(Received for publication, December 17, 1996, and in revised form, February 22, 1997)
From the School of Biological Sciences, University of Manchester,
2.205 Stopford Building Oxford Road, Manchester M13 9PT, United
Kingdom, the § Horticultural Sciences Department, University
of Florida, Gainesville, Florida 32611, the Department of Plant
Sciences, University of Cambridge, Downing Street, Cambridge CB2 3EA,
United Kingdom, and the ** Department of Microbiology, Institute of
Molecular Biological Sciences, Biocentrum Amsterdam, De Boelelaan 1087, 1081 HV Amsterdam, The Netherlands
Signal recognition particles (SRPs) have been identified in organisms as diverse as mycoplasma and mammals; in several cases these SRPs have been shown to play a key role in protein targeting. In each case the recognition of appropriate targeting signals is mediated by SRP subunits related to the 54-kDa protein of mammalian SRP (SRP54). In this study we have characterized the specificity of 54CP, a chloroplast homologue of SRP54 which is located in the chloroplast stroma. We have used a nascent chain cross-linking approach to detect the interactions of 54CP with heterologous endoplasmic reticulum-targeting signals. 54CP functions as a bona fide signal recognition factor which can discriminate between functional and non-functional targeting signals. Using a range of authentic thylakoid precursor proteins we found that 54CP discriminates between thylakoid-targeting signals, interacting with only a subset of protein precursors. Thus, the light-harvesting chlorophyll a/b-binding protein, cytochrome f, and the Rieske FeS protein all showed strong cross-linking products with 54CP. In contrast, no cross-linking to the 23- and 33-kDa proteins of the oxygen-evolving complex were detected. The selectivity of 54CP correlates with the hydrophobicity of the thylakoid-targeting signal and, in the case of light-harvesting chlorophyll a/b-binding protein, with previously determined transport/integration requirements. We propose that 54CP mediates the targeting of a specific subset of precursors to the thylakoid membrane, i.e. those with particularly hydrophobic signal sequences.
The signal recognition particle (SRP)11is a ribonucleoprotein complex which promotes the signal sequence-dependent targeting of nascent precursor proteins to the endoplasmic reticulum (1-3). Mammalian SRP is composed of six polypeptides and a 7 S RNA, although only one of the polypeptides, the 54-kDa subunit (SRP54), binds to the hydrophobic endoplasmic reticulum (ER)-targeting signals (1-2). Functional homologues of SRP54 have been identified in many organisms and appear relatively conserved during evolution (4, 5). These proteins are usually found complexed with a 7 S-like RNA and the minimum requirement for SRP-dependent protein targeting seems to be a ribonucleoprotein particle composed of an SRP54-like protein and a 7 S-like RNA (6-8), together with a cognate receptor for the SRP-precursor protein complex (3, 9, 10). Perturbation of SRP-dependent targeting pathways often leads to the accumulation of secretory proteins. However, in many cases only a subset of precursors accumulate while other proteins continue to be secreted normally (9, 11-13). This suggests that a discrete population of precursors preferentially utilize an SRP-dependent targeting pathway (1, 4, 13).
The delivery of precursor proteins to the thylakoid membrane of chloroplasts is governed by thylakoid-targeting signals. These signals are clearly related to those which target proteins to the ER membrane of eukaroytes and the cytoplasmic membrane of prokaryotes. The key features of all these signals are a charged NH2 terminus followed by a central hydrophobic region that can vary considerably in length and sequence composition (14, 15). The hydrophobic region is often followed by a cleavage site allowing proteolytic removal of the signal. However, many integral membrane proteins have an uncleaved "signal-anchor" sequence which functions as both the targeting signal and the transmembrane anchor (16).
The cloning of 54CP, a cDNA encoding an SRP54-like protein with a
functional chloroplast-targeting signal (transit peptide), suggested
that an SRP-dependent pathway may be involved in targeting precursor proteins from the chloroplast stroma to the thylakoid membrane (17). In fact, several distinct routes for precursor targeting
to the thylakoid membrane have been defined biochemically (15, 18) and
genetically (19). Particular precursors have been proposed to utilize
one of the following pathways: 1) Sec A-dependent (20, 21);
2) SRP-dependent (22); 3) pH-dependent (23,
24); and 4) "spontaneous integration" (25). Pathway selection by a
thylakoid lumen-resident precursor seems to be a function of the
thylakoid-targeting signal that it bears (26, 27). A well characterized
example is the twin-arginine motif which has been shown to be a
critical determinant for entry into the
pH-dependent
pathway (28). The implication of these observations is that there may
be specific signal-binding components that commit subsets of precursors
to these different thylakoid-targeting/integration routes.
In this study we have used a cross-linking assay to establish that 54CP is a major signal-sequence-binding protein present in chloroplast stroma (cf. Ref. 6). Cross-linking analysis with authentic thylakoid precursors established that only a subset of these precursors interact with 54CP. The cross-linking efficiency was correlated to the hydrophobicity of the thylakoid-targeting signal suggesting that this feature is the crucial determinant which promotes 54CP binding. This proposal is supported by a dramatic increase in 54CP binding to a mutant carboxypeptidase Y signal sequence with increased hydrophobic character.
On the basis of these data we propose that 54CP interacts preferentially with thylakoid precursor proteins that have particularly hydrophobic signals. Hence, the SRP dependent targeting route may be particularly important for the delivery of integral membrane proteins to the thylakoid membrane.
Restriction enzymes were from New England
Biolabs, Taq polymerase was from Boehringer Mannheim, and
RNA polymerases were from Promega. The rNTPs and RNasin were obtained
from Pharmacia Biotech Inc. Cycloheximide and 7-methyl-guanosine
5-monophosphate were from Sigma. [35S]Methionine was
purchased from DuPont and cross-linking reagents were from Pierce and
Prochem. The anti-P48 serum was raised against the purified protein (a
gift from I. Sinning, EMBL, Heidelberg). Antibodies against mammalian
SRP54 and 54CP were generous gifts from B. Dobberstein, ZMBH,
Heidelberg, and N. Hoffman, Stanford University, respectively. The wild
type and mutant CPY constructs were kindly provided by Mary-Jane
Gething, University of Melbourne.
The PPL wild type and mutant constructs
have been previously described (6). The coding region for the complete
pea cytochrome f precursor was excised from pT7CF-35 (29) as
a 1.2-kilobase Asp718I-SalI fragment, and
inserted into the SalI site of pJII101 (30) after ligation
of SalI linkers (5-CGTCGACG) to the end-filled fragment.
This construct contains the SP6 promoter and the
translational enhancer from tobacco mosaic virus upstream of the cytochrome f coding region. The coding region of the mature pea Rieske
protein was amplified by PCR from the full-length cDNA in pSP65
(31) and inserted into the SmaI site of pGEM3Zf(
) under
the control of the SP6 promoter.
OE23 Met and OE33 Met are truncated constructs made from the pea iOE23 and wheat iOE33. They are designed to contain twin methionines at their COOH termini and thereby enhance labeling during cell-free translation. OE23 Met was PCR-amplified from iOE23 (32) in pGem 4Z under the control of the SP6 promoter. The PCR product contains the codons for the thylakoid-targeting domain and the first 58 amino acids of the mature protein plus two COOH-terminal methionines, it lacks a stop codon. The PCR product was inserted into the XbaI and HindIII sites of pGem 4Z under the control of the SP6 promoter.
OE33 Met was amplified from wheat iOE33 (33) by PCR. The resulting PCR product contained codons for the lumen-targeting domain and the first 60 amino acids of the mature protein together with two additional methionines at the COOH terminus. No stop codons were present in the product and it was inserted into the SstI and HindIII sites of pGem 4Z under the SP6 promoter.
The pLHCP coding region is the AB80 clone from pea (34) and was inserted into pSP64. The LHCP TM3-PC fusion protein was constructed using PCR splicing by overlap extension with pLHCP from pea and the plastocyanin precursor from Arabidopsis thaliana. The TM3 segment for pLHCP comprised amino acids 189-269 (the COOH terminus) while the plastocyanin segment contained the entire mature plastocyanin sequence plus the cleavage cassette AGNAMA from the lumen targeting domain. The spliced PCR product was ligated into HincII and SstI sites of pGem 3Z under the SP6 promoter. All of the PCR-derived products were sequenced after subcloning, no alterations to the coding region were present in any of the constructs used for the subsequent cross-linking studies.
Transcription TemplatesTemplates for the transcription of truncated mRNAs were prepared either by using restriction sites within the coding region (naturally occurring and introduced) or using PCR (35). In the later case the PCR products were transcribed directly to avoid any selection of PCR-generated mutations. Plasmids encoding PPL and PPL-SSKO were linearized with PvuII, cytochrome f, and TM3-PC constructs were linearized with EcoRI and the cDNA encoding the Rieske protein was linearized with AvaII. Both CPY constructs (36) were linearized with HincII while the OE23 Met and OE33 Met constructs were linearized with HindIII. The LHCP-TM1 and LHCP-TM2 truncations were generated by PCR as described by Nilsson et al. (35). Transcription with T7 or SP6 polymerase was performed as described by the manufacturers (Promega).
Cell Free Translation, Cross-linking, and Product AnalysisTranslation using a wheat germ lysate based system was
performed as described previously (37) and nascent chains were labeled by incorporation of [35S]methionine. After translation
for 15-20 min at 26 °C, samples were pulsed by the addition of 4 mM 7-methyl-guanosine 5-monophosphate, incubated for a
further 10 min and translation stopped by the addition of cycloheximide
to 2 mM and placing the samples on ice. Ribosome bound
nascent chains were then purified by centrifugation through a sucrose
cushion (38). After purification, resuspended samples were incubated
with one-fourth volume of crude stromal extract (7.25 OD280/ml) prepared from pea seedlings (23) or with purified
SRP for 10 min at 26 °C. The cross-linking reagents bis(sulfosuccinimidyl)suberate (BS3), EDC, BMH, or S-MBS, were then
added to a final concentration of 1 mM, samples were
incubated for a further 10 min and then the reaction quenched by the
addition of one-tenth volume of 1 M glycine, 500 mM glycine, and 50 mM
-mercaptoethanol, or
100 mM
-mercaptoethanol as appropriate. Samples were
then split into four equal fractions and either analyzed directly or
after immunoprecipitation with antisera specific for mammalian SRP54,
Escherichia coli P48, or 54CP. Immunoprecipitation was in
the absence of SDS and as described previously (39). All samples were
analyzed by SDS-polyacrylamide gel electrophoresis using 12%
polyacrylamide gels.
After SDS-polyacrylamide gel electrophoresis, gels were exposed to PhosphorImaging plates such that all the signals remained in the linear detection range. The plates were read using a Fujix BAS 2000 Bioimager and the resulting images were quantified using Fuji software. The amount of 54CP cross-linking product is expressed as a percentage of the total label present as nascent chain (i.e. total products plus cross-linking products). In many cases two total samples were run and the average value was then used; likewise the amounts of 54CP cross-linking product present after immunoprecipitation with anti-54CP and anti-P48 were also averaged.
The average signal sequence hydrophobicity was calculated using two scales for relative amino acid hydrophobicity, the von Heijne scale and the Kyte-Doolittle scale (both from Ref. 40). Functional signal sequences with hydrophobic stretches as short as seven amino acids have been identified (41). For this reason the values for the most hydrophobic stretch of six, seven, and eight contiguous amino acids were determined and these were then summed and averaged to give the hydrophobicity values shown in Table I. For the von Heijne scale the most negative number represents the most hydrophobic sequence, while for the Kyte-Doolittle scale the largest number represents the most hydrophobic sequence. Such a simplified analysis does not take into account either the possible effects of flanking residues (i.e. those outside the calculated value), or the fact that the structure of the signal sequence may be important.
|
We adapted an established cross-linking assay
(6) to identify signal sequence-specific binding proteins present in
chloroplast stroma. The previous approach utilized a UV activable probe
incorporated as a modified lysine residue during nascent chain
biosynthesis. In the present analysis we initially investigated the
cross-linking products obtained with different bifunctional
cross-linking reagents (see also Ref. 22). A ribosome bound truncated
preprolactin chain of 86 amino acids (PPL, see Fig. 1)
was mixed with a stromal extract from pea chloroplasts and various
bifunctional cross-linking reagents were added (Fig. 2,
lanes 1-4). PPL86 was used since the
NH2-terminal ER-targeting signal is exposed from the
ribosome and can be efficiently cross-linked to both mammalian and
E. coli SRP (6). As a control, an engineered PPL construct
(PPL-SSKO, see Fig. 1) with a mutated non-functional signal sequence
(6) was analyzed in the same assay (Fig. 2, lanes 5-8). The
addition of the bifunctional primary amine-specific reagent BS3 gave a strong 65-kDa cross-linking product with PPL but not with PPL-SSKO (Fig. 2, lane 2, arrowhead, cf. lane 6). Although less
intense, a 65-kDa cross-linking product was also obtained with BMH
(Fig. 2, lane 3). These 65-kDa cross-linking products were
not observed in the absence of stroma (data not shown). Other
cross-linking products were observed with both PPL and PPL-SSKO
(e.g. the 26-kDa S-MBS-dependent product, Fig.
2, lanes 1 and 5) and were not further characterized.
54CP Binds to Functional ER Targeting Signals and to Authentic Thylakoid Precursors
Immunoprecipitation of the
BS3-dependent PPL cross-linking products obtained in the
presence of stroma showed that the 65-kDa cross-linking product was
efficiently immunoprecipitated with antisera specific for both 54CP
(22) and for P48, the E. coli SRP54 homologue (Fig.
3, lanes 7 and 8). While the
anti-P48 serum immunoprecipitated the 54CP cross-linking products as
efficiently as the anti-54CP serum, antibodies specific for mammalian
SRP54 did not recognize 54CP (Fig. 3, lane 6). A second
weaker cross-linking product of 73 kDa was also immunoprecipitated
under the conditions used (Fig. 3, lanes 7 and 8, asterisk). Both the 65- and 73-kDa products are still
immunoprecipitated with anti-54CP serum after the samples have been
denatured with SDS (data not shown). The different mobilities probably
reflect cross-linking between different lysine residues within the
nascent chain and 54CP. When PPL-SSKO was incubated with stroma and
cross-linking induced with BS3, no 54CP adducts were immunoprecipitated
(data not shown).
Having established that 54CP could bind to heterologous ER-targeting signals we analyzed the interaction of 54CP with authentic thylakoid precursor proteins bearing true thylakoid-targeting signals (see Fig. 1). Cytochrome f is a chloroplast genome-encoded protein with an NH2-terminal cleavable thylakoid-targeting signal (42). Nascent cytochrome f showed a very similar cross-linking pattern to PPL with a major cross-linking product of 66 kDa and a weaker product of 74 kDa (Fig. 3, lane 2). Both products were recognized by antisera specific for 54CP and P48 (Fig. 3, lanes 10 and 11).
The Rieske FeS protein is a nuclear-encoded protein that is synthesized as a precursor with a cleavable chloroplast-targeting signal; the hydrophobic domain toward the NH2 terminus of the Rieske protein functions as the thylakoid-targeting signal (43). A nascent Rieske polypeptide (Fig. 1), lacking the chloroplast-targeting signal and exposing the hydrophobic region outside the ribosome, was analyzed for its interactions with stromal factors. Cross-linking products of a similar size to those seen with cytochrome f were obtained (Fig. 3, lane 3) and shown to be 54CP adducts (Fig. 3, lanes 16 and 17). Cytochrome f and the Rieske protein were also both substrates for mammalian SRP (Fig. 3, lanes 4, 5, 12, and 18).
In some cases a significant amount of nascent chain was
co-immunoprecipitated with 54CP (e.g. Fig. 3, lanes 7, 8, 10, and 11; and Fig. 4, lanes
9 and 10). Such co-precipitation has been previously
described with mammalian SRP54 (37) and presumably reflects the
strength of the interaction between 54CP and particular protein
precursors.
54CP Binds to the Third Transmembrane Domain of LHCP
The light-harvesting chlorophyll a/b-binding protein (LHCP) is, to date, the only thylakoid precursor that has been shown to require 54CP for its integration (22). LHCP is synthesized as a precursor (pLHCP) in the cytosol, imported into the chloroplast, and then integrated post-translationally into the thylakoid membrane. LHCP integration into isolated thylakoids has been reconstituted and requires both 54CP and GTP (22, 44). LHCP and 54CP have also been shown to form a stable complex in vitro using a nondenaturing gel assay (22). Since LHCP lacks a cleavable thylakoid-targeting signal, 54CP presumably binds to a transmembrane region which acts as a signal-anchor sequence. LHCP has three transmembrane (TM) regions (45, 46) and we reasoned that in principle any one of these may bind 54CP and act as a thylakoid-targeting signal (see also Refs. 47 and 48).
Truncated LHCP nascent chains that included either the first transmembrane region alone (LHCP-TM1), or both the first and second transmembrane regions (LHCP-TM1/2) were prepared (see Fig. 1). In addition, a chimera comprised of the third LHCP transmembrane domain placed in front of the mature plastocyanin coding region (TM3-PC) was produced (see Fig. 1 and "Experimental Procedures"). The interaction of 54CP with LHCP-TM1 and TM3-PC was assessed by cross-linking with BS3. The first transmembrane domain showed no 54CP cross-linking products while the third transmembrane domain showed strong 54CP cross-linking adducts (Fig. 4, cf. lanes 3, 6, and 7 with lanes 4, 9, and 10). This suggested that 54CP interacted specifically with the third transmembrane domain of 54CP.
In contrast to all the other signal sequences we had analyzed, the
second transmembrane domain of LHCP has no lysine residues anywhere
near the stretch of hydrophobic amino acids (see Fig. 1). For this
reason we also used a carbodiimide-based cross-linking approach (Fig.
5) which acts to promote a condensation reaction between
free amino and carboxyl groups of adjacent polypeptides. All of the
LHCP transmembrane regions have nearby acidic or basic amino acid
residues suitable for this approach (Fig. 1). EDC-mediated cross-linking of the three LHCP-derived nascent chains confirmed that
only the third transmembrane domain (TM3-PC) was cross-linked to 54CP
(Fig. 5, lanes 11 and 12, arrows). The efficiency
of EDC-mediated cross-linking to TM3-PC was much lower than that
obtained with BS3. A similar reduction in efficiency was seen when
cross-linking products between PPL and 54CP were generated with EDC
(data not shown).
54CP Only Binds to a Subset of Thylakoid Precursors
Several
distinct pathways for the targeting of protein precursors to the
thylakoid have been identified by biochemical approaches (15, 18). We
determined whether the existing classification of precursors could be
correlated with 54CP binding. The 23-kDa protein of the oxygen-evolving
complex (OE23) shows a pH-dependent transport across the
thylakoid membrane (26, 27, 32) and has a twin-arginine motif
characteristic of this pathway (28). In contrast, the transport of the
33-kDa protein of the oxygen-evolving complex (OE33) is dependent upon
ATP (33, 49) and chloroplast SecA (20, 21).
Analysis of OE23 showed no cross-linking of the nascent chain to 54CP
following incubation with stromal extract (Fig. 6,
lanes 2, 9, and 10). Likewise no evidence for any
interaction between OE33 and 54CP was detected (Fig. 6, lanes 4, 15, and 16). When OE23 and OE33 were incubated with
mammalian SRP some cross-linking to SRP54 was observed (Fig. 6,
lanes 5 and 11). This may simply reflect the lack
of specificity which is exhibited by purified mammalian SRP when
incubated with purified nascent chains in the absence of the nascent
chain-associated complex (see Ref. 50). Nevertheless, the cross-linking
of OE23 and OE33 to mammalian SRP54 indicates that, were 54CP to be
bound to these precursors, a cross-linking product could be formed. A
novel, stromal extract-dependent cross-linking product with
OE23 was observed (Fig. 6, lane 2, arrow). The apparent
molecular mass of the cross-linking partner (i.e. after
subtracting the contribution of the nascent chain) was about 70 kDa and
the product was not immunoprecipitated by antisera recognizing 54CP.
Both the identity of this cross-linking partner, and its significance,
remain to be established.
54CP Interacts Preferentially with Hydrophobic Signal Sequences
The observation that 54CP interacts specifically with the third transmembrane domain of LHCP led us to compare the thylakoid and ER-targeting signals we had analyzed. This analysis (see Table I) showed that the efficiency with which 54CP was cross-linked to a nascent precursor could be correlated to the hydrophobicity of the signal sequence. Several different scales were used to calculate hydrophobicity and it soon became apparent that the scale developed by von Heijne (40) gave the best correlation with the cross-linking efficiency between a protein precursor and 54CP. In general, the more hydrophobic the signal sequence, the more efficient the 54CP cross-linking observed. The Kyte-Doolittle scale (see Ref. 40) showed a similar trend but there were obvious anomalies, in particular the apparent lack of hydrophobicity in the third transmembrane region of LHCP.
To test the correlation between signal sequence hydrophobicity and 54CP
cross-linking efficiency we compared two forms of the yeast protein
carboxypeptidase Y in our assay (Fig. 7). Wild-type CPY
has an amino-terminal ER-targeting signal which is functional in
Saccharomyces cerevisiae, but not in mammalian cells (36). The introduction of two point mutations (Gly to Leu) into the CPY
signal sequence increases its hydrophobicity and allows the precursor
to be efficiently targeted and translocated in both mammalian cells and
a rabbit reticulocyte translation system supplemented with canine
pancreatic microsomes (36). These two CPY precursors were ideal
for testing the effect of signal sequence hydrophobicity upon 54CP
cross-linking efficiency. The mutant CPY has a very hydrophobic signal
sequence while the wild-type protein does not (see Table I).
When the interaction of 54CP with wild-type CPY was analyzed it proved to be a very poor substrate for 54CP (Fig. 7, lanes 6 and 7). In contrast, the mutant CPY, differing only at two positions of the signal sequence, was an excellent substrate (Fig. 7, lanes 12 and 13). The very high efficiency of 54CP cross-linking to mutant CPY may reflect the presence of a tract of leucine residues present in a favorable context. When the amount of nascent chain-54CP cross-linking product was determined for each of the precursors used in this study, we observed a good correlation with the calculated hydrophobicity of the signal sequence (see Table I).
In this study we have identified 54CP as a major thylakoid signal-specific factor present in chloroplast stroma. Our initial analysis showed that 54CP can discriminate between functional and non-functional ER-targeting signals present on the model secretory protein PPL. Hence, 54CP behaves like a true signal sequence recognition factor. Other known SRPs are all ribonucleoprotein complexes comprising a minimum of an SRP54-like protein and a 7 S-like RNA. 54CP has been reported to have an apparent molecular mass of 200 kDa (15), suggesting that it may be part of a larger complex. However, this remains to be established.
The analysis of several authentic thylakoid precursor proteins showed that 54CP interacted only with a subset of the precursors. LHCP is the only thylakoid precursor for which direct evidence of 54CP-dependent targeting has been shown (22). We found that 54CP interacts strongly with LHCP, but only with the third transmembrane region. Analysis of 54CP binding to the individual transmembrane regions of LHCP using a native gel assay (cf. Ref. 22) also showed that the interaction was restricted to the third transmembrane region (data not shown). These data support the tentative proposal that the third transmembrane region of LHCP constitutes the thylakoid-targeting signal (47). All three of the transmembrane domains were found to be essential for the correct membrane integration of LHCP (48, 51).
The integration of LHCP into the thylakoid membrane is a post-translational event, and full-length LHCP has been shown to interact with 54CP in a ribosome-independent manner (22).2 We conclude that 54CP promotes LHCP integration by binding to the third transmembrane domain of the full-length protein and mediating its targeting to the thylakoid membrane. Thylakoid precursors which are encoded by nuclear genes, e.g. LHCP, are synthesized by cytosolic ribosomes and then transported across the chloroplast envelope. Thus, these precursors must be integrated into the thylakoid post-translationally.
Like LHCP, the Rieske FeS protein is also nuclear-encoded. When the precursor protein is imported into isolated chloroplasts, the protein is found associated with both Hsp60 and Hsp70 molecular chaperones (52) suggesting that these stromal components may keep the imported the Rieske protein in a "translocation-competent" conformation. Our cross-linking analysis suggests there is also a significant interaction between 54CP and the mature Rieske protein. These data may simply reflect the ability of the Rieske protein to use multiple thylakoid-targeting routes. Alternatively, there may be sequential interactions between imported Rieske protein and various stromal components such as 54CP, Hsp60, and Hsp70. Indeed, the interactions of the Rieske protein with Hsp60 and Hsp70 were found to be time dependent and imported Rieske protein associated first with Hsp60 and then with Hsp70 (52). The use of a short nascent (i.e. ribosome bound) chain may have locked the Rieske polypeptide into a stable interaction with 54CP and prevented any subsequent interactions with other stromal components (see below).
Thylakoid precursors which are encoded by the chloroplast genome and synthesized in the stroma may preferentially interact with 54CP as ribosome-bound nascent polypeptides (i.e. co-translationally). An association of 54CP with nascent chains in vivo is supported by data showing that a fraction of 54CP co-sediments with 70 S chloroplast ribosomes (17). We have used cytochrome f as a representative chloroplast genome-encoded precursor and found it was efficiently cross-linked to 54CP. On this basis we propose that cytochrome f can utilize the 54CP-dependent pathway. This suggestion is further supported by the observation that the cytochrome f and LHCP-thylakoid integration pathways in Chlamydomonas share at least one, genetically defined, component (53) which is not required for OE33 transport. The hydrophobic core of the Chlamydomonas cytochrome f signal sequence was also found to be required for efficient thylakoid integration, again suggesting that cytochrome f is targeted to the thylakoid by 54CP.
The picture is complicated by the observation that, in maize, the tha 1 mutation interferes with both OE33 and cytochrome f targeting, but not with LHCP integration (19). This is consistent with OE33 transport (20, 21) and cytochrome f integration (54, 55) both being SecA-dependent. As with the Rieske protein, there may be sequential interactions between cytochrome f and different stromal factors (i.e. 54CP and chloroplast SecA); alternatively cytochrome f might use multiple thylakoid-targeting pathways. The ability of a single precursor protein to utilize multiple targeting pathways to the ER has recently been established in S. cerevisiae (13).
The presence of a functional SecA homologue in chloroplast stroma is
well established and purified SecA and ATP are the only requirements
for transport of in vitro synthesized OE33 across washed
thylakoid membranes (21, 56). Consistent with these data is our finding
that OE33 nascent chains show no significant cross-linking to 54CP;
equally, no obvious adduct with SecA was observed in the total
cross-linking products. In general, the use of short, ribosome-bound,
nascent chains favors SRP binding when an appropriate signal sequence
is present (cf. Refs. 6 and 57). Interactions between
nascent polypeptides and other stromal components, for example, SecA,
may require longer chains than those used in this study and no evidence
of any interaction between short nascent polypeptides and E. coli SecA was obtained in a recent cross-linking study (57). In
contrast to the other thylakoid proteins used in this study, the OE23
protein has been shown to be transported across the thylakoid membrane
by a distinct, pH-dependent, route (26, 27, 32). As with
OE33, we found no evidence of 54CP being cross-linked to a truncated
OE23 polypeptide. OE23 was shown to be cross-linked to a 70-kDa stromal
factor, this component remains to be identified.
Our analysis of several authentic thylakoid precursor proteins illustrates that 54CP interacts with only a subset of these precursors. The critical factor in determining 54CP binding appears to be the hydrophobicity of the signal sequence. The influence of hydrophobicity was underlined by the dramatic increase in 54CP cross-linking to CPY with a mutated signal sequence. Two point mutations in the signal sequence (36) have a profound effect on both the calculated hydrophobicity and the efficiency of 54CP cross-linking. A role for signal sequence hydrophobicity in recruiting SRP has previously been established in E. coli (57). It was shown that increasing the hydrophobicity of a signal sequence promoted SRP binding in vitro with a concomitant increase in transport efficiency observed in vivo. Likewise in S. cerevisiae, utilization of the SRP-dependent ER-targeting pathway by precursors is dictated by signal-sequence hydrophobicity (13). Thus, 54CP behaves like other characterized SRPs in binding preferentially to particularly hydrophobic signal sequences.
Four biochemically defined thylakoid-targeting pathways have been
identified; the spontaneous integration of proteins such as
CFoII and PSII-W can probably be viewed as special cases
analogous to the insertion of M13 procoat in E. coli (15,
25, 58). Of the remaining three pathways, two have been shown to
require signal sequences with specific properties, i.e.
hydrophobicity for the 54CP-dependent route (this work) and
a twin-arginine motif for the pH-dependent (28). The
relationship between these different pathways remains to be fully
defined. At least some precursors may be able to utilize multiple
targeting routes, for example, using both the
SecA-dependent route and the SRP-dependent
route (cf. Ref. 13). Other precursors may be restricted to
essentially a single targeting pathway. The 54CP-dependent
route might be particularly important in mediating the targeting of
very hydrophobic precursors such as integral membrane proteins.
We are indebted to Colin Robinson (wheat iOE33), Mary-Jane Gething (CPY wild-type and mutant), Claire Anderson (cytochrome f), and Steven Bradshaw (Rieske protein) for the construction and gifts of plasmids. Thanks also to Dr. Neil Hoffman and Prof. Bernhard Dobberstein for sera, Jaqui Knight for help with preparing stromal extract, and Fimme Jan van der Wal for CPY mRNA.