Horticultural Sciences Department and Plant Molecular and Cellular Biology Program, University of Florida, Gainesville, Florida 32611
Transport of proteins to the thylakoid lumen is accomplished by two precursor-specific pathways, the Sec and the unique Delta pH transport systems. Pathway selection is specified by transient lumen-targeting domains (LTDs) on precursor proteins. Here, chimeric and mutant LTDs were used to identify elements responsible for targeting specificity. The results showed that: (a) minimal signal peptide motifs consisting of charged N, hydrophobic H, and cleavage C domains were both necessary and sufficient for pathway-specific targeting; (b) exclusive targeting to the Delta pH pathway requires a twin arginine in the N domain and an H domain that is incompatible with the Sec pathway; (c) exclusive targeting to the Sec pathway is achieved by an N domain that lacks the twin arginine, although the twin arginine was completely compatible with the Sec system. A dual-targeting signal peptide, constructed by combining Delta pH and Sec domains, was used to simultaneously compare the transport capability of both pathways when confronted with different passenger proteins. Whereas Sec passengers were efficiently transported by both pathways, Delta pH passengers were arrested in translocation on the Sec pathway. This finding suggests that the Delta pH mechanism evolved to accommodate transport of proteins incompatible with the thylakoid Sec machinery.
Many proteins of the chloroplast thylakoid membranes are encoded in the nucleus, synthesized
in the cytosol, and localized by a two-step process (for review see Cline and Henry, 1996 Four precursor-specific pathways (or mechanisms) for
protein transport/integration into thylakoids have been
described (Cline and Henry, 1996 The existence of these parallel transport pathways is intriguing. Results of biochemical studies argue that the two
subgroups of proteins are exclusively transported on their
corresponding pathways (Cline and Henry, 1996 Two questions are immediately relevant. First, what elements in LTDs commit a precursor to a specific pathway?
All LTDs have embedded motifs for signal peptides similar to those that direct transport across bacterial and ER
membranes (von Heijne et al., 1989; Pugsley, 1993 Here we used a biochemical approach to identify elements of LTDs and passenger proteins that determine
pathway-specific transport. Our results show that the
transport process can be divided into two steps: targeting
and translocation. Pathway-specific targeting is mediated
solely by the signal peptide motifs of LTDs. In contrast, translocation depends upon passenger protein compatibility with the respective system. Our studies show that the
Sec system is not capable of efficiently translocating passenger proteins normally passed through the Delta pH system, whereas the Delta pH system transports passengers
from both pathways. These results define the molecular elements that govern specific transport on these systems and
further suggest that the novel Delta pH pathway may have
arisen to compensate for translocation limitations of the thylakoid Sec mechanism.
Materials
All reagents, enzymes, and standards were purchased commercially. In vitro transcription plasmids for precursors to OE23 (pOE23), OE33 (pOE33),
the stromal intermediate of OE33 (iOE33), plastocyanin (pPC) from pea,
pPC from Arabidopsis, and the precursor for OE17 (pOE17) from maize
have been described elsewhere (Cline et al., 1993 Construction of Recombinant Precursor Proteins
Coding sequences for all recombinant precursor proteins were constructed by PCR-based methods using the above plasmids as templates, as
well as templates prepared in this study and described below. Most amplifications were performed with Pfu polymerase (Stratagene, La Jolla, CA),
and the remainder with Taq polymerase. PCR products containing restriction sites incorporated into the forward, reverse, or both primers were digested with the appropriate restriction enzymes as noted below and ligated into appropriately restricted pGEM 3Z (Promega, Madison, WI) or
pGEM 4Z in the SP6 direction. When PCR primers lacked restriction
sites, PCR reactions were conducted with Pfu, and PCR products were
blunt-end cloned into the HincII or SmaI sites of pGEM 3Z or 4Z. All
cloned constructs were verified by DNA sequencing. Sequencing was done
with ABI Prism Dye Terminator cycle sequencing protocols developed by
Applied Biosystems (Perkin-Elmer Corp., Foster City, CA) and an Applied Biosystems model 373 Stretch DNA Sequencer (Perkin-Elmer Corp.).
Truncated Precursors.
The coding sequence for the intermediate form
of OE23 (iOE23) was constructed as described previously (Cline et al.,
1993
Chimeric Precursors.
The chimeric precursor t23-PC is an exact fusion
between coding sequences for the t23 signal peptide (MVSRRLAL . . . SPADA) and the mature domain of Arabidopsis PC (MEVLL . . . LTVK).
The t23-PC coding sequence was constructed using 23tPCsel as template and the same forward primer as that used to make tOE23. The PCR product was cloned into XbaI-SstI cut pGEM 3Z. PCn23h/c-PC and 23nPCh/cPC are chimeric truncated precursors in which combinations of N and H/C
domains from OE23 and Arabidopsis PC were fused to the sequence for
mature Arabidopsis PC. PCn23h/c-PC was constructed with t23-PC as
template. The forward primer incorporated the coding sequence for the
tPC N domain and the first 16 bases of the OE23 H/C region. This created
an exact fusion such that the translated protein begins MASLKD-LALSV
. . . SPADA followed by mature PC. The PCR product was cloned into the
HincII-SstI site of pGEM 3Z. 23nPCh/c-PC, which is referred to in the
text as DT-PC, was constructed with Arabidopsis pPC as template. The
forward primer incorporated the coding sequence for the OE23 N domain
and the first 16 bases of the PC H domain such that the translated polypeptide begins MVSRR-FGVIA . . . GNAMA followed by mature PC. The
PCR product was cloned into the SmaI-PstI site of pGEM 4Z.
). In the first
step, cytosolic precursors are imported across the chloroplast envelope membranes into the stroma. In the second step, stromal intermediates are integrated into the thylakoid membrane or transported across it into the lumen.
Precursors destined for the lumen possess bipartite aminoterminal transit peptides. The amino-proximal stroma-targeting domain (STD)1 directs import into the chloroplast
and is removed by a stromal processing protease. The
carboxy-proximal lumen-targeting domain (LTD) governs
transport into the lumen and is cleaved by a lumen-facing thylakoidal processing protease. Thus, the stromal intermediates of luminal proteins are intermediate in size between the full precursor and the mature form.
). In particular, transport
of proteins to the lumen occurs by two distinct pathways:
the Sec and Delta pH translocation systems. Transport of
one subset of lumen-resident proteins by the Sec pathway
is stimulated by a
pH and requires ATP (Hulford et al.,
1994
; Yuan and Cline, 1994
) and CPSecA (Nakai et al., 1994
; Yuan et al., 1994
). CPSecA is a chloroplast homologue of the bacterial SecA protein, a translocation ATPase (Wickner, 1994
). Transport of a second subset of lumen-resident proteins by the Delta pH pathway takes
place uniquely in the absence of nucleotides or soluble factors and is absolutely dependent on the trans-thylakoid
pH (for reviews see Robinson and Klösgen, 1994
; Cline
and Henry, 1996
).
). Studies
of two chimeric proteins indicate that pathway selection is
determined by the respective LTDs. A chimeric precursor
composed of the transit peptide of OE23 (a Delta pH
pathway substrate) and the mature sequence of plastocyanin (PC) (a Sec pathway substrate) was efficiently transported only on the Delta pH pathway (Henry et al., 1994
;
Robinson et al., 1994
). Another chimera that combined
the transit peptide of OE33 (Sec pathway) with the mature
sequence of OE17 (Delta pH pathway) was exclusively
transported on the Sec pathway, although with greatly reduced efficiency (Henry et al., 1994
). These observations suggest that the existence of two transport systems serves
a purpose beyond that of an overflow mechanism.
). Recent work by Chaddock et al. (1995)
indicates that a twin
arginine in the signal peptide is required for Delta pH
transport. However, the role of other elements in LTDs
has not been addressed. A second question: what is the underlying reason for the existence of two transport pathways? It is not related to the specific site of luminal protein function. For example, the OE33 and OE23 subunits
of the photosystem II oxygen evolving complex are transported by the Sec and Delta pH pathways, respectively.
Nor does it correlate with the attachment of prosthetic groups; e.g., the Sec pathway is responsible for transport
of OE33 and PSI-F, which do not possess prosthetic
groups, as well as the copper-binding PC and heme-binding cytochrome f (Voelker and Barkan, 1995
; Nohara et al.,
1996
).
Materials and Methods
). The transcription plasmid for PSII-T and the chimeric precursor between the OE23 transit peptide and the PC mature protein (23tPCsel) were as described (Henry et al.,
1994
). The transcription plasmid for PSI-N from Arabidopsis (accession
No. U32176) was the generous gift of Dr. Paul Sehnke (University of Florida, Gainesville). Escherichia coli-expressed and purified iOE23 was as
described (Cline et al., 1993
).
), except that the forward primer incorporated an XbaI restriction site
for ligating into the XbaI-HindIII site of pGEM 4Z. The coding sequence
for tOE23, an amino-terminal truncated form of iOE23, was constructed
in the same manner as iOE23 except that the initiator methionine incorporated in the forward primer was positioned so that the tOE23 translation
product begins MVSRR (see Fig. 1). A further truncation of tOE23 that
lacks the N domain was similarly constructed; the initiator methionine was
positioned such that the translation product starts MLALSV. The presumed intermediate form of OE17 (iOE17) and an amino-terminal truncated form of iOE17 (tOE17) were amplified using the pOE17 plasmid as
template. The forward primers for iOE17 and tOE17 were designed such
that the translation products started with MASAE and MAGRR, respectively. The PCR products were cloned into the SmaI-XbaI site of pGEM
4Z. Amino-terminal truncated precursors of PC (tPC) from Arabidopsis and pea were amplified from the respective pPC plasmids with forward primers designed such that translation products started MASLKD. The
PCR products were cloned into the HincII-SstI site of pGEM 3Z. An additional truncation of tPC from Arabidopsis to remove the N domain used
a forward primer to give a translation product beginning MFGVIA.
Fig. 1.
LTDs of precursors targeted to the Delta pH or Sec
transport systems in thylakoid membranes. The acidic (A),
charged (N), and hydrophobic (H) regions are shown for LTDs
of proteins transported by the Delta pH pathway or Sec pathway.
OE33 (from pea), OE23 (from pea), and OE17 (from maize) are
the 33-, 23-, and 17-kD subunits of the oxygen evolving complex
of photosystem II. PSII-T (from cotton) is the T subunit of photosystem II. PSI-F (from spinach) and PSI-N (from Arabidopsis)
are the F and N subunits of photosystem I, respectively. The PC
LTD from Arabidopsis is depicted. The cleavage consensus A-X-A
appears on the far right of the sequences. The hydrophobic residues of the H domain are underlined, and charged residues are
indicated by (+) or (). The amino termini for OE23, OE33, and
PC correspond to those determined by Bassham et al. (1991)
.
The precise amino termini for LTDs of OE17, PSI-N, PSII-T, and
PSI-F are not known.
[View Larger Version of this Image (20K GIF file)]
). The DT-33, DT-23, DT-17,
DT-T, or DT-N SOE products were restricted and ligated into pGEM 4Z cut with SstI-XbaI, EcoRI-HindIII, EcoR1, EcoRI-HincII, and EcoRI- HincII, respectively.
Site-directed Mutation of DT-PC. KK-PC is an altered form of DT-PC in which the twin arginine in the signal sequence was changed to a twin lysine. The coding region was amplified with the Arabidopsis pPC plasmid as template and a forward primer that codes for the sequence starting MVSKK-FVGIA. The PCR product was cloned into the HincII-SstI site of pGEM 3Z. DT-PC E/K codes for DT-PC in which the second amino acid (glutamate) of mature PC was changed to a lysine using PCR/SOE. The single amino acid change was made by altering the appropriate bases in the two overlapping internal primers used in the first two PCR reactions. The resulting SOE product was cloned into the SstI site of pGEM 4Z. The same method was used to change a lysine in the 23 C domain to asparagine in PCn23h/c-PC, resulting in PCn23h/c-PC K/N. The amplified SOE product was cloned into the HindIII-SstI site of pGEM 3Z. Several mutants were constructed in which aspartic acid replaced valine residues in the H domain of DT-PC, i.e., amino acids 8, 11, and 18, respectively. These mutations were made by PCR/SOE using the DT-PC as template and incorporating GTC to GAC codon changes in the forward primer for the first set of PCR reactions. The SOE product was cloned into the SstI site of pGEM 4Z.
Constructs for Expression in E. coli.
Coding sequences for t23 and DT23 were prepared for expression in E. coli by amplifying each sequence
from pGEM clones (see above). The forward primer contained an NdeI
site that also encoded the initiator methionine. The PCR products were
digested with NdeI and HindIII and cloned into pETH3c (Cline et al.,
1993).
Preparation of Precursor Proteins
Capped RNA for authentic, chimeric, and mutant precursors was produced in vitro with SP6 polymerase and uncut plasmid; RNA was translated in a wheat germ system in the presence of [3H]leucine (Cline et al.,
1993). Translation products were generally diluted threefold and adjusted
to import buffer (50 mM Hepes/KOH, pH 8.0, 0.33 M sorbitol), containing
30 mM unlabeled leucine.
Preparation of Chloroplasts, Lysates, Thylakoids, and Stromal Extract
Intact chloroplasts were isolated from 9-10-d-old pea seedlings (Laxton's
Progress 9) and were resuspended in import buffer. Lysates and washed
thylakoids were prepared from isolated chloroplasts (Cline et al., 1993).
Stromal extract (SE) for transport assays was prepared from chloroplast
lysate (1.0 mg/ml chlorophyll) by centrifugation for 8 min at 3,200 g to remove the thylakoids, followed by centrifugation at 40,000 g for 30 min to
remove the envelope membranes. Chlorophyll was determined according
to Arnon (1949)
.
Preparation of Purified CPSecA
CPSecA was purified from SE as described by Yuan et al. (1994), except
that studies reported here used CPSecA obtained after the Mono-Q ion
exchange step. The concentration of purified CPSecA was estimated by
Coomassie staining of SDS polyacrylamide gels using BSA as a standard.
Assays for Thylakoid Protein Transport
Transport of radiolabeled proteins into thylakoids was conducted with
chloroplast lysate or washed thylakoids as previously described (Cline et al.,
1993) in 150-µl assays (unless noted otherwise in the figure legend) containing 50 µg chlorophyll and 5 mM Mg-ATP (pH 8.0). Assays were conducted in microcentrifuge tubes in a 25°C water bath illuminated with 70 µE/m2/s of incandescent light. For assays conducted in the presence of inhibitors, chloroplast lysates (50 µg chlorophyll in 100 µl) were preincubated with azide (7 mM final) or a combination of the ionophores nigericin (0.5 µM final) and valinomycin (1.0 µM final) on ice for 15 min before
the addition of Mg-ATP (5 mM final) and radiolabeled precursor. For assays conducted in the absence of ATP, lysate (50 µg chlorophyll in 50 µl)
and diluted translation product (25 µl) were preincubated separately for
10 min at room temperature with 1 U of apyrase. Competition assays for
thylakoid transport were conducted as described previously (Cline et al., 1993
). SE equivalent to 50 µg chlorophyll or purified CPSecA (80 nM final) was added to competition assays as noted in the figure legends. Recovered thylakoids were posttreated with thermolysin, which was terminated
by adding an equal volume of 50 mM EDTA in import buffer, collected by
centrifugation, and dissociated with SDS-PAGE sample buffer.
Analysis of Samples
Samples from the above assays were analyzed by SDS-PAGE followed by
fluorography. Quantification of transport was by scintillation counting of
radiolabeled proteins extracted from excised gel bands (Cline, 1986) and
is reported as a percentage of radiolabeled precursor added to the assay.
Expression of Proteins in E coli
Expression plasmids for tOE23 and DT-23 were introduced into the host
BL21(DE3). 5-ml cultures in Luria-Bertani medium containing 0.1 mg/
ml ampicillin were initiated from overnight colonies and grown at 37°C to
an OD ~0.8 at 600 nm. Expression was induced with 1.0 mM isopropyl
-d-thiogalactoside. For inhibition of SecA-mediated transport, cultures
were adjusted to 2 mM sodium azide at the time of induction. After an additional 2 h of culture, cells (1.4 ml) were pelleted and fractionated into
periplasm and cell contents according to the Novagen (Madison, WI) protocol. Basically, the cell pellet was resuspended in residual media, and 15 µl of chloroform was added, followed after 15 min at room temperature
by 75 µl of 10 mM Tris/HCl, pH 8.0. The periplasm and cell pellet were separated by centrifugation for 15 min at 12,000 g. Residual chloroform was removed from cell pellets, and pellets were resuspended in the same
volume as the periplasm fraction before analysis by SDS-PAGE. For fractionation of cells into soluble and insoluble fractions, 50 ml of culture was
pelleted at 3,000 g for 10 min. The pellet was resuspended in 5 ml of 10 mM
Tris/HCl, pH 8.0, 2 mM EDTA, and 0.1 mg/ml lysozyme. Triton X-100
was added to 0.1%, and the suspension was allowed to sit at room temperature for 15 min. The suspension was passed twice through a Yeda Press
(Cline et al., 1993
). Soluble and insoluble fractions were separated by centrifugation at 12,000 g for 15 min.
Domain Composition of Lumen-targeting Peptides
LTDs for precursors known to be transported on Delta
pH and Sec pathways are shown in Fig. 1. Embedded
within each LTD is a canonical signal peptide motif. In E. coli, the typical signal peptide possesses a five- to six-residue positively charged amino-terminal N domain, followed by an ~12-residue hydrophobic core H domain, and
a more polar cleavage C domain (Izard and Kendall, 1994). The N and H domains are important for transport; the C
domain is necessary only for proteolytic processing (Izard
and Kendall, 1994
). In addition to this minimal signal peptide, nearly all LTDs possess extended amino-terminal regions that are notable for their content of acidic residues,
which are uncommon in transit peptides. In the present
study, these acidic regions are called A domains. Despite
the fact that LTDs govern pathway selection, only one
pathway-related consensus sequence has been identified; Delta pH precursors invariably contain a twin arginine in
their N domains (Fig. 1).
Pathway-specific Transport Requires Only the Signal Peptide Motif of LTDs
Our strategy for identifying essential and pathway-specific elements was to treat each LTD domain as a module, to create deletions and swapping constructs with these modules, and then to test the recombinant precursor proteins in thylakoid transport assays. Based on previous analyses of chimeric proteins, we focused on the precursors for OE23 (Delta pH pathway) and PC (Sec pathway). Transport into isolated thylakoids was selected as an assay because it yields unambiguous assignments for transport pathway. Criteria used to assign precursors to the Sec or Delta pH pathway include: (a) energy requirements unique to each pathway; (b) the requirement for stromal extract of which CPSecA is the essential component; and (c) precursor competition for transport, which relies on the ability to saturate components unique to each transport system with chemical quantities of precursor.
Pathway-specific energy and stroma requirements are illustrated in Fig. 2. Transport of pOE23 (not shown) and
the stromal intermediate form (iOE23) was completely
abolished by ionophores that dissipate the pH (lane 4).
As with other precursors that use the Delta pH pathway,
transport was unaffected by removal of ATP (lane 5), by
the absence of stromal extract (lane 6), or by sodium azide
(lane 7), a SecA inhibitor (Oliver et al., 1990
). In contrast,
pPC transport, which is stimulated only slightly by a
pH
(Yuan and Cline, 1994
), was relatively unaffected by ionophores. Like other precursors that are localized by the Sec system, pPC transport was abolished by removing ATP
(lane 5) and greatly reduced by azide (lane 7) or removing
stromal extract (lane 6), the source of ~90% of the
CPSecA found in chloroplasts.
Deletion of amino-terminal elements of the transit peptide was without effect on transport specificity. OE23 and
PC constructs with minimal signal peptide motifs (tOE23
and tPC, respectively) exhibited transport characteristics
identical to the full-length or intermediate precursors (Fig. 2).
Measurements made during the linear phase of transport
(0-10 min for the Delta pH pathway; 0-30 min for the Sec
pathway) showed that tOE23 was transported at least
twice as efficiently as either pOE23 or iOE23. Similarly, a
truncated form of the OE17 precursor, tOE17 (see Materials and Methods for description), was exclusively transported on the Delta pH pathway with at least twice the
efficiency of either pOE17 or iOE17 (data not shown).
Transport efficiencies of tPC and pPC from pea were comparable (e.g., see Fig. 2). In contrast, transport of Arabidopsis tPC was only 20-50% as efficient as that of Arabidopsis pPC (data not shown). Further deletions that
removed the N domain of OE23 and PC eliminated transport, implying that the N domain is at least a general requirement for transport on either pathway (data not
shown). Fig. 2 also shows that a chimeric precursor protein
containing the minimal OE23 signal peptide fused to mature PC (t23-PC) was transported exclusively on the Delta
pH pathway (Fig. 2), which confirms and extends previous
studies (Henry et al., 1994; Robinson et al., 1994
).
These data show that the OE23 signal peptide motif is both necessary and sufficient for pathway-specific transport on the Delta pH pathway. The A domains of OE23 and OE17 LTDs are required neither for pathway specificity nor the efficiency of Delta pH pathway transport across isolated thylakoids. The data further show that the PC signal peptide is necessary and sufficient to direct PC across the Sec pathway. The role of amino-terminal regions of the PC transit peptide in transport efficiency is presently unclear.
A Dual-targeting Signal Peptide Directs Passenger Proteins to Both Sec and Delta pH Systems
To assess the targeting role of each signal peptide domain,
precursors containing chimeric signals fused to mature PC
were constructed. Energy and soluble factor requirements
served as initial diagnostics for pathway use. The chimeric
precursor PCn23h/c-PC was not transported by either
pathway (Fig. 2). Since the efficiency of thylakoid transport is generally greater in organello (within chloroplasts),
the PC STD was fused to this construct, and the assay was
conducted with intact chloroplasts. The precursor was efficiently imported into chloroplasts but accumulated as an
intermediate in the stroma (data not shown). Another
modification to this construct was made to eliminate the
possibility of incompatibility between the H/C domain and
the amino-terminal region of the mature protein (Laforet
et al., 1989; see Discussion); the first 23 residues of mature
OE23 were inserted immediately after the signal peptide. This precursor also failed to be transported into thylakoids
(data not shown).
The opposite result was obtained with the reciprocal
chimera 23nPCh/c-PC; this precursor was transported by
both pathways and therefore is designated dual-targeting
(DT)-PC. The first indication for dual targeting was that
DT-PC transport requirements were intermediate between
those of exclusively Sec or exclusively Delta pH transport
(Fig. 2, e.g., lanes 4 and 5). To confirm transport of DT-PC
on both pathways, a different set of pathway-specific criteria was used. Transport on the Delta pH pathway was assessed by precursor competition with unlabeled, E. coli-
produced iOE23, and transport on the Sec pathway was
assessed with purified CPSecA. If DT-PC uses both pathways, then saturating concentrations of iOE23 should shift
transport to the Sec pathway, and this transport should be
ATP dependent. Such an experiment is shown in Fig. 3 A. Assays were conducted with thylakoids, stromal extract,
light (to generate a pH), increasing amounts of competitor iOE23, and the presence or absence of 5 mM ATP. As
expected, transport of the Delta pH pathway substrates,
tOE23 and t23-PC, was similarly competed in the presence
or absence of ATP. Transport of the Sec pathway substrate, pPC, was unaffected by iOE23 competitor but virtually eliminated by ATP removal. DT-PC transport exhibited characteristics expected for a dual-targeted substrate.
Transport was reduced ~60-70% by iOE23 competitor in
the presence of ATP, demonstrating that a substantial
amount of DT-PC transport was using the Delta pH mechanism. The residual transport was virtually eliminated by
ATP removal, indicating that DT-PC was also transported by an ATP-dependent mechanism, presumably the CPSecA mechanism.
The experiment shown in Fig. 3 B confirms this presumption regarding CPSecA. Transport assays were conducted in the absence of stromal extract, and Sec pathway
transport was assessed by adding purified CPSecA in the
presence of 2.0 µM iOE23, which virtually eliminated
transport of the Delta pH pathway substrate, tOE23. A
small amount of pPC transport occurred without stromal extract, consistent with residual thylakoid-bound CPSecA
(Yuan et al., 1994); this was unaffected by iOE23 competitor. Addition of stromal extract or purified CPSecA in
the presence of 2.0 µM competitor boosted the transport
of DT-PC as well as pPC, but it had no effect on tOE23
transport (Fig. 3 B). Together with Figs. 2 and 3 A, these
results demonstrate that DT-PC is transported by both the
Sec and Delta pH systems.
As can be seen from Fig. 3 B as well as similar 30-min assays (see below), under conditions allowing only Sec transport (saturating iOE23 competitor and added CPSecA), the DT signal peptide directed transport about as efficiently as the bona fide pPC targeting signal. The efficiency of the DT signal peptide for Delta pH transport was measured separately in 10-min assays with washed thylakoids lacking ATP; DT-PC was transported at least as efficiently as t23-PC. Thus, the DT signal peptide is as effective as authentic pathway-specific signal peptides in governing transport of the same passenger protein on both pathways.
Both the N and H Domains Play a Role in Exclusive Targeting
The results with both chimeric signal peptides suggested that the N and H/C domains have different and distinct roles in pathway-specific targeting. We used DT-PC as a starting point to further identify critical specificity elements and analyze similar regions from other precursors. The DT signal is advantageous in that altering a specificity element is likely to lead to a loss of transport on only one pathway rather than a total loss of transport activity. This is important for distinguishing between a pathway-specific targeting defect and loss of transport due to unrelated effects on precursor structure.
Our initial modification of DT-PC confirmed, in part,
the results of Chaddock et al. (1995), i.e., that a twin arginine
is necessary for transport on the Delta pH pathway. Altering the N domain of DT-PC by replacing both arginines
with lysines (KK-PC) selectively eliminated transport on
the Delta pH pathway. Fig. 4 shows that KK-PC transport
was not inhibited by competitor iOE23 (lanes 5-7), but it
was abolished by removing ATP (lanes 3 and 8) and severely inhibited by removing stromal extract (lanes 4 and
9). In addition, CPSecA was able to replace the stromal requirement for transport of KK-PC (data not shown).
Other changes to DT-PC suggest that exclusive targeting to the Delta pH system involves the H/C domain; i.e., the H/C domains of Delta pH precursors are incompatible with the Sec system. For example, replacing the H/C domain of DT-PC with the OE17 H/C also results in exclusive transport on the Delta pH pathway (Table I). One possibility for this result was that basic residues in the C domain of OE17 as well as OE23 inhibit translocation by the Sec pathway, similar to the effect of basic residues in this region on bacterial Sec transport (Andersson and von Heijne, 1991). However, substituting a lysine for glutamic acid two residues into the PC mature domain (MEV to MKV) of DT-PC (Table I) did not alter its ability to transport on both pathways. Similarly, replacing the lysine in the cleavage site of PCn23h/c-PC (see Fig. 2) with an asparagine did not restore transport (data not shown). Finally, adding the OE23 cleavage domain (KVSPADA) just COOH-terminal to the PC cleavage site only had the effect of reducing but not eliminating transport on the Sec pathway (Table I).
Table I. Effect of Changes to the DT-PC Signal Peptide on Sec and Delta pH Transport |
On the other hand, replacing the PC H domain with the OE23 H domain (DT-PC 23hPCc) inhibited transport on the Sec pathway to below detectable levels, whereas Delta pH-mediated transport was relatively unaffected (Table I). Together, these results imply that a property of the OE23 H domain is incompatible with Sec-dependent transport. We also found that insertion of the OE33 H/C domain (DT-PC 33h/c) resulted in transport only on the Delta pH system (Table I). This was surprising given that OE33 is normally transported by the Sec mechanism. Taken together, these results imply that the Delta pH system is able to tolerate a variety of H/C regions that are incompatible with the thylakoid Sec mechanism. Nevertheless, both transport systems do share a minimal H domain hydrophobicity requirement. Substitution of aspartate residues for valine residues at several locations within the DT-PC H domain virtually eliminated transport (Table I).
Delta pH Passenger Proteins Limit Transport by the Sec-dependent Translocation System
The ability of passenger proteins other than PC to use either pathway was assessed with fusions to the DT signal
peptide. This included all proteins known to be normally
transported by the Delta pH mechanism (OE17, OE23,
PSI-N, and PSII-T; fusions designated DT-17, DT-23,
DT-N, and DT-T, respectively) as well as OE33 (designated DT-33). As shown in Fig. 5, competition by iOE23
severely inhibited transport of tOE23 and all of the DT constructs across buffer-washed thylakoids (compare lanes
1 and 4), indicating that all passenger proteins could be
translocated by the Delta pH mechanism. To assess Sec
pathway transport, purified CPSecA was added in the
presence of 2.0 µM iOE23 competitor. Transport of pPC,
iOE33, and DT constructs with Sec passengers (DT-PC
and DT-33) was stimulated by CPSecA (compare lane 6 to
lane 4). Transport of Delta pH passengers showed virtually no enhancement by the addition of CPSecA (compare
lane 6 to lane 4). This was surprising given that the mature
domain of PSI-N is nearly the same size as mature PC
(~10 kD) and mature PSII-T is only ~3 kD. Transport assays with DT-T yielded essentially the same results as
those with DT constructs with other Delta pH passenger
proteins (data not shown). Thus, although the DT chimeras with Delta pH passengers are competent substrates as
shown by their efficient transport by the Delta pH system,
the Delta pH passenger proteins apparently pose a translocation challenge that the thylakoid Sec mechanism cannot overcome.
E. coli Recognizes Sec But Not Delta pH-targeting Determinants, but Is Capable of Transporting a Delta pH Passenger Protein on the Sec Pathway
In E. coli, the SecA/SecY/SecE/SecG system is the major
route for export of signal peptide-bearing proteins across
the cytoplasmic membrane (Pugsley, 1993; Wickner, 1994
).
Identification of chloroplast homologues of SecA and
SecY (Laidler et al., 1995
) suggests that the thylakoid Sec
system is homologous and mechanistically similar. To test
this assumption, we transformed E. coli with a plasmid harboring the DT-23 coding sequence. Fig. 6 A shows that
after induction with isopropyl
-d-thiogalactoside, mature
OE23 accumulated to a high level and was recovered predominantly in the periplasmic fraction. To determine if
DT-23 was transported by the SecA-mediated system, bacteria were induced in the presence of 2 mM sodium azide (Oliver et al., 1990
). Under these conditions, only DT-23
precursor accumulated and was recovered in the cellular
fraction. This indicates that DT-23 transport was mediated
predominantly, if not exclusively, by the Sec system. When
the bacteria expressed tOE23, which has the authentic
OE23 signal peptide and differs from DT-23 only in the H/
C domain, only tOE23 precursor accumulated and was recovered in the cytoplasmic fraction. Fig. 6 B shows that the
tOE23 was largely soluble and thus accessible to the translocation apparatus. This was subsequently verified by the
observation that tOE23 is efficiently transported and processed in prlA suppressor strains of E. coli (McCaffery,
M., and K. Cline, unpublished results). These findings argue that targeting to the thylakoid and E. coli Sec systems
is similar, but that differences between the two mechanisms allow translocation of the OE23 passenger protein
in E. coli.
In this study we used a biochemical approach to analyze the determinants of signal and passenger proteins that specify transport by the thylakoid Delta pH or Sec pathways. Two relevant questions were addressed: what specific elements in the LTD determine pathway specificity, and what is the underlying reason for the existence of the two separate pathways? The results show that transport can be divided into two steps, targeting and translocation, and make several important points. First, targeting is mediated solely by the signal peptide motif of LTDs. Second, both the N and H domains of the signal peptide are required for pathway-exclusive targeting. Third, translocation depends upon compatibility of the passenger protein with the respective system. We found that all of the Delta pH proteins are incompatible with thylakoid Sec transport, even when assayed in the presence of added CPSecA. These results define the molecular determinants of specific transport on these systems and further suggest a basis for the existence of the Delta pH pathway.
The Targeting Step: Both the N and H Domains Are Critical for Specific and Exclusive Targeting and the A Domain Is Dispensable
Previous studies with chimeric precursors showed that the
LTD determines transport pathway selection (Henry et
al., 1994; Robinson et al., 1994
). Here we showed that
amino-terminal A domains are not required for specific
transport; truncated precursors of OE23, OE17, and PC
possessing only minimal signal peptides were transported
exclusively on the Delta pH (OE23 and OE17) or Sec
(PC) pathway (Fig. 2). These findings are consistent with observations of Ko and Cashmore (1989)
, wherein a chimeric precursor between the STD of a stromal precursor
protein and truncated OE33 lacking the A domain was imported into intact chloroplasts and localized to the thylakoid lumen. Although pathway specificity was not addressed in these early studies, it is likely that the protein
was transported on the Sec pathway.
Deletion of N domains of the signal peptides eliminated transport on both pathways, as did introducing charged residues into the H domain of DT-PC (Table I). This demonstrates a general requirement for each domain in the transport mechanism of both pathways. Pathway-specific targeting is more complex and requires the proper combination of N and H domains. A signal peptide composed of the PC N domain and the OE23 H/C domain failed to transport on either pathway. In contrast, the reciprocal signal peptide with the OE23 N domain and the PC H/C domain directed efficient transport of PC on both pathways (Figs. 2 and 3). The implication of this finding was that the Delta pH pathway has a specific requirement for the N domain, whereas the Sec pathway has a specific requirement for the H domain.
The details regarding N and H specificity were further
explored by modifications of the dual-targeting construct,
DT-PC. An N domain twin arginine (RR) requirement for
the Delta pH pathway was previously reported by Chaddock et al. (1995). Here we confirmed that result by showing that replacing the RR of DT-PC to KK caused loss of
transport only on the Delta pH pathway. Our results further argue that the RR, when combined with a nonspecific
H domain, is sufficient for Delta pH targeting. Additional
sequences are not required, as previously suggested by
Chaddock et al. (1995)
. This is evidenced by the ability of
the MVSRR N domain, when fused to four different H/C
regions as well as one chimeric H/C, to direct Delta pH
transport of PC (Fig. 2; Table I). Furthermore, since the N
domain of the truncated OE17 precursor (MAGRR) differs from MVSRR in all but the RR, it is unlikely that other N
domain residues play a targeting role.
The Sec pathway appears to have a nonspecific N domain requirement. Signal peptides with three different N
domains, MASLKD, MVSRR, and MVSKK, were shown
here to direct PC across the Sec pathway. Clearly, in our
studies, the RR did not mask or repel the precursor from
the Sec system as previously reported (Chaddock et al.,
1995). In fact, DT-PC (containing RR) was transported by
the Sec pathway as efficiently as the natural PC precursor. It occurred to us that the difference between our results
and those of Chaddock et al. (1995)
might reside in the
fact that they used in organello assays with intact chloroplasts. However, we found that a precursor in which the PC
STD was fused to DT-PC (STD-DT-PC) was readily imported into intact chloroplasts and localized to the thylakoids using both pathways. Dual pathway transport in
organello was shown by the inability of a single pathwayspecific inhibitor or competitor to affect thylakoid localization of DT-PC. Inhibition was only achieved with a combination of inhibitors: in this case, ionophores, azide, and a
thylakoid transport-saturating concentration of pOE33
(Carrigan, M., R. Henry, M. McCaffery, and K. Cline, unpublished results). In this regard, we note that in the Chaddock et al. (1995)
experiments, pPC-RR localization was
affected by nigericin as well as by azide, leaving open the possibility that some transport was occurring on the Delta
pH pathway.
The Sec pathway displays a much more stringent H domain requirement than the Delta pH pathway. Four different H/C domains were tested in the DT-PC construct. All of these chimeric precursors were efficiently transported by the Delta pH pathway, but only the precursor with the PC H/C domain was transported across the Sec pathway. In fact, the incompatibility was localized to the hydrophobic core itself, as replacement of only the hydrophobic core with that from OE23 eliminated Sec transport without affecting Delta pH transport (Table I). These results suggest that it is the H domain of Delta pH pathway precursors that prevents them from being targeted to the Sec pathway. Interestingly, even the E. coli Sec machinery discriminated between a Sec-compatible H/C domain and a Delta pH pathway H/C domain (Fig. 6).
The critical H domain feature for thylakoid Sec transport is presently unclear. Mean residue hydrophobicity
and the propensity to adopt an helix in hydrophobic environments have been correlated with transport efficiency
by the E. coli Sec system (for review see Izard and Kendall, 1994
). Mean hydrophobicity values for thylakoid precursors are slightly lower than those for bacterial signal peptides (e.g., as assessed by Doud et al., 1993
), but these
values do not group precursors on the basis of transport
pathway (not shown). Analysis with secondary structure
predictive programs suggests that the H domains of Delta
pH precursors have less of a tendency to adopt an
helix
(Clausmeyer et al., 1993
; unpublished results), but such
putative differences in secondary structure remain to be
experimentally verified with biophysical studies.
One less tangible characteristic of Sec transport in E. coli
was reported by Laforet et al. (1989). In N and H swapping studies carried out with E. coli precursors, they found
that one of three different H domains, that of M13 procoat, failed to functionally substitute for the alkaline phosphatase H domain in vivo. Since transport was partially restored if the procoat C domain and seven residues of the
mature procoat were included, Laforet et al. suggested the
potential for incompatibility between the H domain, C domain, and the amino terminus of the mature protein. If the
potential for such incompatibility is a property of thylakoid Sec transport, it may explain the inability of the
OE33 H/C domain to support Sec transport of PC.
The Translocation Step: Delta pH Passenger Proteins Are Incompatible with the Sec Machinery
The underlying reason for the operation of more than one
transport pathway in thylakoids or any other membrane
system is not known. For thylakoids, the inability of Delta
pH passenger proteins to be translocated by the thylakoid
Sec machinery offers one possible explanation for the existence of a novel pathway. Previous studies have noted
that two Delta pH passenger proteins, OE17 and OE23, impose thylakoid translocation limitations when fused to
Sec pathway transit peptides (Clausmeyer et al., 1993;
Henry et al., 1994
). These studies used in organello assays
with intact chloroplasts, in which thylakoid transport conditions are comparable to those found in vivo. Both chimeric precursors were efficiently imported into chloroplasts but poorly localized to the thylakoid. In the case of
OE17, some thylakoid localization occurred, but transport was inefficient and a considerable portion of the imported
protein accumulated in the stroma (Clausmeyer et al.,
1993
; Henry et al., 1994
). In the case of OE23, none of the
imported protein was transported into the lumen (Clausmeyer et al., 1993
). The present studies confirm and extend those observations. In our thylakoid transport assays,
all four of the known Delta pH passengers proteins were unable to be translocated on the Sec pathway when directed by the DT signal peptide.
One possible explanation for this result is that incompatibility exists between Delta pH passenger proteins and
the DT signal peptide (Laforet et al., 1989). However, we
do not believe this to be the case. The fact that the DT signal concurrently directed efficient transport of all of these
passenger proteins on the Delta pH pathway argues against
any kind of conformational instability that would mask the
signal peptide. In addition, recent studies in our laboratory
have shown that transport on the Delta pH pathway proceeds via a loop mechanism (Fincher, V., and K. Cline,
manuscript in preparation) similar to Sec-mediated transport (Kuhn et al., 1994
), indicating that the signal peptide
and the amino terminus of the mature protein need to be
compatible regardless of the pathway used. Finally, the
fact that the E. coli Sec system very efficiently transported
DT-OE23 argues against incompatibility of the DT signal
peptide and mature OE23 (Fig. 6).
In fact, the differing ability of thylakoid and E. coli Sec
systems to transport OE23 implies that mechanistic differences exist between the two systems. One obvious difference is that, in E. coli, the protonmotive force consists of a
in addition to a
pH, both of which participate in the
translocation process (e.g., Driessen, 1992
). At steady
state, thylakoids generate only a
pH. Thus, it will be interesting to examine the role of a
in E. coli export of
OE23 as well as the ability of E. coli proteins to use the
Sec or Delta pH systems in chloroplasts. Other differences may exist at the level of components that constitute the
two Sec systems, as virtually nothing is known regarding
the composition of the thylakoid Sec pathway translocon.
Taken together, our results suggest that the Delta pH system may exist to compensate for a Sec system that is less robust than the E. coli counterpart. These results also support the need for targeting signals that are capable of exclusive targeting to the Delta pH system, thereby avoiding deleterious effects that are likely to impede thylakoid development. The fact that classical signal peptides are required for transport by both the Sec and Delta pH pathways, as well as studies showing a loop mechanism for Delta pH transport (Fincher, V., and K. Cline, manuscript in preparation), suggest that the two systems may be related, possibly sharing common or homologous components. Unlike proteins localized by the Sec system, no obvious homologues of proteins transported by the Delta pH system are found in cyanobacteria, which are thought to be the present day representative of the endosymbiont that evolved into chloroplasts. This raises the question of where these proteins and their translocation system came from. It is tempting to speculate that Delta pH proteins as well as the translocation system were recruited to the thylakoids after the endosymbiotic event. Studies aimed at identifying components of both systems should shed light on these questions.
R. Henry's present address is Department of Biological Sciences, University of Arkansas, Fayetteville, AR 72701.
Received for publication 3 October 1996 and in revised form 18 December 1996.
Address all correspondence to Kenneth Cline, Horticultural Sciences Department, Fifield Hall, University of Florida, Gainsville, FL 32611. Tel.: (352) 392-4711 ext. 219. Fax: (352) 392-5653. e-mail: KCC{at}icbr.ifas.ufl.eduWe thank Shan Wu for technical assistance, and Vivian Fincher and Liz Summer for critical reading of the manuscript.
This work was supported in part by National Institutes of Health grant R01 GM46951 and National Science Foundation grant MCB-9419287 to K. Cline. 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 Station Journal Series #R-05505.
DT, dual-targeting; LTD, lumen-targeting domain; PC, plastocyanin; SE, stromal extract; SOE, splicing by overlap extension; STD, stroma-targeting domain.