Journal of Experimental Botany, Vol. 52, No. 354, pp. 47-56,
January 2001
© 2001 Oxford University Press
Original Papers |
Chloroplast precursor proteins compete to form early import intermediates in isolated pea chloroplasts
Department of Plant Sciences and Cambridge Centre for Molecular Recognition, University of Cambridge, Downing Street, Cambridge CB2 3EA, UK
Received 14 April 2000; Accepted 29 August 2000
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Abstract |
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In order to ascertain whether there is one site for the import of precursor proteins into chloroplasts or whether different precursor proteins are imported via different import machineries, chloroplasts were incubated with large quantities of the precursor of the 33 kDa subunit of the oxygen-evolving complex (pOE33) or the precursor of the light-harvesting chlorophyll a/b-binding protein (pLHCP) and tested for their ability to import a wide range of other chloroplast precursor proteins. Both pOE33 and pLHCP competed for import into chloroplasts with precursors of the stromally-targeted small subunit of Rubisco (pSSu), ferredoxin NADP+ reductase (pFNR) and porphobilinogen deaminase; the thylakoid membrane proteins LHCP and the Rieske iron-sulphur protein (pRieske protein); ferrochelatase and the
subunit of the ATP synthase (which are both associated with the thylakoid membrane); the thylakoid lumenal protein plastocyanin and the phosphate translocator, an integral membrane protein of the inner envelope. The concentrations of pOE33 or pLHCP required to cause half-maximal inhibition of import ranged between 0.2 and 4.9 µM. These results indicate that all of these proteins are imported into the chloroplast by a common import machinery. Incubation of chloroplasts with pOE33 inhibited the formation of early import intermediates of pSSu, pFNR and pRieske protein. Key words: Chloroplast protein import, LHCP, phosphate translocator, precursor protein, Rieske protein, Rubisco, translocation.
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Introduction |
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Most chloroplast proteins are synthesized in the cytosol as higher molecular weight precursor proteins (Keegstra et al., 1989
; de Boer and Weisbeek, 1991) which contain an N-terminal extension called the transit sequence. Import of precursor proteins is post-translational and can be divided into several stages (reviewed by Chen and Schnell, 1999). The transit sequence interacts first with outer envelope lipids (van't Hof and de Kruijff, 1995a) which probably causes a conformational change leading to recognition by proteinaceous components of the chloroplast protein import machinery. The initial binding of precursor proteins to the import machinery is energy independent and reversible (Perry and Keegstra, 1994). The receptor consists of Toc159, formerly Toc86 (Perry and Keegstra, 1994; Schnell et al., 1997; Chen and Schnell, 1999), in concert with Toc75 (Ma et al., 1996) and Toc34 (Kouranov and Schnell, 1997). In the presence of 100 µM ATP and GTP the precursor binds irreversibly to the import apparatus (Cline et al., 1985; Friedman and Keegstra, 1989) to Toc159 and Toc75 as well as an Hsp70 (Com70) on the outer face of the outer envelope, an Hsp70 on the inner face of the outer envelope and two inner envelope proteins, Tic20 and Tic22 (Waegemann and Soll, 1991; Hirsch et al., 1994; Kessler et al., 1994; Schnell et al., 1994; Wu et al., 1994; Seedorf et al., 1995; Tranel et al., 1995; Ma et al., 1996; Kouranov and Schnell, 1997; Kouranov et al., 1998). Thus a stably bound precursor protein actually spans the outer envelope and is in contact with the inner envelope. It is now referred to as an early import intermediate. Toc75 is thought to comprise the protein conducting channel of the outer envelope since its predicted structure is very similar to that of the bacterial porins whereas Toc159 and Toc34 are thought to present the precursor protein to Toc75 using the energy derived from GTP hydrolysis. The outer envelope Hsp70s probably serve to maintain the precursor in an import-competent form. In the presence of 1 mM ATP in the stroma, the precursor protein is translocated across the outer and inner chloroplast envelopes (Theg et al., 1989); this involves a 110 kDa component of the inner envelope, Tic110 (Schnell et al., 1994; Wu et al., 1994; Lubeck et al., 1996) in addition to Tic20 and Tic22 (Kouranov and Schnell, 1997; Kouranov et al., 1998) and Tic36 (Schnell et al., 1994). Once the precursor protein is inside the stroma, the transit sequence is removed by the stromal processing peptidase (vanderVere et al., 1995).
This description of chloroplast protein import suggests that there is only one import machinery which imports all of the different precursor proteins. The Toc and Tic components were identified using chimaeric precursors derived from three different precursor proteins, namely pSSu, the precursor of ferredoxin, and pOE33 (Perry and Keegstra, 1994; Waegemann and Soll, 1991; Schnell et al., 1994; Wu et al., 1994). Competition studies have also been carried out which showed that various precursor proteins are imported via the same import machinery. Incubation of chloroplasts with synthetic peptides corresponding to 20-amino-acid-residue sections of the transit peptides of the precursors of the light-harvesting chlorophyll a/b-binding protein (pLHCP) and the small subunit of Rubisco (pSSu) inhibited the import of pSSu, pLHCP and the precursors of ferredoxin (pFd) and plastocyanin (pPc) (Buvinger et al., 1989; Perry et al., 1991), whereas peptides corresponding to the C-terminal 30 amino acid residues of the transit peptides of pSSu and pFd blocked import of pSSu and pFd into chloroplasts (Schnell et al., 1991). A synthetic peptide corresponding to the entire transit sequence from the precursor of the subunit of the chloroplast ATP synthase (pAtpC) from Chlamydomonas reinhardtii inhibited the import of pSSu into pea chloroplasts (Theg and Geske, 1992). These studies indicated that pSSu, pLHCP, pFd, pPc, and pAtpC are all imported via the same import machinery.
Import competition studies have also been carried out using entire chloroplast precursor proteins which were produced by expression in Escherichia coli. Incubation of chloroplasts with pLHCP inhibited the import of pSSu into the chloroplasts (Oblong and Lammpa, 1992). Similarly the precursor of the 23 kDa protein of the oxygen-evolving complex (pOE23) competed with pSSu, pLHCP, pPc, pOE17, pOE23, and pOE33 (precursors of the 17, 23 and 33 kDa subunits of the oxygen-evolving complex, respectively) for import into the chloroplast (Cline et al., 1993). The group of chloroplast precursor proteins shown to utilize this common import machinery has been extended still further to include the precursor of the phosphate translocator (pPT) and the maize bt 1-encoded protein (Keegstra et al., 1995, although the data was not shown), the precursor of the 96 kDa protein of the inner envelope, pIEP96 (Hirsch and Soll, 1995), the precursor of Toc75 (Tranel et al., 1995), and the precursor of SecA (Berghöfer et al., 1995).
More recently, the view that there is only one general chloroplast protein import apparatus has been challenged by the finding that there are two homologues of Toc34 in Arabidopsis, namely Toc33 and Toc34 (Jarvis et al., 1998) and three of Toc159 (Toc120 and Toc132 in addition to Toc159; Bauer et al., 2000). Disruption of Toc33 by T-DNA insertion led to the formation of a mutant with an impaired ability to import chloroplast precursor proteins, called ppi1, for plastid protein import 1 mutant (Jarvis et al., 1998). These plants have a general import defect, including the inability to import protochlorophyllide oxidoreductase, a non-photosynthetic protein. Western blotting of an Arabidopsis mutant with a disruption in Toc159 revealed reduced expression of the large and small subunits of Rubisco and LHCP, suggesting a defect in the general import pathway which resulted in transcriptional down-regulation of these genes (Bauer et al., 2000). The expression of a plastid protein which is not specific to chloroplasts, namely chorismate mutase, was unaffected as were the expression and processing of Toc75 and Tic110. This suggests that Toc159 functions in the import of precursors of photosynthetic chloroplast proteins, but perhaps that Toc120 and Toc132 might function in import of precursors of non-photosynthetic proteins.
In this paper, import competition experiments were carried out between either pOE33 or pLHCP and a wide variety of chloroplast precursors, of both photosynthetic and non-photosynthetic proteins, in order to examine whether the proteins are imported via the common import machinery. In each case, the concentration of pOE33 or pLHCP causing half-maximal inhibition of import (the I50) was determined. In addition, experiments were carried out to determine whether pOE33 would compete for the formation of early import intermediates with a subset of these precursors.
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Materials and methods |
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Plasmids encoding precursor proteins
Bacterial plasmids for the synthesis of precursor proteins in radiolabelled form by transcription and translation in vitro have been described previously. pSMS64 encodes pea pSSu (Anderson and Smith, 1986
), pSAB80XD/4 encodes pea pLHCP (Cline et al., 1989), pPPT.8 encodes pea pPT (Knight and Gray, 1995), pSP-5PC3 encodes pea pPc (Helliwell and Gray, 1995), pGEF 3–1 encodes pFerrochelatase from Arabidopsis (Smith et al., 1994) and pAc222 encodes Arabidopsis pPBGD (Lim et al., 1994). Also used were plasmids encoding pea pFNR (Newman and Gray, 1988), pea pRieske protein (Salter et al., 1992) and tobacco pAtpC (Larsson et al., 1992). The plasmids for expression of pOE33 and pLHCP in E. coli consisted of the cDNA encoding Arabidopsis pOE33 (Ko et al., 1989) or the intronless gene encoding pea pLHCP (Cashmore, 1984) which had been inserted into the expression plasmid pET-3d (Studier et al., 1990) under the transcriptional control of the T7 promoter.
Expression in E. coli
Expression of pOE33 and pLHCP was carried out using E. coli strain BL21 (DE3) (Studier and Moffatt, 1986). Inclusion bodies of pOE33 and pLHCP were isolated and purified exactly as described previously (Paulsen et al., 1990) using E.coli cells of strain BL21 (DE3) harbouring either pET-3d::pOE33 or pET-3d::pLHCP. The purity of the inclusion body protein was checked by SDS-PAGE (Laemmli, 1970) followed by staining of the proteins with Coomassie Brilliant Blue R-250. The typical yield of pLHCP and pOE33 inclusion body protein was 0.3–0.5 mg protein from 50 ml bacterial culture, as determined using a Bio-Rad protein assay with bovine serum albumin as standard.
Transcription and translation in vitro
In order to prepare radiolabelled precursor proteins, cDNAs encoding chloroplast precursor proteins were transcribed using SP6 polymerase and the RNA was translated in a wheat germ extract in the presence of [35S] methionine as described (Knight and Gray, 1995). [35S] methionine (1000 Ci mmol-1) was purchased from Amersham International (Little Chalfont, Buckinghamshire).
Chloroplast isolation and protein import
Chloroplasts were isolated from peas (Pisum sativum L. cv. Feltham First) sown in Levington M2 compost (Fisons) and grown for 7–10 d in a greenhouse with an ambient temperature of 15–25 °C with supplementary artificial lighting providing a PAR of 150 µmol photons m-2 s-1 on a 16 h photoperiod as described earlier (Kirwin et al., 1989). Chlorophyll content was determined by measuring A652 after acetone extraction (Arnon, 1949).
Import competition experiments were carried out according to a modification of the method described previously (Cline et al., 1993) in which the conditions for chloroplast protein import were those described earlier (Newman and Gray, 1988). Inclusion bodies of pOE33 or pLHCP containing 200 µg protein were dissolved in 75 µl 6 M urea, 8 mM DTT at 25 °C for 4 h with occasional mixing. The protein solution was centrifuged at 20 000 g for 10 min at room temperature to remove any insoluble protein and the protein concentration was determined by carrying out a Bio-Rad protein assay using BSA as the standard. Chloroplasts containing 40 µg chlorophyll were incubated with 5 mM ATP, 1 mM methionine, 330 mM sorbitol, 25 mM HEPES-KOH pH 8.0, 6 mM DTT, and either 0–10 µM pOE33 or 0–5 µM pLHCP in a total volume of 140 µl. More 6 M urea and 8 mM DTT were added as necessary so that the concentration of urea and DTT would be the same in each of the reactions. The urea concentration was not allowed to exceed 0.5 M, which does not affect import (Cline et al., 1993). The pOE33 and pLHCP solutions in urea were added last and the concentrations referred to are concentrations in the final import reaction. The reactions were incubated at 25 °C for 10 min under room lights and 10 µl translation mix containing 35S-labelled protein were added and the reactions incubated again at 25 °C for 20 min. The chloroplasts were treated with thermolysin to degrade precursor proteins which had not been imported, and reisolated as described previously (Knight and Gray, 1995). The chloroplast pellets were analysed by SDS-PAGE and fluorography.
Formation of early import intermediates
Competition assays for the formation of early import intermediates were carried out in Eppendorf tubes which had been siliconized by dipping them in a 2% solution of dimethyldichlorosilane in 1,1,1-trichloroethane (BDH Ltd, Poole, Dorset) and allowing them to dry in the fume cupboard for approximately 1 h. Inclusion bodies of pOE33 were solubilized in 6 M urea as described above. Chloroplasts containing 40 µg chlorophyll were incubated with 200 µM ATP, 400 nM nigericin, 1 mM methionine, 330 mM sorbitol, 25 mM HEPES-KOH pH 8.0, and 1–10 µM pOE33 (final concentrations given) in a total volume of 140 µl on ice for 10 min. Translation mix containing 35S-labelled protein (10 µl) was added and the samples were incubated on ice for a further 20 min. Intact chloroplasts were reisolated and analysed by SDS-PAGE and fluorography.
In order to chase precursors bound as early import intermediates into chloroplasts, pellets of chloroplasts with associated early import intermediates were resuspended in 5 mM ATP, 1 mM methionine, 330 mM sorbitol, and 25 mM HEPES-KOH pH 8.0 in a volume of 150 µl. The samples were incubated at 25 °C under room lights for 15 min and intact chloroplasts were reisolated as described above. The chloroplasts were analysed by SDS-PAGE and fluorography.
Electrophoresis and fluorography
Proteins were separated by SDS-PAGE through 15% polyacrylamide gels (Laemmli, 1970). Chloroplast import assays were analysed by SDS-PAGE, loaded on an equal chlorophyll basis. Following electrophoresis, the gels were soaked in boiling 5% trichloroacetic acid for 5 min to hydrolyse methionyl-tRNA. The gels were rinsed in water, stained in 0.0025% (w/v) Coomassie Brilliant Blue R-250, 10% (v/v) ethanol, and 5% (v/v) acetic acid for 1 h and scanned with a Molecular Dynamics 300S laser scanning densitometer to check that equivalent amounts of each import sample had been loaded. The gels were neutralized in 2 M TRIS (unbuffered) for 5 min and subjected to fluorography by soaking for 30 min in Amplify (Amersham International). The gels were dried onto filter paper and exposed against Fuji RX film at -80 °C. The bands on the fluorograms corresponding to bound or imported precursor proteins were scanned with a Molecular Dynamics 300S laser scanning densitometer using volume integration.
I50 values
I50 values were calculated as follows. The percentage import remaining in the presence of a given concentration of precursor protein was calculated by expressing the density of the band of imported protein in that sample as a percentage of the density of the band in the control. Subtraction of this value from 100 gave the percentage inhibition of import in each sample. Values of I50 for the effect of a competing protein on import of a second protein were calculated either from a graph of [competing protein]/percentage inhibition of import against [competing protein] (Hanes, 1932) or from a direct linear plot (Eisenthal and Cornish-Bowden, 1974).
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Results and discussion |
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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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The results of import competition experiments between pOE33 and pLHCP and a range of chloroplast precursor proteins which have already been shown to enter the chloroplast via a common import site are shown in Fig. 1
. Incubation of chloroplasts with increasing concentrations of pOE33 inhibited the import of pSSu, pAtpC and pLHCP into chloroplasts since the intensity of the band of mature protease-protected protein decreased in each case with increasing concentrations of pOE33. Quantitation of the fluorograms to show the percentage import remaining in the presence of each concentration of competing pOE33, relative to the control lacking pOE33, is presented in Fig. 1b.
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The fluorogram in Fig. 1a
(iv) showing the effect of incubation of chloroplasts with increasing concentrations of pOE33 on the import of pPc into the chloroplasts contains, in addition to mature Pc, bands corresponding to pPc and two forms of Pc which are intermediate in size between pPc and mature Pc, referred to as iI and iII. Incubation of chloroplasts with increasing concentrations of pOE33 resulted in an accumulation of the precursor and intermediate forms of Pc, whilst the amount of mature Pc decreased. This effect has been documented (Cline et al., 1993) and results from competition between imported pOE33 and pPc for transport into the thylakoids. Quantitation and calculation of the total amount of Pc protein which was present in the chloroplasts in one form or another (Fig. 1b) shows that pOE33 competed with pPc for import into chloroplasts.
Incubation of chloroplasts with increasing concentrations of pLHCP also inhibited the import of pSSu, pAtpC, pLHCP, and pPc into chloroplasts (Fig. 1c, d). Comparison of the import competition experiments involving pOE33 (Fig. 1a, b) with those involving pLHCP (Fig. 1c, d) show that, in each case, pOE33 was more efficient than pLHCP at inhibiting chloroplast protein import of precursor proteins. This is reflected in the fact that the concentrations of pOE33 which cause half-maximal inhibition of import (I50 values, presented in Table 1) are in general lower than the I50 values for pLHCP. The results presented in Fig. 1 show that pOE33, pLHCP, pSSu, pAtpC, and pPc are all imported into the chloroplast via the common import machinery, confirming previous findings.
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Results of competition experiments between pOE33 or pLHCP and a variety of other precursor proteins not known to follow the general import pathway are presented in Fig. 2
. Both pOE33 and pLHCP competed for import with labelled precursors of ferredoxin NADP+ reductase (FNR), porphobilinogen deaminase (PBGD), which like FNR is a stromal protein (Shashidara et al., 1992), ferrochelatase, which is associated with the thylakoids and the inner envelope (Roper and Smith, 1997), the Rieske iron–sulphur protein (pRieske protein) of the thylakoid membrane and the phosphate translocator (PT) protein of the inner envelope. Thus each of these precursors shares a common saturable component of the import machinery with pOE33 and pLHCP. The lower band in the import experiments involving pPBGD has been observed previously (Lim et al., 1994) and may be a product of import of incomplete translation products or proteolytic degradation of imported pPBGD. FNR and Rieske protein are photosynthetic proteins whereas PBG deaminase, ferrochelatase and the phosphate translocator are non-photosynthetic proteins, suggesting that pOE33 and pLHCP compete for import with both photosynthetic and non-photosynthetic proteins.
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Comparison of the curves showing the effect of pOE33 on import of pFNR, pPBGD, pFerrochelatase, pRieske protein, and pPT in Fig. 2b
with those showing the effect of pLHCP on the import of these precursors (Fig. 2d) shows that, in general, pOE33 was more efficient than pLHCP at inhibiting the import of precursor proteins into chloroplasts. This may result from pLHCP having a lower affinity for the import machinery than pOE33. Indeed the transit sequence of pSSu was shown to be better than that of pLHCP at directing either SSu or LHCP to the chloroplast (Hand et al., 1989). It is also possible, however, that the lower efficiency of pLHCP at inhibiting precursor protein import compared to pOE33 reflects improper folding of a proportion of the pLHCP molecules in vitro. Synthesis of 35S-labelled pOE33 and pLHCP followed by chloroplast protein import assays revealed that the pOE33, but not the pLHCP, was import competent (data not shown). Moreover, pLHCP which had been synthesized by expression in E.coli, dissolved in 6 M urea and then dialysed against 4.5 M urea, 3.0 M urea, 1.5 M urea, and finally 20 mM TRIS-HCl pH 8.0, competed with pSSu for import into chloroplasts (Oblong and Lamppa, 1992). Only 55% of this pLHCP could be processed by the stromal processing peptidase, however, and none of it was monomeric.
The concentrations of pOE33 and pLHCP giving 50% inhibition (I50 values) of import of the precursor proteins in Figs 1 and 2 were derived graphically, using Hanes or direct-linear plots, and are presented in Table 1. The I50 value is an estimate of the effectiveness of the competitor protein under the conditions of the individual protein import assay. Because the absolute concentrations of the 35S-labelled precursor proteins are unknown, and are likely to be different for each individual precursor protein, it is necessary to be careful when comparing I50 values for different precursors. In addition, several of the I50 values determined are lower than the lowest concentration of the competitor protein used in the competition import experiment, adding to the need for caution in interpretation. However, for most of the precursor proteins, the I50 values for competition by pOE33 (0.2–3.3 µM) were lower than the values for competition by pLHCP (0.2–4.9 µM). In general, these values are similar to those in the literature, ranging between 0.1 µM for the inhibition of pSSu import by pLHCP (Dabney-Smith et al., 1999) and 5 µM for the inhibition of pSSu import by the transit peptide from Chlamydomonas pAtpC (Theg and Geske, 1992).
To assess whether precursor proteins compete for the formation of early import intermediates, chloroplasts were incubated with various concentrations of pOE33 in the presence of 200 µM ATP and 400 nM nigericin on ice for 10 min. This has the effect of reducing the stromal ATP concentration since nigericin inhibits photophosphorylation (Grossman et al., 1980a; Cline et al., 1985) whereas carrying out the reactions at 4 °C (Grossman et al., 1980b; Friedman and Keegstra, 1989) prevents uptake of ATP into the stroma (Leheny and Theg, 1994). 35S-labelled pSSu, pFNR or pRieske protein were added, followed by incubation for a further 20 min and analysis by SDS-PAGE and fluorography. Under these conditions the 35S-labelled precursor proteins bound to the chloroplasts, but were not imported and processed to the mature form (Fig. 3), a state known to represent the formation of early import intermediates in which the precursor protein is associated with the import components of the outer envelope and spans the outer envelope to make contact with some proteins of the inner envelope (Schnell et al., 1994; Ma et al., 1996; Kouranov and Schnell, 1997). pOE33 competed with pSSu, pFNR and pRieske protein for the formation of early import intermediates (Fig. 3a(i), c(i), e(i)).
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In addition, duplicate reactions were set up in which chloroplasts with pSSu, pFNR or pRieske protein bound as early import intermediates were re-isolated and incubated with 5 mM ATP for 15 min in order to chase the precursor protein along the import pathway into the chloroplast. In this way, it was possible to examine the effect of pOE33 on association of pSSu, pFNR and pRieske protein with the import machinery in the absence of non-specific binding of these precursors to other components of the chloroplast envelope, since the non-specifically bound precursors would not be imported. pOE33 inhibited the association of pSSu, pFNR and pRieske protein with the import machinery, but did not affect non-specific binding. The I50 values obtained were 0.1 µM, 0.2 µM and 0.5 µM for pSSu, pFNR and pRieske protein, respectively, which are similar to the I50 values for the effect of pOE33 on import of pSSu (0.2 µM) and pFNR (0.4 µM).
The percentages of chloroplast-associated pSSu, pFNR and pRieske protein which could be imported into chloroplasts in a chase reaction, of 83%, 87% and 62%, respectively, agree well with the values quoted in the literature for pSSu of 50–83% (Cline et al., 1985; Friedman and Keegstra, 1989; Olsen et al., 1989). The fact that the chase of pRieske protein into chloroplasts was less efficient than that of pSSu or pFNR may be explained by the finding that only 30–50% of bound pLHCP could be imported into chloroplasts (Cline et al., 1985) and suggests that precursors of chloroplast membrane proteins might have a higher non-specific binding to the chloroplast envelope than those of soluble chloroplast proteins.
The initial energy-independent binding of precursors to the chloroplast envelope involves Toc159, Toc75 and Toc34 (Perry and Keegstra, 1994; Ma et al., 1996; Kouranov and Schnell, 1997). The early import intermediate which is formed in the presence of low levels of ATP and GTP is associated with Toc159, Toc75, Com70, Hsp70, Tic20, and Tic22, with Toc75 being the major target of crosslinking to the transit sequence of pSSu at this stage in import (Ma et al., 1996). Any one of these outer envelope components, or a combination of several of them, might be the component for which the precursor proteins compete. The fact that the I50 for the effect of pOE33 on the formation of an early import intermediate with pSSu (0.1 µM) was very similar to both the Km for initial energy-independent binding of a chimaeric precursor consisting of pSSu fused to protein A, pS-Prot A (Schnell and Blobel, 1993) and the Km for the formation of an early import intermediate with pS-Prot A (Kouranov and Schnell, 1997) at around 50–100 nM suggests that the site of competition might be the complex of Toc159, Toc75 and Toc34.
However, there is also some evidence that of these three Toc proteins, Toc75 might be the site of competition, by analogy to the situation in mitochondrial import in which precursor proteins compete for insertion into the general insertion site of the outer membrane even after being recognized by the same initial import receptor (Pfaller et al., 1988). Import of the isolated transit peptide of the ferredoxin precursor was not affected by pretreatment of the chloroplasts with proteases, although it did exhibit saturation kinetics (van't Hof and de Kruijff, 1995b). Saturation kinetics indicate the involvement of a proteinaceous component and yet most of the outer envelope import components are destroyed by thermolysin treatment so that protease-treated chloroplasts cannot import whole precursor proteins (Cline et al., 1985; Friedman and Keegstra, 1989). Toc75, however, is known to be protease resistant (Tranel et al., 1995) suggesting that the ferredoxin transit peptide may bypass Toc159 and Toc34 (which may be involved in the unfolding of precursor proteins) to interact directly with Toc75. Since the ferredoxin transit peptide competes with pSSu for a saturable component of the chloroplast protein import machinery (Pilon et al., 1992), this suggests that the site of competition might be Toc75. This might also explain the finding that incubation of chloroplasts with the transit peptide of pSSu and pAtpC caused accumulation of whole pSSu precursor on the surface of chloroplasts (Pinnaduwage and Bruce, 1996; Theg and Geske, 1992). This might represent competition between the transit peptide and the whole precursor protein for Toc75 and subsequent accumulation of precursor protein on Toc159 and Toc34. The finding that pOE33 and pLHCP inhibited the import of precursors of both photosynthetic and non-photosynthetic proteins suggests that although the different homologues of Toc159 might be responsible for recognizing different subsets of precursors, there is only one Toc75, the putative site for competition, and all precursors must pass through this one Toc75.
To summarize, the range of chloroplast precursor proteins which are known to use the general import machinery have been extended to include pFNR, pPBGD, pFerrochelatase, pRieske protein, and pPT and it has been shown that precursor proteins compete for the formation of early import intermediates.
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Acknowledgments |
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We thank Neil Hoffman for the gift of E. coli strains which express pOE33 and pLHCP; Jenny Roper for the gift of pGEF3.1, and for help with the experiment to determine the effect of pOE33 and pLHCP on import of pFerrochelatase into chloroplasts; Alison Smith for the gift of pAc222; Chris Helliwell for the gift of pSP-5PC3 and Jacqui Knight for the gift of pPPT.8. PER also thanks Hal Dixon for his explanation of enzyme kinetics, Jacqui Knight, Paco Madueño, Simon Barnes, Renuka Sornarajah, Aliki Kapazoglou, Sam Haward, and Ruth Mould for helpful discussions, and Rainer Duden for help in preparing the figures. PER thanks the Science and Engineering Research Council and St John's College, Cambridge for financial support.
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Notes |
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1 Present address: Department of Clinical Biochemistry and Cambridge Institute for Medical Research, University of Cambridge, Wellcome Trust/MRC Building, Addenbrooke's Hospital, Hills Road, Cambridge CB2 2XY, UK.
2 To whom correspondence should be addressed. Fax: +44 1223 333953. E-mail: jcg2{at}mole.bio.cam.ac.uk
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Abbreviations |
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DTT, dithiothreitol; I50, the concentration of precursor protein which causes a 50% inhibition of import of a second precursor protein; pAtpC, precursor of the
subunit of the ATP synthase; pFc, precursor of ferrochelatase; pFd, precursor of ferredoxin; pFNR, precursor of ferredoxin-NADP+ reductase; pLHCP, precursor of the light-harvesting chlorophyll a/b-binding protein; pOE17, pOE23, pOE33, precursors of the 17, 23 and 33 kDa subunits of the oxygen-evolving complex, respectively; pPBGD, precursor of porphobilinogen deaminase; pPc, precursor of plastocyanin; pPT, precursor of the phosphate translocator; pRieske protein, precursor of the Rieske iron-sulphur protein; pSSu, precursor of the small subunit of Rubisco; Rubisco, ribulose 1, 5-bisphosphate carboxylase/oxygenase. Enzymes: thermolysin (EC 3.4.24.27). |
References |
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