures." The deletion strain is
viable under aerobic respiratory or fermentative growth conditions, indicating that the gene does not encode an essential protein. However,
most of the proposed (15) substrates for the Sec-independent translocase in E. coli are components of anaerobic
respiratory pathways. We therefore tested the effects of the TatC
mutation on the localization of five such proteins.
tatC mutant fails to grow on the nonfermentable carbon
source glycerol with either TMAO or Me2SO as sole terminal
electron acceptor, indicating a defect in respiration involving these
oxidants. Analysis of the mutant strain cultured on a fermentable
carbon source shows that the TMAO and Me2SO reductase
activities are both mislocalized to the cytoplasmic compartment (Table
I). Whereas the vast majority (83%) of
TorA is found in the periplasmic fraction in the wild-type cells, over
96% of the enzyme activity is cytoplasmically located in the
tatC mutant. The localization of Me2SO
reductase is affected to a similar extent; the enzyme is almost
exclusively located in the membrane fraction in wild-type cells,
whereas over 94% is found in the cytoplasm in the tatC
strain. These data indicate a severe defect in the export of these
enzymes.
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tatC mutant strain, and each accumulates instead in
enzymatically active form in the cytoplasm (Fig. 2, a and
b, lanes 6).
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Pulse-Chase Analysis of a TorA-23K Construct--
The above data
clearly show that multiple twin-arginine precursor proteins are
mislocalized in the tatC strain. To directly demonstrate
that the mutation affects the kinetics of twin-Arg precursor export and
processing, we carried out pulse-chase tests. However, the
twin-arginine precursors examined above are either membrane-associated,
which complicates the analysis of export, or are relatively large
proteins for which it is difficult to detect presequence processing by
a change in electrophoretic mobility. For these reasons we carried out
the pulse-chase experiments on a simplified construct, TorA-23K, in
which the transfer peptide of TorA is fused to the mature 23-kDa
protein (23K) of the plant photosystem II oxygen-evolving complex. 23K
is targeted exclusively by the Sec-independent pathway in chloroplasts
(9, 11, 12) and was predicted to be tolerated by the corresponding
system in bacteria. TorA-23K expression was placed under the control of
an inducible promoter, and Fig.
3a shows that induction of synthesis results in efficient export of the protein. A mixture of
precursor protein and mature 23K is apparent immediately after pulse-labeling of wild-type cells (lane 0), but only mature
23K is detected after chase times of 5 and 15 min. When spheroplasts were generated from these cells (lane Sp) a substantial
proportion of the mature protein is lost, suggesting a periplasmic
location, and this is confirmed by the finding that the residual mature 23K is completely sensitive to added proteinase K. In contrast, no
mature protein whatsoever can be detected in the tatC
deletion strain, even after a 15-min chase, and we observe instead only the precursor protein, which runs as a doublet, possibly indicating partial degradation. The precursor protein is resistant to proteinase K
digestion, indicative of a cytoplasmic location. We conclude that
export of the TorA-23K construct is completely blocked in the
tatC deletion strain.
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tatC strain (Table I). Furthermore, Fig.
3b shows that the export and processing of a typical Sec substrate, pro-OmpA, is unaffected in the mutant strain. Only a single
OmpA band is detected under standard pulse-chase conditions, and this
can be confirmed as mature size because the precursor protein becomes
apparent when azide is included during the pulse-chase to inhibit ATP
hydrolysis by SecA (28) and hence block Sec pathway export (lanes
Az). Export and processing of pro-OmpA are equally affected in the
wild-type and tatC strains. The tatC deletion thus has no measurable effect on export via the Sec pathway.
We have also been able to exclude the possibility that the
tatC mutation causes a defect in cofactor insertion, rather
than in export. The enzymes nitrate reductase-A and fumarate reductase contain cytoplasmically located subunits binding cofactors (MGD and
iron-sulfur clusters) of the type found in twin-arginine transfer peptide-dependent periplasmic proteins. We demonstrated
that the activities of these enzymes are undiminished in the mutants
(Table I). Furthermore, hydrogenases 1 and 2 and TorA accumulate in the
cytoplasm of the tatC mutant in active forms, indicating
that cofactor insertion has taken place. Finally, export of the
TorA-23K is blocked even though this construct does not bind
cofactors.
Severity of the tatC Phenotype--
These data confirm an
essential and specific role for the TatC protein in the translocation
of proteins with twin-arginine signal peptides. Our previous study (18)
on the roles of TatA and TatE (the two Hcf106 homologues) indicated
that these proteins play important roles because deletion strains are
severely affected in Sec-independent protein export. However, neither
strain is totally defective in this export pathway. A complete block in the export of four proteins is observed when both genes are disrupted, suggesting overlapping functions for the two gene products, but the
membrane localization of hydrogenase-2 is still not completely blocked
even in this strain. In contrast, in this report we have shown that the
tatC deletion leads to a complete block in the export of all
five proteins tested indicating a critical role in the translocation
process.
tatC Homologues Are Also Present in Plastids and Mitochondria-- TatC homologues are present in all fully sequenced prokaryotic genomes, including the archaeaon Archaeoglobus fulgidus, that code for proteins with twin-arginine signal peptides, strongly suggesting a central role in the Sec-independent export of proteins in these species. TatC homologues are furthermore present in the plastid genomes of the eukaryotic algae Porphyra purpurea and Odontella sinensis. Although these genes are absent from the plastid genomes of higher plants, it is highly likely that such genes are present in the nuclear DNA of these species because the plastid genomes of these algae are notable for containing genes (including, for example, secY and secA) that have been transferred to the nucleus in all higher plant species analyzed to date. The presence of TatC homologues in chloroplasts is not unexpected given the presence of a Sec-independent thylakoid import system in these organelles. Much more intriguing is the presence of TatC-like proteins coded by the mitochondrial genomes of four higher plants including Arabidopsis thaliana, the liverwort Marchantia polymorpha, and the nonphotosynthetic protist Reclinomonas americana. The function of these mitochondrial homologues is not yet clear, but it may be involved in either the import of folded proteins and/or their export from the matrix into the intermembrane space. We have been unable to identify potential substrates for this system that contain a twin-Arg presequence, but the system may well differ in certain respects from the bacterial/plastid systems, and the targeting signal may also have been modified during the course of evolution. The predicted topological organization of the TatC homologues is the same in all cases with six predicted transmembrane helices arranged such that the N terminus of the proteins is at the N-side (that is, cytoplasmic in prokaryotes) of the membrane.
In summary, we have identified a key component of a novel bacterial Sec-independent export system that may be used primarily for the translocation of folded proteins. The available evidence suggests that TatC-dependent systems operate also in chloroplasts and mitochondria, raising the possibility that this type of system may be almost ubiquitous in nature.| |
ACKNOWLEDGEMENTS |
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We thank Drs. Margaret Wexler and Gary Sawers for discussions and reagents, Prof. David Boxer for providing antibodies to hydrogenase-2 and formate dehydrogenase-N, Gunnar von Heijne and Jan-Willem de Gier for help with the pulse-chase analysis, and Ralf Bernd Klösgen for providing antibodies to 23K.
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FOOTNOTES |
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* This work was supported by the Biotechnology and Biological Sciences Research Council, The Royal Society, and The Norwich Research Park.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.
To whom correspondence should be addressed. Tel.:
44-1603-452571; Fax: 44-1603-454970; E-mail:
tracy.palmer{at}bbsrc.ac.uk.
1 The abbreviations used are: PCR, polymerase chain reaction; TMAO, trimethylamine N-oxide; MGD, molybdopterin guanine dinucleotide; Fdn, formate dehydrogenase-N.
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REFERENCES |
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Abstract
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Procedures
Results & Discussion
References
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