THE JOURNAL OF BIOLOGICAL CHEMISTRY
© 2000 by The American Society for Biochemistry and Molecular Biology, Inc.
Vol. 275, No. 31, Issue of August 4, pp. 23483–23490, 2000
Printed in U.S.A.
The Thylakoid ⌬pH-dependent Pathway Machinery Facilitates
RR-independent N-Tail Protein Integration*
Received for publication, May 15, 2000
Published, JBC Papers in Press, May 17, 2000, DOI 10.1074/jbc.M004137200
Elizabeth J. Summer, Hiroki Mori, A. Mark Settles, and Kenneth Cline‡
From the Horticultural Sciences and Plant Molecular and Cellular Biology Program, University of Florida,
Gainesville, Florida 32611
The thylakoidal ⌬pH-dependent and bacterial twin arginine transport systems are structurally and functionally related protein export machineries. These recently
discovered systems have been shown to transport folded
proteins but are not known to assemble integral membrane proteins. We determined the translocation pathway of a thylakoidal FtsH homologue, plastid fusion/
protein translocation factor, which is synthesized with a
chloroplast-targeting peptide, a hydrophobic signal
peptide, and a hydrophobic membrane anchor. The twin
arginine motif in its signal peptide and its sole integration requirement of a ⌬pH suggested that plastid fusion/
protein translocation factor employs the ⌬pH pathway.
Surprisingly, changing the twin arginine to twin lysine
or deleting the signal peptide did not abrogate integration capability or characteristics. Nevertheless, three
criteria argue that all three forms require the ⌬pH pathway for integration. First, integration was competed by
an authentic ⌬pH pathway precursor. Second, antibodies to ⌬pH pathway component Hcf106 specifically inhibited integration. Finally, chloroplasts from the
hcf106 null mutant were unable to integrate Pftf into
their thylakoids. Thus, ⌬pH pathway machinery facilitates both signal peptide-directed and N-tail-mediated
membrane integration and does not strictly require the
twin arginine motif.
Export type pathways target proteins to the bacterial plasma
membrane, the endoplasmic reticulum, the mitochondrial inner membrane, and the chloroplast thylakoid membrane (1).
Where known, component and mechanistic similarity support
the hypothesis that the organelle pathways are descendent
from those of the prokaryotic endosymbiont. This is most evident when comparing export pathways of chloroplasts and
bacteria. Chloroplasts share with bacteria at least four distinct
pathways: a Sec pathway, a ⌬pH/Tat1 pathway, a signal rec* This work was supported in part by National Institutes of Health
Grant R01 GM46951 (to K. C.). This manuscript is Florida Agricultural
Experiment Station Journal series no. R-07593. The costs of publication
of this article were defrayed in part by the payment of page charges.
This 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: Horticultural Sciences Dept., Fifield Hall, University of Florida, Gainesville, FL 32611.
Tel.: 352-392-4711 (ext. 219); Fax: 352-392-5653; E-mail: kcline@
ufl.edu.
1
The abbreviations used are: Tat, twin arginine transport; SRP,
signal recognition particle; Pftf, plastid fusion/protein translocation
factor; PCR, polymerase chain reaction; LHCP, the light harvesting
chlorophyll a/b protein; OE33, OE23, and OE17, the 33-, 23-, and
17-kDa subunits of the photosystem II oxygen-evolving complex;
PSII-T, the T subunit of the photosystem II complex; p, i, and m,
precursor, intermediate precursor, and mature forms of the thylakoid
proteins described in the text, respectively; t, truncated precursor;
This paper is available on line at http://www.jbc.org
ognition particle (SRP) pathway, and a spontaneous insertion
pathway (2– 4). Chloroplasts and bacteria also appear to employ homologues of the mitochondrial Oxa1p export protein (5).
The ⌬pH/Tat pathway in thylakoids and bacteria is the most
recently recognized protein translocation pathway. The ⌬pH
pathway was first described as a system that transported a
subset of thylakoid lumenal proteins using only the thylakoidal
⌬pH as an energy source (6). Substrates of this pathway possess cleavable, amino-terminal signal peptides with an invariant twin arginine motif amino-terminal to the hydrophobic core
(7). Where examined, substitution of one or both arginines
abolishes ⌬pH pathway transport (7, 8). Identification of a
component of the ⌬pH pathway, Hcf106 (9), and the recognition
that a subset of bacterial periplasmic proteins possesses twin
arginine-containing signal peptides (10, 11) led to the description of a homologous bacterial pathway. Disruption or mutations of Hcf106 homologues impaired transport of a range of
twin arginine-bearing precursors (12, 13). In addition, disruption of a Tat operon gene for the multispanning membrane
protein TatC also impaired protein transport (14). A plant
chloroplast TatC homologue has been identified and shown to
be necessary for transport of ⌬pH pathway substrates in vitro.2
Recent studies from several laboratories indicate that the ⌬pH
and Tat systems do not employ components of the Sec system
and vice versa (15–18).
An intriguing question is why two pathways, Sec and ⌬pH/
Tat, co-evolved to target proteins to the same location. One
possible explanation is that unique capabilities of each system
match the translocation problems of the respective substrates.
For example, substrates of the ⌬pH and Tat pathways appear
unable to utilize the Sec pathways, even when provided with a
Sec signal peptide (8, 19, 20). This may be because substrates
of the ⌬pH/Tat pathways appear to be transported in a folded
conformation (20, 21, 22), whereas the Sec pathway requires its
substrates to be unfolded during translocation (23). Conversely, the Sec pathway is able to recognize transmembrane
domains and integrate them into the lipid bilayer, whereas
previously identified ⌬pH pathway precursors are extrinsic
proteins, suggesting that the ⌬pH pathway might be unable to
insert membrane proteins.
In this paper, we present evidence that the ⌬pH pathway can
integrate membrane proteins. Pftf is an integral thylakoid
membrane protein and a AAA family member related to mitochondrial and bacterial zinc-dependent proteases, e.g. FtsH,
that chaperone protein biogenesis (24). The Pftf precursor has
two hydrophobic segments, similar to other FtsH homologues
(Fig. 1). However, the first hydrophobic segment (H1) of the
Pftf precursor is relatively short, like a signal peptide and is
PAGE, polyacrylamide gel electrophoresis.
2
H. Mori, E. J. Summer, V. Fincher, and K. Cline, manuscript in
preparation.
23483
23484
N-Tail Translocation by the ⌬pH-dependent Machinery
preceded by a twin arginine. In fact, all known plant chloroplast Pftf homologues possess an RRXFLK motif (25), a conserved motif among bacterial Tat pathway precursors (10).
Here, we show that Pftf is integrated into thylakoid membranes with characteristics indistinguishable from other ⌬pH
pathway substrates. Pftf integration required only the thylakoidal ⌬pH as energy source. It was competed by overexpressed
precursor substrates of the ⌬pH pathway and inhibited by
antibodies to components of the ⌬pH pathway. Furthermore,
Pftf integration was blocked in hcf106 null mutant
chloroplasts.
Nevertheless, Pftf integration does not strictly require the
typical targeting elements of the ⌬pH pathway, as they are
currently understood. Evidence for this conclusion is that substitution of KK for the signal peptide RR did not change integration characteristics. Surprisingly, deleting the entire signal
peptide did not alter integration characteristics of the resulting
mPftf, including its competition by ⌬pH precursors and inhibition by antibodies to Hcf106 and Tha4. Thus, Pftf lacking the
⌬pH targeting elements still requires components of the ⌬pH
machinery for insertion. These results significantly expand the
substrate range of the ⌬pH pathway and suggest a re-evaluation of the nature of the ⌬pH pathway and the cis elements
thought to govern its targeting.
EXPERIMENTAL PROCEDURES
Preparation of Radiolabeled Precursors—Plasmids for pPftf (pepper),
pLHCP, pOE17 (maize), iOE23 (pea), pOE33 and iOE33 (wheat), and
pPSII-T (cotton) used for in vitro transcription and translation have
been described (8, 26 –28). The sequence of Pftf, originally reported by
Hugueney et al. (29), was amended and can be found at GenBankTM
accession number AJ012165. Cloning and analysis of DNA products
were by standard molecular biology procedures (30). Deletion clones of
pPftf were constructed by PCR with Pfu polymerase (Stratagene) using
the cloned pPftf as template. The forward primer for synthesis of mPftf
was 5⬘-CTCACAATGGCTGATGAGCAAGGTGTTTCTAACTCAAGGTTGTCT-3⬘, which gave an amino-terminal sequence of MADEQGVSNSRLSYSI. Besides adding an initiator methionine to the predicted
amino terminus, the methionine at position 12 of the mature protein
was changed to leucine to prevent internal initiation in vitro. The reverse primer for amplifying mPftf was 5⬘-GTTCAGGGTCTGTTCTCTTTCATC-3⬘. pKKPftf was constructed by splicing by overlap extensionPCR (31) in which the internal primers contained sequences to modify
arginine residues 60 and 61 of pPftf to lysines. The internal forward
primer was 5⬘-CGGATGAAGGAAAAAAGGCTTTCTTAAATTATTG-3⬘
and the reverse primer was the exact complement of the forward
primer. The flanking forward and reverse primers, used for the second
round of PCR, were 5⬘-AAGAGCTCATATGGCTACTTCATCAG-3⬘ and
the above reverse primer used for mPftf amplification, respectively.
mKK Pftf was constructed by SOE-PCR with mPftf (above) as template.
The forward internal primer 5⬘-CTTTTCCTGCTATCAAAGAAGTCTAACGGAGG-3⬘ and reverse internal primer 5⬘-CCTCCGTTAGACTTCTTTGATAGCAGGAAAAG-3⬘ contained sequences to modify the two
arginines carboxyl proximal to the membrane anchor (H2) to two lysines. All PCR products were digested with EcoICRI and XhoI and
ligated into the EcoICRI/XhoI-digested pPftf plasmid and transformed
into XLI-Blue (Stratagene) cells, and the resulting clones were sequenced entirely on both strands by the University of Florida Interdisciplinary Center for Biotechnology Research DNA sequencing core. In
vitro transcription with SP6 RNA polymerase (Promega) and translation with rabbit reticulocyte lysate (Promega) or coupled transcription/
translation with wheat germ TnT (Promega) in the presence of
[3H]leucine (NEN Life Science Products) was performed following the
manufacture’s guidelines. Unless otherwise stated, translation products were diluted with one volume of 60 mM leucine in 2⫻ import buffer
(1⫻ ⫽ 50 mM HEPES, KOH, pH 8.0, 0.33 M sorbitol) prior to use.
Chloroplast Protein Import and Thylakoid Protein Transport and
Integration Assays—Chloroplasts were isolated from 9 to 10-day-old pea
seedlings (Pisum sativum cv. Laxton’s Progress 9) as described (32).
Maize plants were grown at 26 °C in a 16 h light/8 h dark cycle for 7–10
days. Mutant hcf106 mum3 maize seedlings were selected by their pale
green phenotype and confirmed by high chlorophyll fluorescence with a
hand-held UV lamp. Maize chloroplasts were isolated by essentially the
same procedure as pea chloroplasts except that intact chloroplasts were
purified on a step density gradient consisting of 8 ml of 75% Percoll and
15 ml of 35% Percoll in GR buffer. The gradients were centrifuged at
3200 ⫻ g for 15 min, and intact chloroplasts were collected at the
35%/75% interface. Chloroplast lysate, washed thylakoids, and stromal
extract were prepared from intact chloroplasts as described (26).
Import of precursors into chloroplasts was as described (26). Briefly,
in vitro translated precursors were incubated with intact chloroplasts
(0.33 mg chlorophyll/ml) and 5 mM MgATP in import buffer at 25 °C in
a light bath (70 microeinstein/m2/s) for the times indicated in the figure
legends. Reactions were initiated by the addition of translation products equivalent to one-sixth the assay volume and terminated by transfer to an ice bath. Intact chloroplasts were recovered, with or without
protease treatment, by centrifugation through a 35% Percoll cushion.
For fractionation, recovered chloroplasts were lysed by resuspension in
20 mM Hepes, KOH, pH 8, and incubation on ice for 5 min. The
thylakoid membranes were separated from the stromal fraction by
centrifugation for 8 min at 3200 ⫻ g. Thylakoids were either washed
with import buffer, extracted with 0.1 M NaOH, or treated with thermolysin as described (32) except that for protease treatment, samples
were resuspended in import buffer containing 0.2 mg/ml thermolysin
for 60 min at 4 °C followed by washing in import buffer containing 50
mM EDTA.
Assays for protein translocation into isolated thylakoids were essentially as described (26). Assays contained 0.33 mg/ml chlorophyll equivalent of thylakoids, lysate, or reconstituted lysate, 1/6 volume radiolabeled translation product, and where indicated, 5 mM MgATP. Reaction
mixtures were incubated for the indicated times at 25 °C in the presence or absence (foil-wrapped tubes) of light. Reactions were terminated
by transfer to 0 °C, and the thylakoids were either washed in import
buffer plus 10 mM MgCl2, extracted with 0.1 M NaOH, or treated with
thermolysin as described above.
Competition for integration into isolated thylakoids was performed
essentially as described (26). Inclusion bodies of Escherichia coli-expressed tOE23 were dissolved in fresh 10 M urea, 10 mM dithiothreitol
to 90 ␮M protein for 3 h at room temperature, diluted to appropriate 3⫻
stocks in 0.5 M urea, 0.5 mM dithiothreitol, import buffer plus 10 mM
MgCl2, and added to otherwise complete reaction mixtures. All reactions contained 0.33 mg of chlorophyll/ml of chloroplast lysate, 5 mM
MgATP, radiolabeled translation product, 0.167 M urea, and 0.16 mM
dithiothreitol in import buffer plus 10 mM MgCl2. Reactions were
started immediately after the addition of competitor by incubation at
25 °C for 20 min in the light. The assays were terminated by the
addition of nigericin and valinomycin (0.5 and 1.0 ␮M final concentration, respectively) and transferred to ice. Recovered thylakoids were
then protease-treated.
In organello competition was conducted essentially as described (26).
Inclusion bodies of pOE23 or pOE33 were dissolved in fresh 10 M urea,
10 mM dithiothreitol for 3 h at room temperature. Chloroplasts in
import buffer plus 5 mM MgATP were preincubated with unlabeled
competitors for 7 min in the light at 25 °C. Competitors were aliquotted
from stocks, such that the final competitor concentration was 0.75 ␮M
pOE23, 0.6 ␮M pOE33, or no competitor, and the urea concentration
was 0.25 M in all assays. Radiolabeled precursors (1/6 volume) were
then added, and the incubation was continued for an additional 10 min.
Competition assays were stopped by adding an equal volume of 1.5 ␮M
nigericin and 3 ␮M valinomycin in import buffer and transferred to ice.
Chloroplasts were then repurified by centrifugation through Percoll
cushions.
Use of Inhibitors and Antibody Inhibition of Thylakoid Protein
Transport—Integration assays were conducted in the presence of
apyrase by pretreatment of thylakoids, lysate, and translation products
with 0.04 units apyrase/ml as described (6). Import and integration
assays were conducted in the presence of ionophores nigericin and
valinomycin at 0.5 ␮M and 1.0 ␮M final concentration, respectively, as
described (6). Thylakoids were treated with anti-maize Hcf106 and
anti-pea Tha4 IgGs in the presence or absence of antigens as described
(17). Thylakoids were treated with anti-pea Hcf106 and anti-pea plant
chloroplast TatC homologue as described (17).2
Miscellaneous—Chlorophylls were determined according to Arnon
(33). Proteins were quantified using the BCA method (Pierce) with
bovine serum albumin as standard. Immunoblotting was by the ECL
method as described (17). Unless indicated, chemicals were purchased
from Sigma.
RESULTS
Pftf Is Imported into Isolated Intact Chloroplasts and Properly Integrated into Thylakoids by a Two-step Mechanism—
N-Tail Translocation by the ⌬pH-dependent Machinery
23485
FIG. 1. Topology of pPftf and mPftf and constructs. pPftf is
synthesized with two hydrophobic domains, H1 from 66 to 82 and H2
from 169 to 193 (28). The predicted stromal processing site, 49 –50, and
thylakoid processing site, 86 – 87, are indicated by gaps. pKKPftf was
constructed by mutating the arginines at positions 60 and 61 (in the
RRXFLK motif) to lysines. mPftf was constructed by deleting the entire
bipartite transit peptide, i.e. residues 1– 86 as described under “Experimental Procedures.” mKKPftf was constructed by replacing the two
arginines following the H2 of mPftf to two lysines. The topology of mPftf
in the thylakoids was deduced from experimental results presented in
this paper. Charged residues in the lumenal tail are indicated with a
“⫹” (K or R) or “⫺” (D or E).
Upon incubation with chloroplasts, the 74-kDa Pftf precursor
(pPftf) (Fig. 2, lane 1) was imported into the organelles and
processed to an ⬃65-kDa protein (mPftf) that was protected
from protease treatment of the intact chloroplasts (Fig. 2A,
lanes 2 and 3). Upon subfractionation of the chloroplasts following import, mPftf was found exclusively in the thylakoid
fraction (lane 5). It was resistant to extraction with 0.1 M
NaOH, demonstrating that it was integrally associated with
the bilayer (Fig. 2A, compare lanes 5 and 7) and partially
degraded by protease treatment of thylakoids (lane 6), resulting in an ⬃13-kDa degradation product (Fig. 2B, lane 2). These
characteristics are identical to those previously determined for
the endogenous protein of chromoplast internal membranes
and pea thylakoid membranes (28 and data not shown). Furthermore, they indicate that the ⬃13-kDa protease resistant
fragment is a suitable indicator of properly integrated Pftf.
If pPftf possesses a bipartite transit peptide consisting of a
stromal targeting domain followed by a lumen-targeting signal
peptide (Fig. 1), then pPftf would be processed in two steps,
first by a stromal processing protease to form a species intermediate in size, and second by a thylakoidal protease to generate mPftf. Two lines of evidence support this model. The first
is the result of conducting a chloroplast import assay in the
presence of ionophores that dissipate the proton motive force.
Chloroplast protein import requires ATP, but not ion or pH
gradients, whereas most thylakoid translocation is stimulated
by the thylakoidal pH gradient (see Ref. 2). When pPftf was
incubated with chloroplasts, ATP, and ionophores, it accumulated in chloroplasts as a protein slightly larger than mPftf
(Fig. 2A, compare lanes 8 and 7). This intermediate-sized Pftf
(iPftf) was predominantly associated with thylakoids, but it
was not integrated into the membrane because it was largely
extracted by 0.1 M NaOH (Fig. 2A, lane 13, compare with lane
7) and degraded by protease treatment (Fig. 2B, lane 3). These
characteristics are similar to those of lumenal proteins previously shown to localize by a two-step pathway, except that
lumenal intermediates generally accumulate in the stromal
fraction (e.g. see Ref. 26). A second line of evidence is that pPftf
was processed by clarified stromal extract to a species inter-
FIG. 2. Import and thylakoid integration of pPftf occurs in two
steps in vitro. A, intact pea chloroplasts were incubated with radiolabeled pPftf translation product (tp) for 10 min in light with 5 mM ATP
in the absence or presence of nigericin and valinomycin (N/V) as indicated above the panel and described under “Experimental Procedures.”
Following import, chloroplasts were repurified without (C) or with (CP)
protease post-treatment. Untreated chloroplasts were lysed and subfractionated into stroma (S) and thylakoids (T). Equivalent aliquots of
thylakoids were extracted with 0.1 M NaOH (TN) or treated with protease (TP). The locations of precursor (p), intermediate (i), and mature
form (m) are indicated on the right of the panel. The samples were
analyzed by SDS-PAGE and fluorography on a 7.5% gel. B, the protease-protected fragment of Pftf (Pftf-DP) was resolved in samples from
lanes 6 and 12 in A by 12.5% SDS-PAGE and fluorography. Translation
products (tp, 0.15 ␮l) and assay samples resulting from incubation with
1.6 ␮l of translation product were loaded on the gel lanes.
mediate in size (data not shown) and processed by washed
thylakoids to mPftf (see Fig. 3). These data support the idea
that the H1 domain of pPftf is a signal peptide that is removed
upon integration into thylakoids and that the mature sized
mPftf has a single transmembrane anchor and a lumenal tail.
Additional evidence for this point will be presented below.
Pftf Requires Neither the Twin Arginine Nor the Entire Signal Peptide for Integration into Thylakoids—Preliminary results demonstrated that pPftf integrates into isolated thylakoids in a ⌬pH-dependent manner. This suggested that Pftf
employs the ⌬pH pathway for integration. One means of addressing whether Pftf is using the ⌬pH pathway is to modify
the cis topogenic elements for ⌬pH pathway targeting. The two
arginines just prior to H1 in pPftf were replaced with two
lysines (Fig. 1; pKKPftf). As a more severe test, the entire
signal peptide of pPftf was removed producing mPftf. These
modified Pftf proteins were assayed with intact chloroplasts as
well as with isolated thylakoids. As expected, pKKPftf was
imported into isolated chloroplasts (Fig. 3A, lanes 7 and 8), but
mPftf remained on the outside of the chloroplasts where it was
degraded by protease (Fig. 3A, lanes 12 and 13). Imported
pKKPftf became integrally associated with the thylakoid membranes, similar to pPftf, as determined by resistance to extraction with NaOH and by the production of the characteristic
23486
N-Tail Translocation by the ⌬pH-dependent Machinery
FIG. 4. pPftf, pKKPftf, and mPftf integration require the thylakoidal proton motive force but not stromal factors or NTPs.
Integration (pPftf, pKKPftf, and mPftf) and transport (pOE17 and
iOE33) reactions were conducted with pea thylakoids under conditions
designed to test requirements for stroma, ATP, and proton motive force.
Assays were conducted for 15 min at 25 °C. Assay conditions, depicted
above the panels, included the absence or presence of stromal extract
(i.e. assays with washed thylakoids or reconstituted lysate), the absence
or presence of white light, 5 mM ATP, nigericin and valinomycin (N/V),
and apyrase to scavenge all residual ATP and GTP. Thylakoids recovered following incubation were protease-treated and analyzed by 12.5%
SDS-PAGE and fluorography. Gel lanes contained 1 ␮l of translation
products (tp) and thylakoids recovered following incubation with 6.25 ␮l
of translation product.
FIG. 3. Analysis of the cis-acting elements required for Pftf
integration. A, import and localization of pPftf, pKKPftf, and mPftf.
Intact pea chloroplasts were incubated with in vitro translated pPftf
(lanes 1–5 and 14), pKKPftf (lanes 6 –10 and 15), or mPftf (lanes 11–13)
and ATP for 30 min. Following incubation chloroplasts were repurified
without (C) or with (CP) protease treatment. Repurified chloroplasts
were extracted with 0.1 M NaOH (CN) or used to prepare total membranes, which were treated with protease in the absence (TP) (lanes 5
and 10) or presence (PD) (lanes 14 and 15) of 1% Triton X-100. B and C,
thylakoid integration assays with pPftf, pKKPftf, and mPftf. Washed
pea thylakoids were incubated with in vitro translated pPftf (lanes 1, 4,
and 5), pKKPftf (lanes 2, 6, and 7), or mPftf (lanes 3, 8, and 9) for 20 min
in the light. Thylakoids recovered from assays were divided into equal
aliquots. B, one aliquot was washed with import buffer (T) and the
second was extracted with 0.1 M NaOH (TN). The sample in lane 10 was
identical to lane 4 of A and was included as a standard for mPftf
obtained from a chloroplast import assay. C, the remaining aliquots
were treated with protease. Following proteolysis, samples were
washed with buffer (P) or extracted with 0.1 M NaOH (PN). Samples
were analyzed by SDS-PAGE and fluorography on either 12.5% gels (A
and C) or a 7.5% gel (B). In the figure, translation products (tp) and
samples deriving from assays with pPftf, pKKPftf, and mPftf are designated as p, pKK, and m, respectively, above the panels.
13-kDa degradation product following lysis of the chloroplasts
and protease treatment of the membranes (Fig. 3A, lanes 9 and
10 compare with lanes 4 and 5). This indicates that the twin
arginine motif is not essential for Pftf integration.
The ability of pKKPftf and mPftf to integrate into isolated
thylakoid membranes was also examined. When incubated
with washed thylakoids (Fig. 3B), pKKPftf was processed to
mature size and became integrally associated with thylakoids
as judged by resistance to extraction with NaOH, similar to
pPftf (Fig. 3B, lanes 6 and 7). mPftf was also integrated into
washed thylakoids (lanes 8 and 9) and produced the 13-kDa
protease degradation product (Fig. 3C, lane 5), which was further resistant to base extraction, indicating that it was an-
chored in the membrane (Fig. 3C, lane 6). These data indicate
that Pftf does not require the signal peptide for membrane
targeting or integration. A time course of integration determined that mPftf integrates into washed thylakoids at approximately the same rate as pPftf, whereas pKKPftf integrates at
only about 50% the rate of pPftf (data not shown).
pPftf, pKKPftf, and mPftf Share Identical Requirements for
Integration into Thylakoids—To assess whether pKKPftf and
mPftf utilize the same mechanism for integration as pPftf, the
stromal and energetic requirements of pKKPftf and mPftf were
determined in simultaneous assays with pPftf, pOE17, and
iOE33 (Fig. 4). Like pPftf integration (Fig. 4A), pKKPftf (Fig.
4B) and mPftf (Fig. 4C) integration did not require stromal
factors (lanes 2 and 7) or ATP (lanes 3 and 7). Integration was
dependent on the proton motive force as it was inhibited by
ionophores in the light (lane 6) and was ATP-dependent in the
dark (lanes 4 and 5), i.e. via reverse action of the ATP synthase
(6). In another experiment, the proton motive force requirement of Pftf was further dissected by conducting assays in the
presence of increasing levels of nigericin or valinomycin separately. The protonophore nigericin inhibited Pftf integration,
whereas the electrogenic ionophore valinomycin had no effect
on Pftf integration at levels as high as 1 ␮M (data not shown).
This indicates that it is the ⌬pH component of the proton
motive force, rather than the ⌬⌿ component, that is required
for Pftf integration. Thus all forms of Pftf assayed exhibited the
identical translocation requirement, a pH gradient but not
ATP, similar to the ⌬pH substrate pOE17 (Fig. 4D) and very
different from the Sec pathway substrate iOE33 (Fig. 4E).
Thylakoid Integration of pPftf, pKKPftf, and mPftf Are Competed by the ⌬pH Precursor OE23—Considering the fact that all
three Pftf constructs still required only a pH gradient for translocation, it was important to subject them to other criteria used
to assess utilization of the ⌬pH pathway. Previous studies (26)
N-Tail Translocation by the ⌬pH-dependent Machinery
23487
FIG. 5. Integration of pPftf, mPftf, and pKKPftf is competed by saturating levels of ⌬pH pathway precursors. A, competition for
integration into isolated thylakoids. Integration of radiolabeled pPftf and mPftf and transport of pOE17 and iOE33 into isolated pea thylakoids was
conducted in the presence of increasing concentrations of unlabeled E. coli-synthesized tOE23 (depicted above the panels) as described under
“Experimental Procedures.” Recovered thylakoids were protease-treated prior to analysis by 12.5% SDS-PAGE/fluorography. The radiolabeled
substrate in each reaction is designated to the left of each fluorogram. Gel lanes contained 0.5 ␮l of translation product, and thylakoids recovered
following incubation with 3.12 ␮l of translation product. B, in organello competition for thylakoid transport and integration. Intact pea chloroplasts
were incubated with 5 mM ATP and 0.75 ␮M unlabeled pOE23, 0.6 ␮M pOE33, or without competitor (Control) for 7 min at 25 °C in the light.
Radiolabeled precursors (shown to the left of the panels) were then added to the reaction mixtures, and the incubation was continued for another
10 min. Parallel assays were conducted with radiolabeled precursors and nigericin/valinomycin (N/V)-treated chloroplasts for 10 min for
comparison with thylakoid integration blocked by dissipating the ⌬pH. Chloroplasts were recovered from the assays by centrifugation through
Percoll cushions and were analyzed directly (C) or subfractionated into stroma (S) and thylakoids (T). For assays with pOE17 and pOE33, only the
recovered chloroplasts are shown. Assays were analyzed by SDS-PAGE and fluorography on 7.5% gels (Pftf and pKKPftf) or 11.5% gels (pOE17 and
pOE33). Gel lanes contain the equivalent of 0.25-␮l translation products and samples following incubation with 2.2-␮l translation products.
established conditions by which saturating concentrations of
unlabeled overexpressed precursors competed for translocation
of precursors employing the same pathway. Fig. 5 shows the
results of two competition experiments. In Fig. 5A, translocation into isolated thylakoids was competed with E. coli-produced tOE23, an efficient substrate of the ⌬pH pathway (8).
Insertion of pPftf was partially competed by tOE23 (lanes 3–5).
Interestingly, mPftf integration was much more strongly competed; elevated concentrations of tOE23 virtually eliminated
mPftf integration. The response of the controls, pOE17 for the
⌬pH pathway and iOE33 for the Sec pathway, verified the
specificity of this competitor.
Competition experiments were also carried out in organello,
wherein competition for thylakoid translocation occurs within
intact chloroplasts. In these assays, competitive precursors are
imported into the chloroplasts at a rate much faster than
thylakoid transport, allowing saturating levels of intermediates to accumulate. In organello competition more closely represents physiological conditions as all competing intermediates
have first been imported across the plastid envelope and are
completely competent for thylakoid transport (26). The results
of in organello competition of pPftf and pKKPftf are shown in
Fig. 5B. Competition with the ⌬pH pathway precursor pOE23
inhibited Pftf integration as evidenced by the accumulation of
about 50% of the imported Pftf as iPftf (lanes 5–7). A significant
percentage of iPftf accumulated in the stromal fraction (lane 6).
pOE23 competition of pKKPftf virtually eliminated thylakoid
integration and nearly all of the resulting iKKPftf accumulated
in the stroma (lanes 5–7). Interestingly, iKKPftf also accumulated in the stroma when thylakoid integration was blocked
with ionophores (lanes 11–13). As can be seen, thylakoid localization of Pftf and KKPftf was unaffected by the Sec pathway
competitor pOE33 (lanes 8 –10) compared with the “no competitor” control (lanes 2– 4). Competition experiments with radio-
labeled pOE17 and pOE33 (Fig. 5B, lower panels) verified the
efficacy and specificity of the competitors.
Antibodies to Components of the ⌬pH Pathway Machinery
Inhibit pPftf and mPftf Integration—We recently showed that
antibodies to Hcf106 and Tha4, two components of the ⌬pH
pathway, specifically inhibit translocation of ⌬pH pathway
substrates but not those of the Sec or SRP pathway (17). As
shown in Fig. 6, antibodies to either Hcf106 (lane 2) or Tha4
(lane 5) inhibited integration of pPftf or mPftf. Inhibition was
prevented by including 20 ␮M of the respective antigens,
hcf106sd (lane 3) or tha4sd (lane 7), during the antibody-binding step. Inhibition was greater for mPftf than pPftf. The
controls for this experiment, iOE23 and pPSII-T for the ⌬pH
pathway and pLHCP for the SRP pathway, demonstrated the
specificity and efficacy of the treatments. In other experiments
(data not shown), integration of pPftf, pKKPftf, and mPftf was
unaffected by antibodies to cpSecY under conditions that virtually eliminated transport of Sec pathway substrates in parallel assays.
Pftf Was Imported into hcf106 Chloroplasts, but Failed to
Integrate into the Membranes—Hcf106 was originally identified through genetic experiments in which mutant maize
plants were specifically defective in thylakoid transport of ⌬pH
pathway precursor proteins both in vivo and in vitro (9, 34). As
an additional test of whether Pftf employs ⌬pH machinery,
chloroplasts were isolated from hcf106 maize seedlings and
used in chloroplast import assays (Fig. 7). The hcf106 chloroplasts were devoid of the Hcf106 protein, but contained normal
amounts of Tha4 (Fig. 7B). pPftf was imported into wild type
maize chloroplasts and accumulated as the integrated form, as
determined by NaOH extraction (Fig. 7A). In contrast, although pPftf was imported normally into hcf106 chloroplasts, it
was not integrated into thylakoids as it was extracted from the
thylakoid fraction with NaOH (Fig. 7A). In the experiment
23488
N-Tail Translocation by the ⌬pH-dependent Machinery
shown in Fig. 7, iPftf was not clearly resolved from mPftf.
However, in other experiments, it was apparent that only iPftf
accumulated in hcf106 chloroplasts, whereas it was largely
mPftf that accumulated in wild type chloroplasts (data not
shown). The ⌬pH pathway control for this experiment, pOE17,
was imported into wild type chloroplasts and accumulated normally as the mature, lumenal form, whereas it accumulated in
hcf106 chloroplasts primarily as the stromal intermediate
iOE17 (Fig. 7A, lower panel). The Sec pathway control, pOE33,
was imported and accumulated as the mature, lumenal form in
both hcf106 and wild type chloroplasts (Fig. 7A, lower panel).
A Conserved Twin Arginine Carboxyl Proximal to the H2
Anchor Is Not the ⌬pH Pathway cis-Targeting Element—Examination of Pftf sequences revealed the presence of an RR flanking the hydrophobic anchor on the carboxyl side (Fig. 1). We
assessed the possibility that the ⌬pH machinery can recognize
a twin arginine motif on either end of a hydrophobic segment
by constructing a modified mPftf (mKKPftf) in which the carboxyl flanking RR was changed to KK. mKKPftf integration
was analyzed in the absence or presence of ionophores and with
thylakoids preincubated with antibodies to ⌬pH pathway components (Fig. 8). Similar to mPftf, mKKPftf integration was
prevented by dissipation of the thylakoidal pH gradient (Fig. 8,
lanes 2 and 3). We recently prepared antibodies to pea Hcf106
and pea chloroplast TatC. Preincubation of thylakoids with
either of these antibodies specifically inhibits the transport of
⌬pH pathway substrates but does not affect the proton gradient.2 As shown in Fig. 8, either antibody inhibited mPftf or
mKKPft integration (lanes 5 and 7). Specificity of the antibody
effect is evident by the lack of effect of pre-immune IgGs (lanes
4) and prevention of antibody inhibition by co-incubation with
the corresponding antigens (lanes 6 and 8). In addition, parallel
assays verified the lack of effect of antibodies on transport of
the Sec pathway substrate OE33 and the SRP pathway substrate LHCP (data not shown).
DISCUSSION
FIG. 6. Antibodies to zmHcf106 and psTha4 inhibit integration
of pPftf and mPftf. Pea thylakoids were incubated in import buffer
plus 10 mM MgCl2 and 1% bovine serum albumin with or without
anti-pea Tha4 IgG (0.5 mg/ml) or anti-maize Hcf106 IgG (1 mg/ml) in
the presence or absence of 20 ␮M antigen, tha4sd, or hcf106sd, for 1 h on
ice as described (17) and depicted above the panels. Thylakoids were
then washed with import buffer plus 10 mM MgCl2 and assayed for
insertion of pPftf, mPftf, iOE23, pPSII-T, or pLHCP in a reaction
containing ATP and stroma in the light at 25 °C for 30 min. Reactions
were stopped on ice, and the samples were subjected to protease posttreatment. Samples were analyzed by 12.5% (pPftf, mPftf, LHCP, and
iOE23) SDS-PAGE and fluorography. PSII-T assay samples were acetone-extracted prior to analysis by 16.5% tricine gel electrophoresis. Gel
lanes contain equivalent quantities of thylakoids recovered from the
assays.
In this work we analyzed Pftf integration into thylakoids and
found that it employs the ⌬pH pathway machinery. This is the
first membrane protein found to be a substrate for the ⌬pH
pathway; all other known substrates are soluble proteins of the
thylakoid lumen. Several lines of evidence argue for this conclusion. First Pftf integration did not require stromal factors
and relied entirely on the pH gradient as an energy source for
integration (Fig. 4). Second, integration was competed by saturating quantities of ⌬pH pathway precursors but not by Sec
pathway precursors (Fig. 5). Third, antibodies to the ⌬pH machinery specifically inhibited Pftf integration (Figs. 6 and 8).
Finally, hcf106 mutant chloroplasts were unable to integrate
Pftf into thylakoids, even though they efficiently imported the
precursor into the organelle (Fig. 7). These results indicate
FIG. 7. Insertion of Pftf is inhibited in hcf106 mutant plants. Chloroplasts were isolated from wild type and hcf106-mum3 maize leaves and
described under “Experimental Procedures.” A, chloroplasts were assayed for import and localization of pPftf, pOE17, and pOE33. Intact
chloroplasts were repurified following the assays by centrifugation through Percoll cushions and analyzed directly (C) and also subfractionated into
stroma (S) and thylakoids (T). Thylakoids were also extracted with 0.1 M NaOH (TN). For assays of pOE17 and pOE33 import, only the recovered
chloroplasts are shown. B, chloroplasts from mutant hcf106 and wild type seedlings were subjected to immunoblotting with antibodies to Hcf106
and Tha4. Each lane contained chloroplasts equivalent to 1.75 ␮g of chlorophyll.
N-Tail Translocation by the ⌬pH-dependent Machinery
FIG. 8. A conserved RR carboxyl proximal to the H2 anchor is
not required for ⌬pH pathway integration of mPftf. Integration
mPftf and mKKPftf was assayed with washed thylakoids or thylakoids
preincubated with IgGs. Thylakoids were pretreated with 1 mg/ml
preimmune IgGs (PI), 0.2 mg/ml anti-psHcf106 IgGs (␣106), or 1 mg/ml
anti-pea plant chloroplast TatC homologue IgGs (␣TatC), respectively,
as described under “Experimental Procedures” and depicted above the
panels. Where designated, thylakoid preincubation was conducted in
the presence of 20 ␮M antigen, either pshcf106sd or plant chloroplast
TatC homologue peptide. Assays were conducted for 25 min in the
absence or presence of nigericin and valinomycin (N/V) as designated
above the panels. Assays were terminated with nigericin and valinomycin, and the recovered thylakoids were treated with thermolysin. Analysis was by SDS-PAGE and fluorography on a 12.5% gel. Gel lanes
contain equivalent quantities of thylakoids recovered from the assays.
that, like the Sec system, the ⌬pH pathway can distinguish
between segments of hydrophilic peptide to be completely
transported to the lumen and segments of hydrophobic peptide
to be released into the bilayer.
One surprising and novel feature of Pftf integration is that it
required neither the RR motif of the signal peptide nor even the
hydrophobic core domain. pKKPftf integrated faithfully into
thylakoids (Figs. 3 and 4) and exhibited all of the hallmarks of
⌬pH pathway translocation. mPftf, which lacks the entire signal peptide, was similarly integrated into thylakoids by the
⌬pH pathway (Figs. 3 and 4). Nevertheless, several observations indicate that the RR motif and the hydrophobic core of the
signal peptide contribute to efficient integration. pKKPftf integrated into isolated thylakoids with only ⬃50% of the efficiency of pPftf. Although mPftf exhibited the same rate of
integration into isolated thylakoids, as did pPftf, other characteristics demonstrate a weaker interaction with the ⌬pH pathway machinery. For example, mPftf and pKKPftf were competed by lower concentrations of overexpressed ⌬pH pathway
precursors than pPftf (Fig. 5). Because effective concentrations
for competition are related to Km values of the competing
substrates, this indicates that mPftf and pKKPftf interact with
⌬pH pathway machinery more weakly than pPftf. In support of
this conclusion, when pKKPftf was imported into chloroplasts
under conditions that prevented integration, iKKPftf accumu-
23489
lated in the stroma, rather than bound to the membrane surface as did iPftf (Fig. 5B). We interpret these data to mean that
the RR-containing signal peptide of pPftf promotes a strong
interaction with the ⌬pH pathway machinery but is not essential for integration. This differs from previous analyses of lumenal protein substrates, where conservative substitution of
the RR eliminates transport (7, 8). Studies of the bacterial Tat
pathway have shown that conservative substitution of a single
arginine of some substrates reduces but does not eliminate
transport (11, 35). However, this is the first example where
deleting the entire signal peptide did not eliminate
translocation.
The fact that mPftf integrates with a reasonable efficiency
suggests the presence of a second targeting signal. This could
be a redundant signal that serves to simply increase the interaction of the precursor with the machinery or it might be a
cryptic signal that is unmasked by deletion of the signal peptide. The most likely second signal is the H2 membrane anchor
of Pftf, which presumably would promote translocation of the
amino terminus into the lumen (N-tail translocation). Interestingly, chloroplast Pftf proteins possess conserved twin arginines flanking H2 on the carboxyl proximal side. Although RR
has never been shown to function on the carboxyl side of a
hydrophobic core, we examined integration of a modified mPftf
in which the RR was replaced with two lysines. mKKPftf was
still integrated into thylakoids by the ⌬pH pathway (Fig. 8).
Thus, the role of H2 in targeting remains to be dissected in
future studies.
We also considered the possibility that the mPftf-targeting
element allows Pftf to access another translocation pathway
that conceivably shares ⌬pH pathway components. Antibody
inhibition experiments argued against the involvement of the
Sec translocon. The fact that mPftf must translocate a large
N-tail to the lumen suggested that the chloroplast Oxa1p homologue might be involved. In mitochondria, Oxa1p has been
shown to facilitate N-tail translocation of moderately sized
amino-flanking peptides across the inner membrane (5, 36, 37),
and antibody inhibition studies show that the chloroplast homologue is involved in integration of the membrane protein
LHCP into thylakoids (38). However, recent experiments in our
lab did not find an effect of antibodies to cpOxa1p on mPftf
integration.3
We therefore conclude that the ⌬pH protein transport system is an exceptionally versatile translocation system, i.e. it
can transport signal peptide-containing precursors, large
folded polypeptide domains, and large, charged N-tails. In addition to significantly expanding the range of known capabilities of the ⌬pH system, our studies refocus attention on the
puzzling multiplicity of export systems across prokaryote-derived membranes.
Acknowledgments— We thank Dr. Alice Barkan for the generous gift
of antibodies to maize Tha4. We also thank Ralph Henry and Steve
Theg for critical review of this manuscript and helpful suggestions and
Mike McCaffery for excellent technical assistance.
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