Báo cáo khóa học: Fidelity of targeting to chloroplasts is not affected by removal of the phosphorylation site from the transit peptide doc

Fidelity of targeting to chloroplasts is not affected by removal of the phosphorylation site from the transit peptide Kerry-Ann Nakrieko, Ruth M. Mould and Alison G. Smith Department of Plant Sciences, University of Cambridge, UK Phosphorylation of the transit peptide of several chloroplast- targeted proteins enables the binding of 14-3-3 proteins. The complex that forms, together with Hsp70, has been demonstrated to be an intermediate in the chloroplast pro- tein import pathway in vitro [May, T. & Soll, J. (2000) Plant Cell 12, 53–63]. In this paper we report that mutagenesis (in order to remove the phosphorylation site) of the transit peptide of the small subunit of ribulose bisphosphate carb- oxylase/oxygenase did not affect its ability to target green fluorescent protein to chloroplasts in vivo. We also found no mistargeting to other organelles such as mitochondria. Similar alterations to the transit peptides of histidyl- or cysteinyl-tRNA synthetase, which are dual-targeted to chloroplasts and mitochondria, had no effect on their ability to target green fluorescent protein in vivo. Thus, phos- phorylation of the transit peptide is not responsible for the specificity of chloroplast import. Keywords: amino acyl-tRNA synthetase; confocal micros- copy; dual targeting; GFP; Rubisco. Most chloroplast and mitochondrial proteins are nuclear- encoded and are synthesized in the cytosol. Correct targeting of these proteins to the organelles is thus essential for cellular function and for the biogenesis of the individual organelles. In most cases, this is achieved by the presence of an N-terminal extension, called a transit peptide or pre- sequence [1–3]. Analysis of the primary amino acid sequence has revealed that there is little conservation either in composition or in length, although some general features have been identified [4,5]. Chloroplast transit peptides have few acidic residues and are rich in hydroxylated residues; plant mitochondrial presequences share these characteristics but also frequently form amphipathic a-helices, with positive charges clustered on one side [3–5]. This character- istic has been shown to be important for targeting to mitochondria in rice [6]. The transit peptide is necessary and sufficient for fidelity of targeting to the chloroplast or mitochondrion, as shown most elegantly by the fact that they are able to target passenger proteins to the appropriate organelle. The receptor machinery on the outer membranes of chloroplasts and mitochondria is able to discriminate between bona fide precursors and those of the other organelle. For example, precursors for the light-harvesting chlorophyll a/b-binding protein and the 33 kDa photosystem II protein are not imported into plant mitochondria [7,8]. Likewise, the transit peptide of the b-subunit of the F 1 -ATPase (preF 1 b) will target proteins to plant mitochondria in vitro [7,8] and in vivo [9] but not to chloroplasts. On the other hand, there are some dual-targeted proteins, in particular a number of the amino acyl-tRNA synthetases, where the transit peptides direct import into both mitochondria and chloro- plasts with equal efficiency both in vitro and in vivo [10–12]. Despite the importance of these transit peptides in determining the specificity of import, the mechanism of this specificity remains uncertain, although a number of studies have addressed this question. In one investigation, several chloroplast precursors, including the small subunit of ribulose bisphosphate carboxylase/oxygenase (Rubisco) from tobacco and the 23 kDa and 33 kDa oxygen-evolving polypeptides from pea, were incubated with pea cytosol in the presence of [ 32 P]ATP. It was found that they were phosphorylated on a specific serine or threonine residue within the transit peptide [13]. The consensus phosphory- lation sequence (Fig. 1A) resembled the motif for binding of 14-3-3 proteins, and 14-3-3 proteins were shown to bind to the phosphoserine/phosphothreonine site en route to the chloroplast envelope [14], although before import into the chloroplast dephosphorylation occured [13]. Subsequently, it was demonstrated that phosphorylated precursors form a complex with 14-3-3 proteins and a heat shock protein, Hsp70 isoform. This complex was found to increase the rate of translocation into the chloroplast by three to four-fold compared to the free precursor [14], implying that this may act as a Ôguidance complexÕ during the translocation process. In contrast, the mitochondrial precursor preF 1 b and the precursor for peroxisomal malate dehydrogenase were not phosphorylated [13], nor did they associate with 14-3-3 Correspondence to A. G. Smith, Department of Plant Sciences, University of Cambridge, Downing Street, Cambridge, CB2 3EA, UK. Fax: + 44 1223 333953, Tel.: + 44 1223 333952, E-mail: alison.smith@plantsci.cam.ac.uk Abbreviations: GFP, green fluorescent protein; Rubisco, ribulose bisphosphate carboxylase/oxygenase; TSSU.tp.wt, transit peptide of the small subunit of Rubisco from tobacco; PSSU.tp.wt, transit peptide of the small subunit of Rubisco from pea; CtRS.tp.wt, transit peptide of cysteinyl-tRNA synthetase from Arabidopsis thaliana; HtRS.tp.wt, transit peptide of histidyl-tRNA synthetase from Arabidopsis thaliana; CoxIV.tp, transit peptide of cytochrome c oxidase from yeast; wt, wild-type. (Received 1 September 2003, revised 19 November 2003, accepted 1 December 2003) Eur. J. Biochem. 271, 509–516 (2004) Ó FEBS 2004 doi:10.1046/j.1432-1033.2003.03950.x proteins [14]. Furthermore, phosphorylatable precursor proteins translated in a wheat-germ system were stably associated with 14-3-3 proteins, but those translated in a reticulocyte system were not. This provided an attractive possibility as a means of preventing mistargeting of chloroplast precursors to other organelles such as mito- chondria [13]. In this paper we describe experiments conducted to investigate this possibility in vivo.Thetransit peptide of the small subunit of Rubisco (SSU) precursor from tobacco (Nicotiana tabacum) (TSSU.tp.wt), identical to that used in the in vitro studies [13,14], was fused to the green fluorescent protein (GFP) from Aequorea victoria [15], and the phosphorylation site was mutated. After transfor- mation of the construct into plant cells by particle bombardment, the targeting of the GFP by the transit peptide was viewed directly in living cells by confocal microscopy. Similar experiments were performed with the transit peptide of SSU from pea (Pisum sativum) (PSSU.tp.wt), and those of Arabidopsis thaliana cysteinyl- and histidyl-tRNA synthetases (CtRS.tp.wt and HtRS.tp.wt, resepectively), which have been shown to be dual-targeted to chloroplasts and mitochondria [11,12]. Materials and methods Materials Restriction enzymes, T4 DNA ligase and polymerase, and dNTPs came from GibcoBRL, Life Technologies (Paisley, UK) or New England BioLabs Inc. (Hitchin, UK). GFP- containing plasmids (pOL-GFP.LT, pCoxIV-GFP, pCtRS- GFP and pHtRS-GFP) encoding GFP, yeast CoxIV-GFP and Arabidopsis thaliana cysteinyl-tRNA synthetase-GFP and histidyl-tRNA synthetase-GFP, were obtained from I. Small (INRA, Evry, France), together with a reverse primer for GFP to enable sequencing of the border of the fusion constructs. The QuikChange Site-Directed Mutagen- esis Kit was from Stratagene (La Jolla, CA, USA). Tungsten microcarriers, macrocarriers, stopping screens and rupture disks were purchased from Bio-Rad Laboratories Ltd. (Hemel Hempstead, UK). Components for in vitro tran- scription, wheat germ extract and amino acids were obtained from Promega (Madison, WI, USA). L -[ 35 S]methi- onine/cysteine PRO-Mix TM was purchased from Amersham Pharmacia (Chalfont St.Giles, Bucks, UK). Oligonucleotide primers were synthesized by MWB-Biotech AG (Ebersberg, Germany) or Invitrogen Life Technologies (Paisley, UK). Generation of fusion-protein constructs and site-directed mutagenesis The SSU transit peptide of tobacco ([16]; accession number PSRBCS3A) was fused in frame with GFP (encoding solubility-modified red-shifted GFP) [17], into the KpnI/SalI sites of pOL-GFP.LT [12], yielding pTSSU.tp.wt-GFP (Table 1). Similarly, the pea SSU transit peptide sequence ([18]; accession number P07689) was inserted in frame into the KpnI/SphI sites of pUC18-GFP, yielding pPSSU.tp.wt- GFP (Table 1). Constructs were verified by DNA sequence analysis. Site-directed mutagenesis of these chloroplast transit peptides, together with those for histidyl-tRNA synthetase (HtRS) and cysteinyl-tRNA synthetase (CtRS) was designed and performed according to the guidelines sug- gested in the manual of the Stratagene QuikChange TM Site- Directed Mutagenesis Kit. For each transit peptide, a single mutant was constructed, in which the phosphorylated serine or threonine residue was altered to an alanine, and also a double mutant where the upstream serine was also changed to an alanine (Table 1). All mutations were verified by DNA sequence analysis. Further details of cloning and primers used for mutagenesis are available on request from A. G. Smith (University of Cambridge). Import assays into isolated chloroplasts in vitro For chloroplast import experiments in vitro, the constructs encoding the wild-type and modified transit peptides fused to GFP were subcloned into pBluescript, such that the genes were under the control of the T7 or T3 promoter. Methods Table 1. Plasmids generated in this study. Name Encoding Mutation pTSSU.tp.wt-GFP Transit peptide of tobacco SSU fused to GFP None pTSSU.tp.S34A-GFP As above Serine 34 changed to alanine pTSSU.tp.S31A/S34A-GFP As above Serine 31 and serine 34 changed to alanine pPSSU.tp.wt-GFP Transit peptide of pea SSU fused to GFP None pPSSU.tp.T34A-GFP As above Threonine 34 changed to alanine pPSSU.tp.S32A/T34A-GFP As above Serine 32 and threonine 34 changed to alanine pCtRS.tp.wt-GFP Transit peptide of cysteinyl-tRNA synthetase fused to GFP None pCtRS.tp.S22A-GFP As above Serine 22 changed to alanine pCtRS.tp.S21A/S22A-GFP As above Serine 21 and serine 22 changed to alanine pHtRS.tp.wt-GFP Transit peptide of histidyl-tRNA synthetase fused to GFP None pHtRS.tp.S52A-GFP As above Serine 52 changed to alanine pHtRS.tp.S50A/S52A-GFP As above Serine 50 and serine 52 changed to alanine 510 K A. Nakrieko et al.(Eur. J. Biochem. 271) Ó FEBS 2004 for in vitro transcription, in vitro translation and isolation of chloroplasts were as described [19]. Expression of GFP-fusion constructs in vivo For transient expression in plant tissues of the GFP-fusion protein constructs in pUC18-GFP or pOL-GFP.LT, tobacco (Nicotiana tabacum) and pea (Pisum sativum)plants were grown as described [19], and onion (Allium cepa)was obtained from a local market. The constructs were intro- duced into the plant material by biolistic transformation, and the location of GFP fluorescence was determined by confocal microscopy as described [19]. Results Generation of fusion constructs and mutagenesis of the phosphorylation signal The consensus phosphorylation site in chloroplast transit peptides has been identified as (P/G)X n (K/R)X n (S/ T)X n (S*/T*) [13], where the asterisk indicates the site of phosphorylation and n is a spacer of 0–3 residues (Fig. 1A). Figure 1B indicates the phosphorylation motifs in the transit peptides of tobacco and pea preSSU, and the dual- targeted CtRS and HtRS from Arabidopsis. For each transit peptide, two mutant forms were generated – one in which the phosphorylated threonine or serine was altered to an alanine, and a double mutant, where the upstream serine was also altered to an alanine. This serine has been suggested to affect the efficiency of phosphorylation [13], and might also be able to be phosphorylated itself. The sequences encoding the wild-type and mutant forms of the transit peptides were fused in frame to the cDNA encoding GFP such that the fusion proteins were under the control of the ubiquitous CaMV 35S promoter and nopaline synthase (nos) terminator (Fig. 1C). Import of TSSU.tp-GFP fusion proteins into isolated chloroplasts in vitro Mutation of the phosphorylated serine in the transit peptide of tobacco SSU did not alter the efficiency of import into isolated chloroplasts in vitro [13]. We wanted to ensure that our constructs, in which the mature small subunit had been replaced with GFP, behaved similarly, before we carried out our experiments in vivo. Accordingly, the constructs enco- ding the fusion proteins were subcloned into a vector for transcription in vitro, and radiolabelled fusion proteins were made by translation into a wheat germ system in the presence of [ 35 S]methionine and [ 35 S]cysteine. The radio- labelled precursors were incubated with isolated pea chlo- roplasts, followed by reisolation of the organelles, and the proteins were analysed by SDS/PAGE and fluorography (Fig. 2). Figure 2A shows the results for GFP alone. The translation product of 27 kDa, corresponding to the size of GFP, does not associate with chloroplasts (+ Cp). In contrast, the 33 kDa precursor of pTSSU.tp.wt-GFP is imported into the chloroplast and processed to the size of GFP alone (Fig. 2B). The two mutant forms of the tobacco SSU transit peptide (Fig. 2C,D) behave like the wild-type, andineachcase, 2% of added precursor is imported (estimated by densitometry). We performed time-course experiments to investigate the rate of import, where we observed no difference between the constructs encoding the single and double mutants and the wild-type transit peptides (data not shown). Using the same approach, we tested the wild-type and mutant forms of the pea SSU transit peptide. Again, identical import reactions in vitro were observed for all three constructs. From these experiments we can conclude that the presence of the GFP as passenger protein, rather than the mature SSU protein, did not affect the ability of the SSU transit peptide to target proteins to chloroplasts in vitro. Furthermore, mutations in the transit peptide had no significant effect on this ability. Fig. 1. Identification of a phosphorylation motif in the transit peptides of tobacco SSU, pea SSU and dual-targeted Arabidopsis CtRS and HtRS. (A) The consensus phosphorylation motif described by Waegeman and Soll [13]. (B) The transit peptides of tobacco and pea SSU and Arabidopsis CtRS and HtRS, with the phosphorylation motif underlined and the predicted site of phosphorylation marked with an asterisk. (C) Schematic of the GFP-fusion constructs with the individual transit peptides (TP), such that the genes were under the control of the CaMV 35S promoter and nopaline synthase (nos)terminator. Ó FEBS 2004 Chloroplast targeting fidelity (Eur. J. Biochem. 271) 511 Targeting of GFP in vivo by wild-type and mutated transit peptides of tobacco SSU The ability of the SSU transit peptides to target GFP in vivo was investigated by transient expression in plant tissues. The constructs in pUC18-GFP or pOL-GFP.LT were intro- duced into four-week old tobacco leaves by biolistic transformation, and after 16–24 h in the dark at 25 °C, cells exhibiting GFP fluorescence were identified by epiflu- orescence. These were examined further by confocal micro- scopy (Fig. 3). For each construct, the GFP fluorescence, chlorophyll autofluorescence, and the overlay of the two, are shown in a single guard cell; the other cell of the pair was not transformed. As expected, when GFP alone is expressed, it is found throughout the cytosol and in the nucleoplasm, but is excluded from the chloroplasts (Fig. 3A). In contrast, GFP expressed as a fusion protein with the wild-type tobacco SSU transit peptide is found exclusively in chloroplasts, as evidenced by the exact superposition of GFP and chlorophyll fluorescence in the overlay (Fig. 3B). The single and double mutants of this transit peptide, TSSU.tp.S34A-GFP and TSSU.tp.S31A/ S34A-GFP give essentially identical patterns (Fig. 3C,D); all the GFP fluorescence was localized in chloroplasts, and none was seen in either the cytosol or other organelles, such as mitochondria. A punctate pattern of GFP fluorescence in much smaller organelles, as demonstrated in Fig. 3E, is seen where the cell is expressing mitochondrially targeted CoxIV.tp-GFP [12]. In order to ensure that this pattern was representative of the targeting properties of the transit peptides in other cells, the targeting properties of TSSU.tp.wt, TSSU.tp.S31A and TSSU.tp.S31A/S34A were observed in onion epidermal cells, which are nonphoto- synthetic (Fig. 4). Although there is no chlorophyll fluor- escence, the plastids can be identified by the virtue of stromules [20], clearly visible as long protrusions (arrowed) from the plastids in the higher magnification pictures (Fig. 4B,D,F). Effect of alteration of phosphorylation site on GFP-targeting by transit peptides of pea SSU and dual-targeted CtRS & HtRS Our results with the tobacco SSU transit peptide constructs were reproducible and clearly demonstrated that alteration of the phosphorylation signal had no effect on the efficacy or specificity of targeting in vivo. To determine if this were true for other transit peptides, we chose three others to investigate using the same approach. The transit peptide for pea SSU has 64% sequence identity to the tobacco SSU, with the phosphorylation motif at an identical position. In addition, we chose the transit peptides of two amino Fig. 3. Targeting of tobacco SSU-GFP fusion proteins (pTSSU.tp.wt- GFP, pTSSU.tp.S34A-GFP and pTSSU.tp.S31A/S34A-GFP) in tobacco guard cells in vivo. In each panel, the left column is a false- colour image of the GFP channel, the middle column is chlorophyll autofluorescence and the right column is the GFP and chlorophyll channels superimposed. All images are multiprojections of six or eight scans of the depth of a whole tobacco guard cell. (A) GFP alone. (B) pTSSU.tp.wt-GFP. The GFP is clearly targeted to the chloroplasts as the GFP fluorescence overlays precisely with that of the chlorophyll autofluorescence. (C) Mutant transit peptide pTSSU.tp.S34A-GFP. (D) The double mutant transit peptide pTSSU.tp.S31A/S34A-GFP. (E) A mitochondrial-targeted CoxIV-GFP [12] is shown where the typical punctate pattern of these smaller organelles is apparent. The scale bar in all images is 10 lm. Fig. 2. Import experiment with isolated chloroplasts and tobacco SSU- GFP fusion proteins. (A) Incubation of the translation product of GFP (27 kDa), and with isolated pea chloroplasts (+ Cp). (B) Incubation of the 33 kDa precursor of pTSSU.tp.wt-GFP (Twt), and with isolated chloroplasts. In this case, the precursor is imported into chloroplasts and processed to the size of GFP alone. (C) and (D) Incubation of GFP fused to the two mutant forms of the tobacco SSU transit peptide (pTSSU.tp.S34A-GFP and pTSSU.tp.S31A/S34A), and with isolated pea chloroplasts. Import is essentially the same as for the wild-type transit peptide. 512 K A. Nakrieko et al.(Eur. J. Biochem. 271) Ó FEBS 2004 acyl-tRNA synthetases, CtRS and HtRS, from A. thaliana. These proteins have been shown to be dual-targeted both in vivo and in vitro [10–12], so the transit peptides (65 and 73 residues long, respectively) must contain targeting informa- tion for both mitochondria and chloroplasts. They both contain phosphorylation motifs, but these are not in equivalent positions: in CtRS it is in the first third of the sequence (residues 17–22), whereas in HtRS it is towards the end, at position 48–52 (Fig. 1B). For each of these three transit peptides, both the phosphoacceptor residue and the upstream serine were mutated to alanine to generate single and double mutants (Table 1). These constructs were introduced into pea or tobacco guard cells by biolistic transformation and the location of the GFP fluorescence viewed by confocal microscopy (Fig. 5). The results with the pea SSU transit peptides were identical to those for tobacco SSU transit peptides. Alteration of the phosphorylation site did not impede targeting of the passenger GFP to the chloroplasts, nor was there any mistargeting to mitochondria. The pattern of GFP fluorescence after targeting by either CtRS.tp.wt or HtRS.tp.wt differed from that with SSU.tp. As well as being in large round organelles that coincided with the chlorophyll fluorescence, it was also seen in small punctate organelles that correspond to mitochondria (com- pare with Fig. 3E). Again, modification of the phosphory- lation motif had no effect on the efficacy of chloroplast targeting, or indeed to mitochondria. Identical results were obtained using Arabidopsis leaf material for biolistic trans- formation (data not shown). Discussion In this paper, we have used the ability to image GFP fluorescence in living plant tissue by confocal microscopy, in order to test the role of a phosphorylation motif in the transit peptides of several precursor proteins. This phos- phorylation motif has been shown to be necessary to form a complex with 14-3-3 proteins and Hsp70, which make the precursor more import-competent [14]. This charac- teristic has been proposed as a possible means of ensuring specificity for chloroplast import. However, our results demonstrate that removal of the phosphorylation motif from the transit peptides of tobacco and pea SSU did not Fig. 4. The effect of mutagenesis of the phosphorylation site in the transit peptide of tobacco SSU on the targeting of GFP fusion proteins in onion epidermal cells. The tobacco SSU.tp-GFP constructs were transiently expressed in nonphotosynthetic onion epidermis. The figures are multi- projections of 14 or 16 scans through two adjacent cells expressing the fusion proteins, superimposed on a single bright field scan, allowing the outline of the cells to be visualized easily. (A) and (B) pTSSU.tp.wt-GFP. (C and D) pTSSU.tp.S34A-GFP. (E and F) pTSSU.tp.S31A/S34A-GFP. For each construct, GFP is localized in plastids, which are easily identified as such by the presence of stromules [20], indicated by the arrows in the higher magnification images. The scale bar is 50 lm in the images on the left, and 10 lm in the higher magnification images from regions in (A), (C) and (E), shown on the right. Ó FEBS 2004 Chloroplast targeting fidelity (Eur. J. Biochem. 271) 513 prevent accurate targeting of the passenger GFP to plastids in either leaf cells (Figs 3 and 5A) or nonphoto- synthetic cells (Fig. 4). Similarly, this caused no alteration in the dual-targeting to chloroplasts and mitochondria by the transit peptides of A. thaliana CtRS and HtRS (Fig. 5B,C). We therefore conclude that this signal is not involved in determining the specificity of import into chloroplasts. Instead, the guidance complex may be important to ensure high rates of translocation for highly expressed chloroplast proteins, or to prevent the accumulation of nonimport competent protein in the cytosol. Cytosolic chaperones, mitochondrial import stimulating factor [21] (now known to be a 14-3-3 protein [22]) and presequence binding factor [23], have been proposed to prevent aggre- gation of mitochondrial precursors in the cytosol [24]. Interestingly, although many chloroplast-targeted proteins have the motif, it is not present in all plastid-targeted transit peptides. For instance, it is not present in the transit peptides for light-harvesting chlorophyll a/b binding proteins from pea and Arabidopsis, although as these are very hydro- phobic proteins they might be a special case. It is absent from the transit peptide of ferredoxin from Silene pratensis, a soluble stromal protein, whereas ferredoxins from other higher plants do contain the motif [25]. A notable group of proteins that do not contain the motif are the type-2 ferrochelatases (the terminal enzyme of haem biosynthesis), which are targeted exclusively to chloroplasts in vitro [26,27]. In contrast, the type-1 ferrochelatases, which are imported into both chloroplasts and mitochondria in vitro [26,28], contain the phosphorylation motif. In fact, import into some isolated plant mitochondria has been shown to not be robust, as photosynthetic protein precursors like plastocyanin are imported and processed [19,29]. In an attempt to overcome this apparent lack of specificity, a competitive import assay was developed [30], in which isolated mitochondria and chloroplasts from pea are mixed together and incubated with the precursor proteins, and then the organelles are re-separated. The authors report that this allowed them to distinguish genuinely dual- targeted precursors from chloroplast- or mitochondria- destined precursors. In this study we have taken an alternative approach using GFP as a marker to track targeted proteins in vivo. The fact that GFP can be used to image the location of targeted proteins in living tissue avoids the potential artefacts of in vitro systems, in particular their lack of specificity. Furthermore, the stability of GFP ensures that problems of degradation of mistargeted proteins, which is characteristic of the in vitro system, do not occur. Fig. 5. Effect of mutagenesis of the phosphorylation site in transit peptides of pea SSU, and Arabidopsis CtRS and HtRS on their ability to target GFP in vivo. All images are overlays of the GFP channel and the red chlorophyll fluorescence channel. They are multiprojections of six or eight scans of the depth of the guard cell(s) expressing the GFP-fusion constructs. (A) The targeting of pPSSU.tp.wt-GFP (wild-type), pPSSU.tp.T34A-GFP (single mutant) and pPSSU.tp.S32A/T34A-GFP (double mutant). (B) CtRS.tp.wt-GFP (wild-type), CtRS.tp.S21A-GFP (single mutant) and CtRS.tp.S21A/S22A-GFP (double mutant). (C) Wild-type HtRS (HtRS.tp.wt-GFP), single mutant (HtRS.tp.S50A-GFP) and double mutant (HtRS.tp.S50A/S52A). The scale bar in all images is 10 lm. 514 K A. Nakrieko et al.(Eur. J. Biochem. 271) Ó FEBS 2004 As well as the role of the transit peptide itself, several other processes may play a role in the specificity of targeting to organelles, including interactions with organellar surface lipids, subcellular location of translation, and the receptors at the outer organellar translocon. Lipids that are present at the chloroplast outer envelope and not on the outer membrane of the mitochondria, such as digalactosyl diacylglycerol [31], represent a possible means for chloro- plast precursor discrimination. In yeast, an mRNA-binding protein has been shown to bind to transcripts of mito- chondrial preproteins, directing them to ribosomes in closer proximity to mitochondria [32]. Also in fungi, the acidity of Tom22 at the mitochondrial surface is thought to provide a binding site for basic mitochondrial presequences [33]. The lack of Tom22 in higher plant mitochondria has been proposed as a means of preventing chloroplast precursors from entering mitochondria [33]. 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