COMMUNICATION
A Signal Peptide That Directs Non-Sec Transport in Bacteria Also
Directs Efficient and Exclusive Transport on the Thylakoid Delta pH
Pathway*
Hiroki
Mori and
Kenneth
Cline
From the Horticultural Sciences Department and Plant Molecular and
Cellular Biology Program, University of Florida, Gainesville, Florida
32611-0690
 |
ABSTRACT |
Signal peptides that specifically direct
precursor proteins to the thylakoid Delta pH pathway possess an N
domain RR motif. Signal peptides that direct transport of bacterial
proteins across a non-Sec export pathway possess an N domain
RRXFLK consensus motif. Recent genetic studies suggest an
evolutionary link between these two protein translocation pathways. To
further explore this relationship, we examined the thylakoid targeting
capability of the signal peptide for Escherichia coli
hydrogenase 1 small subunit (HyaA) by linking it to plastocyanin and
assaying the chimeric protein in an in vitro thylakoid
transport assay. The chimeric precursor was transported across
thylakoids with high efficiency. Transport was characteristic of the
Delta pH but not the Sec pathway, i.e. it was eliminated by
ionophores that dissipate the
pH but occurred in the absence of
stromal extract or ATP. This result was confirmed by competition with
chemical quantities of a Delta pH pathway precursor. This indicates
that the HyaA signal peptide has the necessary elements for efficient
and exclusive targeting to the Delta pH pathway and further supports
the notion that the alternate targeting pathways in prokaryotes and
plant thylakoids are analogous.
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INTRODUCTION |
Many thylakoid lumen-resident proteins of plant chloroplasts
are synthesized in the cytosol as larger precursors with bipartite amino-terminal extensions called transit peptides (see Ref. 1 for
review). The stroma-targeting domain of the transit peptide governs
import into the chloroplast stroma; the lumen-targeting domain directs
subsequent transport into the thylakoid lumen. Two precursor-specific
pathways for protein transport into the thylakoid lumen have been
identified by in vitro and genetic studies (1). The
thylakoid Sec pathway requires a chloroplast SecA protein (cpSecA) and
ATP (1) and appears analogous to the bacterial Sec system. The Delta pH
pathway operates independently of ATP and soluble factors, requiring
only a thylakoidal pH gradient (1). Targeting specificity for the two
pathways is determined primarily by the lumen-targeting domains, which
contain motifs of bacterial signal peptides, i.e. an
amino-terminal charged N domain, a hydrophobic H domain, and a
carboxyl-terminal cleavage C domain (2). Precursors targeted to the
Delta pH pathway invariably contain an essential N domain twin arginine
that provides access to the Delta pH pathway (Fig.
1) (3). In addition, Delta pH pathway
precursors have H and/or C domains that are nonfunctional for Sec
pathway transport (4, 5). These latter elements have been termed
"Sec-avoidance" elements (4).

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Fig. 1.
Lumen-targeting domains of precursors
targeted to the Delta pH pathway and signal peptides of precursors to
bacterial redox cofactor-binding proteins. The acidic
(A), N-terminal charged (N), hydrophobic
(H), and C-terminal cleavage (C) domains are
shown for lumen-targeting domains of precursors transported by the
Delta pH pathway. These are compared with signal peptides of several
bacterial precursor proteins that bind redox cofactors. Bold type R
shows a conserved arginine in the N domain; the H domain is
underlined. Signal peptides for hydrogenase small subunits
of D. vulgaris Hildenborough and E. coli contain
two twin arginine motifs. The first twin arginine motif is not required
for export (6).
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A non-Sec protein export pathway also appears to operate in bacteria.
Nivière et al. (6) showed that a chimeric precursor containing the Desulfovibrio vulgaris hydrogenase small
subunit signal peptide fused to
-lactamase was exported efficiently
only under anaerobic conditions and this export depended upon a
critical N domain twin arginine motif. Export of Pseudomonas
stutzeri nitrous oxide reductase also depends upon an N domain RR
(7). Recently, it was shown that trimethylamine N-oxide
reductase, which bears an N domain twin arginine, is exported by a
mechanism independent of SecA, SecY, or SecE, but dependent on the
transmembrane
µH+ (8). Berks (9)
pointed out that many precursors for bacterial proteins that bind redox
cofactors share a conserved N domain (S/T)RRXFLK motif (Fig.
1) and suggested that the export system for such proteins may be
related to the thylakoid Delta pH pathway. Strong support for such a
notion was recently provided by Settles et al. (10). Maize
Hcf106 mutant chloroplasts are selectively defective in the Delta pH
pathway. The Hcf106 protein has striking homology to several open
reading frames from bacterial genomes. In the case of Azotobacter
chroococcum, mutation of the Hcf106 homologue results in
mislocalization of hydrogenase (10).
Here we show that the signal peptide of Escherichia coli
hydrogenase 1 small subunit (HyaA) is functionally equivalent to a
lumen-targeting domain for chloroplast Delta pH pathway precursors. This indicates that the HyaA signal peptide has the essential elements
that both engage the Delta pH pathway and avoid the Sec pathway.
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EXPERIMENTAL PROCEDURES |
Materials--
All reagents, enzymes, and standards were
purchased commercially. In vitro transcription plasmids for
the stromal intermediate of
OE331 (iOE33) from wheat
(11), precursors to plastocyanin (pPC) from Arabidopsis and
LHCP (12), and the chimeric precursors t23-PC and DT-PC (5) were
previously described. For tOE23 expression in E. coli, the
coding sequence was amplified from the transcription plasmid with a
forward primer that contained an NdeI site encompassing the
initiator methionine codon and a reverse primer that also contained a
HindIII restriction site. The PCR product was cloned into
the NdeI/HindIII sites of pETH3c (12). Expression
of tOE23 in E. coli strain BL21 (DE3) and isolation of
inclusion bodies were as described (12). The HyaA coding sequence (13)
was amplified by PCR with E. coli genomic DNA as template
and cloned into pGEM-3z. PCR splicing by overlap extension (SOE) (14)
was used to construct a chimeric precursor, Hya-PC, which is an exact fusion between coding sequences for the HyaA signal peptide and the
mature domain of Arabidopsis PC. DNA fragments corresponding to the signal peptide and to PC were amplified separately and spliced
in a second round of PCR. Forward and reverse primers for the SOE
reaction contained restrictions sites for HindIII and
SstI sites, respectively, and the SOE product was cloned
into the HindIII and SstI sites of pGEM-3z. The
sequences of all PCR-cloned constructs were confirmed by Taq
DyeDeoxy Terminator cycle sequencing.
Assays for Thylakoid Protein Transport--
Capped RNA for the
various precursors was produced in vitro with SP6 polymerase
and uncut plasmid. Precursors were translated in a wheat germ system in
the presence of [3H]leucine and adjusted to import buffer
(50 mM HEPES-KOH, pH 8.0, 0.33 M sorbitol)
containing 30 mM unlabeled leucine prior to use (12).
Chloroplasts and thylakoids were prepared from pea seedlings as
described (15). Transport of radiolabeled proteins into thylakoids was
conducted with chloroplast lysates or washed thylakoids in 75-µl
assays (12). Precursors and recovered thylakoid membranes were analyzed
by SDS-polyacrylamide gel electrophoresis and fluorography. Quantification was accomplished by scintillation counting of
radiolabeled proteins extracted from excised gel bands (15).
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RESULTS AND DISCUSSION |
Transport of a Chimeric Precursor Hya-PC into the Thylakoid
Lumen--
We initially assayed transport of the HyaA precursor
protein into isolated thylakoids. A low level of transport was achieved as determined by expected proteolytic maturation and protection from
exogenous protease (data not shown). However, this low level of
transport was insufficient for a full examination of the targeting properties. Because the focus here was on the targeting capability of
the signal peptide of HyaA, we constructed a chimeric precursor protein
(Hya-PC) possessing the HyaA signal peptide fused to the mature domain
of Arabidopsis PC. PC was chosen as a passenger protein
because it can be transported on the Sec pathway and the Delta pH
pathway when linked to appropriate signal peptides (4, 5).
Fig. 2 shows a thylakoid transport assay
with Hya-PC. Incubation of Hya-PC with isolated thylakoids produced a
smaller product at the location of mature PC (lane 2) that
was resistant to thermolysin treatment of the membranes (lane
3). Mature PC was recovered in the lumen subfraction when the
recovered thylakoids were sonicated to release the lumenal contents
(lane 7). Mature PC was not produced when assays were
conducted in the presence of ionophores (lane 4), and the
membrane-associated precursor was degraded by thermolysin (lane
5). Fig. 2 also shows control assays for translocation/integration of authentic pPC and the membrane protein LHCP. These assays
demonstrate that the HyaA signal peptide directs transport into the
thylakoid lumen. Average transport of Hya-PC for three experiments was
32% of the added precursor. This compares very favorably with the 16%
of t23-PC transport (Fig. 3, lane
3) for the same three experiments. t23-PC is a fusion protein
between the core targeting peptide for the Delta pH substrate OE23 and
the mature domain of Arabidopsis PC (5).

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Fig. 2.
Transport of a chimeric precursor Hya-PC into
the thylakoid lumen. Transport/integration assays were conducted
for 30 min at 25 °C with chloroplast lysate and 5 mM
MgATP. Assays were conducted in the light to generate a
thylakoidal pH, which was dissipated in assays containing 0.5 µM nigericin and 1 µM valinomycin
(lanes 4 and 5). After assay, the thylakoids were
recovered by centrifugation, resuspended in import buffer with or
without thermolysin, incubated for 40 min on ice, washed, and then
resuspended in SDS sample buffer. For thylakoid subfractionation,
thermolysin-treated thylakoids were sonicated at 10 watts three times
for 10 s. After centrifugation for 30 min at 65,000 rpm with
Beckman TLA100.3 rotor, the membrane fraction (lane 6) was
resuspended in SDS sample buffer, and lumenal proteins in the
supernatant (lane 7) were precipitated with 10%
trichloroacetic acid. Lanes were loaded with recovered thylakoids,
membrane, or lumen fraction equivalent to 20% of each assay. The
radiolabeled precursor (TP) represents 2% of the amount in
each assay (lane 1). The precursors used are designated to
the left of the fluorogram. The positions of the precursor
(p) and mature (m) forms of the proteins are
marked. DP designates a characteristic protease degradation
product of membrane-inserted LHCP.
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Fig. 3.
Transport requirements suggest that Hya-PC is
transported by the Delta pH pathway. Transport of precursors
across thylakoid membranes was conducted for 30 min at 25 °C with
chloroplast lysate to provide stromal extract (SE) or washed
thylakoids. Energy was provided in the form of ATP and/or light to
generate a pH. Assay conditions examined the requirement for a pH
(lane 4), ATP (lane 5), stromal extract
(lane 6), and sensitivity to azide (lane 7).
Final concentrations were 5 mM ATP, 10 mM
sodium azide, 0.5 µM nigericin (nig),
and 1 µM valinomycin (val). Apyrase (0.5 unit
per 75 µl) was used to eliminate residual ATP in lysate and
translation products for assays conducted in the absence of ATP. These
conditions are designated above the panel. Recovered thylakoids were
post-treated with thermolysin. Analysis and designations were as in
Fig. 2.
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Transport Requirements of Hya-PC Are Consistent with Delta pH
Pathway Transport--
Assays in Fig. 3 were conducted under a variety
of conditions designed to assess energy and stroma requirements that
are characteristic for thylakoid translocation pathways. As with t23-PC
(a Delta pH pathway substrate), transport of Hya-PC was completely
abolished by addition of ionophores that dissipate the thylakoidal
pH (lane 4), but was unaffected by the addition of sodium
azide (lane 7), a SecA inhibitor (16), or by removal of the
stromal extract (lane 6). Depletion of ATP with apyrase in
these experiments diminished, but did not abolish, Hya-PC transport
(lane 5). A similar reduction of t23-PC in the presence of
apyrase also occurred. Such an effect was not previously recognized for
t23-PC (5) but appears to be related to the PC mature domain because in
parallel assays, tOE23 transport was not reduced by apyrase (data not
shown). In contrast, transport of the Sec pathway substrate pPC was
only marginally affected by ionophores (lane 4), virtually
eliminated by removal of ATP or of stromal extract, the source of
~90% of the cpSecA (lanes 5 and 6), and
inhibited by azide (lane 7). These requirements suggest that
Hya-PC is transported on the Delta pH pathway rather than the Sec
pathway. In addition, Hya-PC transport requirements rule out the
participation of two other pathways that are responsible for insertion
of membrane proteins (1); i.e. membrane integration by the
chloroplast SRP is absolutely dependent on the presence of stroma and
NTPs, whereas membrane integration by a spontaneous mechanism occurs
even in the absence of a thylakoidal
pH.
Competition Assays Verify That Hya-PC Is Targeted to the Delta pH
Pathway but Not to the Sec Pathway--
To further clarify the pathway
utilized by Hya-PC, competition assays were conducted with bacterially
synthesized tOE23 (Fig. 4A).
Increasing concentrations of tOE23 progressively competed transport of
t23-PC and Hya-PC. At 2 µM tOE23, transport of Hya-PC was
reduced to ~5% of that achieved in the absence of competitor. In
contrast, transport of Sec pathway substrates pPC and iOE33 was
unaffected by tOE23 competitor.

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Fig. 4.
Competition assays verify that Hya-PC is
exclusively transported on the Delta pH pathway. A,
competition assays were conducted with chloroplast lysates in the
presence of 5 mM ATP with increasing concentration of
unlabeled tOE23 as described previously (12). The final concentration
of urea in each assay was 167 mM. The precursors used are
designated to the left of the fluorograms. B, assays were
conducted with chloroplast lysates to provide cpSecA (lanes
2, 3, and 8-10) or washed thylakoids
(lanes 4-7) with increasing concentration of unlabeled
tOE23. For the assay in the absence of ATP (lanes 3 and
9), chloroplast lysates and in vitro translation
products were treated with apyrase. The final concentration of urea in
each assay was 167 mM. Sodium azide (10 mM
final) was added to verify the utilization of cpSecA (lane
10). The precursors used are designated to the left of
the fluorogram panels. These conditions and final concentration of
tOE23 (µM) are designated above the fluorogram
panels.
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To determine if the residual Hya-PC transport in the presence of 2 µM tOE23 competitor was mediated by the Sec pathway, the effect of ATP and stromal extract were assessed in the presence of 2 µM tOE23 (Fig. 4B). DT-PC transport served as
a positive control for this experiment. DT-PC is a chimeric precursor
protein that contains the N domain of the OE23 precursor and the H/C
and mature domains of PC. Importantly, DT-PC is transported by both the
Sec and the Delta pH pathways (5). In the absence of stromal extract,
tOE23 competitor substantially reduced transport of Hya-PC and DT-PC
(lanes 4-7). As expected, addition of stromal extract greatly stimulated the DT-PC residual transport (lane 8),
and this stimulation was largely eliminated by removal of ATP
(lane 9) or inclusion of sodium azide (lane 10).
These three effects are characteristic of Sec transport as exemplified
by the control assays with iOE33. In contrast to these results, the
residual Hya-PC transport was unaffected by the addition of stroma
extract, the removal of ATP, or the inclusion of azide. This
demonstrates that Hya-PC is not transported by the
cpSecA-dependent mechanism even when the transport on the
Delta pH pathway is virtually eliminated by competition. Furthermore,
these results indicate that the reduction of transport of Hya-PC and
t23-PC by apyrase seen in Fig. 3 does not reflect the involvement of
the cpSecA translocation ATPase.
Taken together, these results indicate that Hya-PC is exclusively
targeted to the Delta pH pathway and does not utilize the Sec pathway
(or other thylakoidal pathways). Thus, the signal peptide for HyaA is
functionally equivalent to the Delta pH pathway lumen-targeting domain
and contains the two essential elements required for exclusive
targeting, an N domain twin arginine (3) that provides access to the
Delta pH pathway and an element that prevents engagement by the Sec
pathway. It was uncertain whether the N domain twin arginine in the
HyaA signal peptide would be functional for the Delta pH pathway
because it is followed by several amino acids, SFLK, prior to the H
domain. For most Delta pH pathway precursors, the twin arginine
immediately precedes the first amino acid of the H domain. Furthermore,
a chimeric construct that placed an asparagine between the twin
arginine motif and the H domain was nonfunctional for Delta pH pathway transport (4). However, the HyaA signal peptide was twice as efficient
as the authentic core signal peptide for Delta pH pathway precursor
OE23. It is likely, but remains to be demonstrated, that the thylakoid
Sec avoidance element in the HyaA signal peptide resides in the H/C
domain.
Because of the endosymbiotic origin of chloroplasts from an ancestral
cyanobacterium, it was speculated that protein transport into plant
thylakoids would be evolutionarily related to prokaryotic transport
mechanisms. Work during the past several years has shown that three of
the four known mechanisms of thylakoid protein translocation can be
ascribed to prokaryote mechanisms. These include a
SecA-dependent pathway, an SRP-like pathway for insertion
of a membrane protein, and a spontaneous protein insertion mechanism
(1). Although characteristics of the thylakoid Delta pH pathway suggest
a prokaryote-like mechanism, i.e. a classical signal peptide
and initiation via a loop mechanism (17), no analogous system had been
identified in bacteria. The recent report of the identity of the Hcf106
protein involved in Delta pH pathway, and the existence of the
bacterial homologues (10) now suggests the identity of the bacterial
counterpart. Our results further support the notion that the pathway
associated with export of redox cofactor-binding proteins in bacteria
is related to the Delta pH pathway of chloroplast thylakoids.
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ACKNOWLEDGEMENT |
We thank Shan Wu for excellent technical
assistance.
 |
FOOTNOTES |
*
This work was supported in part by National Institutes of
Health Grant R01 GM46951 and National Science Foundation Grant
MCB-9419287 (to K. C.). DNA sequencing was conducted by the University
of Florida Interdisciplinary Center for Biotechnology Research (ICBR) DNA Sequencing Core, which is supported by funds supplied by the Division of Sponsored Research and the ICBR at the University of
Florida. This paper is Florida Agricultural Experiment Station Journal
Series R-06240.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.: 352-392-4711 (Ext. 219); Fax: 352-392-4711; E-mail: KCC{at}nervm.nerdc.ufl.edu.
1
The abbreviations used are: OE23 and OE33, the
23- and 33-kDa subunits of the photosystem II oxygen evolving complex,
respectively; p, i, and t, full-length precursor, intermediate
precursor, and truncated precursor form, respectively; PC,
plastocyanin; HyaA, E. coli hydrogenase 1 small subunit; DT,
dual-targeting; LHCP, the light-harvesting chlorophyll
a/b protein; SOE, splicing by overlap extension;
PCR, polymerase chain reaction.
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