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

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 a

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 pre-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 F1-ATPase (preF1b) 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 [32P]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, Hsp70isoform 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 preF1b 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)

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 The transit

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-pCtRS-GFP) encoding pCtRS-GFP, yeast CoxIV-pCtRS-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-MixTMwas 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 QuikChangeTM 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 chloroplastsin 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.

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

for in vitro transcription, in vitro translation and isolation

of chloroplasts were as described [19]

Expression of GFP-fusion constructsin 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)Xn(K/R)Xn(S/

T)Xn(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 chloroplastsin 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 [35S]methionine and [35S]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, and in each case,  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.

Targeting of GFPin 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 SSU-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.

acyl-tRNA synthetases, CtRS and HtRS, from A thaliana.

These proteins have been shown to be dual-targeted both

in vivoand 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.

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.

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] These few examples

illustrate clearly that a complete understanding of organelle

targeting requires a multifaceted approach in order to

integrate studies of transit peptide structure and

character-istics with those of the import machinery and other cellular

factors The approach described in this paper provides a

quick, versatile and unambiguous means of testing the effect

of altering the components of the targeting and import

process

Acknowledgements

We thank Dr Ian Small (INRA, Evry, France) for the GFP plasmids

and Professor Colin Robinson (University of Warwick, UK) for help

with in vitro import experiments We are grateful to the Royal Society

for funding K.-A N was in receipt of a studentship from the

Cambridge Commonwealth Trust.

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