molecular components required for the targeting of pex7 to peroxisomes in arabidopsis thaliana

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Molecular components required for the targeting of PEX7 to peroxisomes in Arabidopsis thaliana Tanuja Singh 1 , Makoto Hayashi 1,2 , Shoji Mano 1,2 , Yuko Arai 1 , Shino Goto 1,2 and Mikio Nishimura 1,2,* 1 Department of Cell Biology, National Institute for Basic Biology, Okazaki 444-8585, Japan, and 2 Department of Basic Biology, The Graduate University for Advanced Studies (Sokendai), Okazaki 444-8585, Japan Received 21 May 2009; revised 21 June 2009; accepted 24 June 2009; published online 24 July 2009. * For correspondence (fax +81 564 55 7505; e-mail [email protected]). SUMMARY PEX7 is a soluble import receptor that recognizes peroxisomal targeting signal type 2 (PTS2)-containing proteins. In the present study, using a green fluorescent protein (GFP) fusion protein of PEX7 (GFP-PEX7), we analyzed the molecular function and subcellular localization of PEX7 in Arabidopsis thaliana. The overexpres- sion of GFP-PEX7 resulted in defective glyoxysomal fatty acid b-oxidation, but had no significant effect on leaf peroxisomal function. Analysis of the subcellular localization of GFP-PEX7 in transgenic Arabidopsis showed that GFP-PEX7 localizes primarily to the peroxisome. Transient expression of a C- or N-terminal fusion protein of PEX7 and yellow fluorescent protein (YFP) (PEX7-YFP and YFP-PEX7, respectively) in leek epidermal cells, using the particle bombardment technique, confirmed that fluorescent protein-tagged PEX7 localizes to peroxisomes in Arabidopsis. Immunoblot analysis revealed that GFP-PEX7 accumulates primarily in peroxisomal membrane fractions, whereas endogenous PEX7 was distributed evenly in cytosolic and peroxisomal membrane fractions, which indicated that both endogenous PEX7 and GFP-PEX7 are properly targeted to peroxisomal membranes. The results of bimolecular fluorescence complementation (BiFC) and yeast two-hybrid analyses showed that PEX7 binds directly to PTS2-containing proteins and PEX12 in the peroxisomal membrane. We used red fluorescent protein (tdTomato) fusion protein of PEX7 (tdTomato-PEX7) in several Arabidopsis pex mutants to identify proteins required for the targeting of PEX7 to peroxisomes in planta. The results demonstrated that pex14, pex13 and pex12 mutations disrupt the proper targeting of PEX7 to peroxisomes. Overall, our results suggest that the targeting of PEX7 to peroxisomes requires four proteins: a PTS2-containing protein, PEX14, PEX13 and PEX12. Keywords: Arabidopsis, peroxisome, peroxisomal targeting signal, PEX7, protein targeting, bimolecular fluorescence complementation. INTRODUCTION Peroxisomes are single-membrane-bound organelles that house important metabolic reactions. Peroxisomes in higher plant cells differentiate into at least three different classes: glyoxysomes, leaf peroxisomes and unspecialized peroxi- somes (Beevers, 1979). Each class of organelle contains a unique set of enzymes that carries out specialized functions in the various organs of higher plants. Glyoxysomes are present in the cells of storage organs, such as endosperms and cotyledons, during the post-germinative growth of oil- seed plants, as well as being present in senescent organs (Nishimura et al., 1996). They contain enzymes for fatty acid b-oxidation and the glyoxylate cycle, and play a pivotal role in the conversion of seed-reserved lipid into sucrose. It has been suggested that fatty acids produced from lipids are exclusively degraded in glyoxysomes during germination and post-germinative growth in plants (Beevers, 1982). By contrast, leaf peroxisomes are widely distributed in the cells of photosynthetic organs. Some of the enzymes responsible for photorespiration are localized in leaf peroxisomes, even though the entire photorespiratory process involves a combination of enzymatic reactions that occur in chlorop- lasts, leaf peroxisomes and mitochondria (Tolbert, 1982). Peroxisomal proteins are encoded by nuclear genes, are synthesized on free polysomes in the cytosol, and are then post-translationally imported into the peroxisome (Lazarow and Fujiki, 1985; Lazarow, 2003). Protein sorting to peroxi- somes is mediated by specific peroxisomal targeting signals (PTSs). Two common PTSs, types 1 and 2, have been identified on peroxisomal matrix proteins (Heiland and Erdmann, 2005; Hayashi and Nishimura, 2006; Platta and 488 ª 2009 The Authors Journal compilation ª 2009 Blackwell Publishing Ltd The Plant Journal (2009) 60, 488–498 doi: 10.1111/j.1365-313X.2009.03970.x

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Page 1: Molecular components required for the targeting of PEX7 to peroxisomes in Arabidopsis thaliana

Molecular components required for the targeting ofPEX7 to peroxisomes in Arabidopsis thaliana

Tanuja Singh1, Makoto Hayashi1,2, Shoji Mano1,2, Yuko Arai1, Shino Goto1,2 and Mikio Nishimura1,2,*

1Department of Cell Biology, National Institute for Basic Biology, Okazaki 444-8585, Japan, and2Department of Basic Biology, The Graduate University for Advanced Studies (Sokendai), Okazaki 444-8585, Japan

Received 21 May 2009; revised 21 June 2009; accepted 24 June 2009; published online 24 July 2009.*For correspondence (fax +81 564 55 7505; e-mail [email protected]).

SUMMARY

PEX7 is a soluble import receptor that recognizes peroxisomal targeting signal type 2 (PTS2)-containing

proteins. In the present study, using a green fluorescent protein (GFP) fusion protein of PEX7 (GFP-PEX7), we

analyzed the molecular function and subcellular localization of PEX7 in Arabidopsis thaliana. The overexpres-

sion of GFP-PEX7 resulted in defective glyoxysomal fatty acid b-oxidation, but had no significant effect on leaf

peroxisomal function. Analysis of the subcellular localization of GFP-PEX7 in transgenic Arabidopsis showed

that GFP-PEX7 localizes primarily to the peroxisome. Transient expression of a C- or N-terminal fusion protein

of PEX7 and yellow fluorescent protein (YFP) (PEX7-YFP and YFP-PEX7, respectively) in leek epidermal cells,

using the particle bombardment technique, confirmed that fluorescent protein-tagged PEX7 localizes to

peroxisomes in Arabidopsis. Immunoblot analysis revealed that GFP-PEX7 accumulates primarily in

peroxisomal membrane fractions, whereas endogenous PEX7 was distributed evenly in cytosolic and

peroxisomal membrane fractions, which indicated that both endogenous PEX7 and GFP-PEX7 are properly

targeted to peroxisomal membranes. The results of bimolecular fluorescence complementation (BiFC) and

yeast two-hybrid analyses showed that PEX7 binds directly to PTS2-containing proteins and PEX12 in the

peroxisomal membrane. We used red fluorescent protein (tdTomato) fusion protein of PEX7 (tdTomato-PEX7)

in several Arabidopsis pex mutants to identify proteins required for the targeting of PEX7 to peroxisomes in

planta. The results demonstrated that pex14, pex13 and pex12 mutations disrupt the proper targeting of PEX7

to peroxisomes. Overall, our results suggest that the targeting of PEX7 to peroxisomes requires four proteins: a

PTS2-containing protein, PEX14, PEX13 and PEX12.

Keywords: Arabidopsis, peroxisome, peroxisomal targeting signal, PEX7, protein targeting, bimolecular

fluorescence complementation.

INTRODUCTION

Peroxisomes are single-membrane-bound organelles that

house important metabolic reactions. Peroxisomes in higher

plant cells differentiate into at least three different classes:

glyoxysomes, leaf peroxisomes and unspecialized peroxi-

somes (Beevers, 1979). Each class of organelle contains a

unique set of enzymes that carries out specialized functions

in the various organs of higher plants. Glyoxysomes are

present in the cells of storage organs, such as endosperms

and cotyledons, during the post-germinative growth of oil-

seed plants, as well as being present in senescent organs

(Nishimura et al., 1996). They contain enzymes for fatty acid

b-oxidation and the glyoxylate cycle, and play a pivotal role

in the conversion of seed-reserved lipid into sucrose. It has

been suggested that fatty acids produced from lipids are

exclusively degraded in glyoxysomes during germination

and post-germinative growth in plants (Beevers, 1982). By

contrast, leaf peroxisomes are widely distributed in the cells

of photosynthetic organs. Some of the enzymes responsible

for photorespiration are localized in leaf peroxisomes, even

though the entire photorespiratory process involves a

combination of enzymatic reactions that occur in chlorop-

lasts, leaf peroxisomes and mitochondria (Tolbert, 1982).

Peroxisomal proteins are encoded by nuclear genes, are

synthesized on free polysomes in the cytosol, and are then

post-translationally imported into the peroxisome (Lazarow

and Fujiki, 1985; Lazarow, 2003). Protein sorting to peroxi-

somes is mediated by specific peroxisomal targeting signals

(PTSs). Two common PTSs, types 1 and 2, have been

identified on peroxisomal matrix proteins (Heiland and

Erdmann, 2005; Hayashi and Nishimura, 2006; Platta and

488 ª 2009 The AuthorsJournal compilation ª 2009 Blackwell Publishing Ltd

The Plant Journal (2009) 60, 488–498 doi: 10.1111/j.1365-313X.2009.03970.x

Page 2: Molecular components required for the targeting of PEX7 to peroxisomes in Arabidopsis thaliana

Erdmann, 2007a). Most peroxisomal matrix proteins are

targeted by PTS1, a C-terminal tripeptide. In higher plant

cells, permissible combinations of tripeptide sequences for

PTS1 include (C/A/S/P)-(K/R)-(I/L/M) (Hayashi et al., 1996).

PTS2 is within a cleavable N-terminal presequence that has

the consensus sequence (R)-(A/L/Q/I)-X5-(H)-(L/I/F) (where X

represents any amino acid) (Kato et al., 1996). Recently it

was reported that the Arabidopsis genome contains almost

300 genes that encode proteins with either PTS1 or PTS2

sequences (Kamada et al., 2003; Reumann et al., 2004,

2007).

PEX5 and PEX7 are cytosolic import receptors that

recognize PTS1 and PTS2, respectively (Subramani, 1998;

Baker and Sparkes, 2005; Hayashi and Nishimura, 2006;

Brown and Baker, 2008). Yeast two-hybrid and co-immuno-

precipitation analyses have demonstrated that PEX5 binds

directly to PTS1, that PEX7 binds directly to PTS2, and that

there is a direct interaction between PEX5 and PEX7 in

Arabidopsis. From these results, we proposed the model of

protein import into plant peroxisomes, in which a receptor–

cargo complex is formed in the cytosol, consisting of PEX5,

PEX7, a PTS1-containing protein and a PTS2-containing

protein (Nito et al., 2002). The receptor–cargo complex is

then captured by the interaction of PEX14 with PEX5 on the

peroxisomal membrane (Hayashi et al., 2000; Nito et al.,

2002). This model suggested that the import of PTS1-

containing proteins depends only on PEX5, whereas the

import of PTS2-containing protein depends on PEX5 and

PEX7 (Nito et al., 2002; Woodward and Bartel, 2005). In

support of this model, the Arabidopsis pex5 mutant exhibits

defects in the import of both PTS1- and PTS2-containing

proteins, whereas the pex7 mutant exhibits a defect in the

import of PTS2-containing proteins (Hayashi et al., 2005). In

mammals, two isoforms of PEX5, the long and short

isoforms, are produced from two splice variants of the

PEX5 gene. The targeting of PTS2-containing proteins by

PEX7 requires binding to the long isoform of PEX5 (Braver-

man et al., 1998; Matsumura et al., 2000; Otera et al., 2000).

By comparison, Arabidopsis PEX5, the PEX7 binding partner

in plants, is similar to that of the long form of mammalian

PEX5. It is noteworthy that Arabidopsis has a single form of

PEX5 (Mullen et al., 2001). In fungi, PEX7 does not require

PEX5 for targeting to peroxisomes, but requires other

proteins, such as PEX18 and PEX21 in Saccharomyces

cerevisiae (Purdue et al., 1998) and PEX20 in Neurospora

crassa (Sichting et al., 2003), Yarrowia lipolytica (Einwachter

et al., 2001) and Pichia pastoris (Leon et al., 2006).

Following the docking of receptor–cargo complexes on

the peroxisomal membrane, and the delivery of cargo into

peroxisomes, it is thought that the two receptors, PEX5 and

PEX7, are recycled back to the cytosol (the ‘simple shuttle’

mechanism; Dodt and Gould, 1996; Marzioch et al., 1994).

However, recent studies in yeast and mammals support an

‘extended shuttle’ mechanism, in which the receptors enter

the peroxisome together with their cargo, release the cargo

in the lumen of the peroxisome and are then recycled back

to the cytosol to participate in further rounds of import. The

details of the targeting/recycling mechanism are still under

debate; however, a commonly agreed upon feature is the

requirement of peroxisomal membrane proteins, the

so-called peroxins, which are encoded by a number of PEX

genes (Dammai and Subramani, 2001; Nair et al., 2004;

Lazarow, 2006; Thomas and Erdmann, 2006; Platta and

Erdmann, 2007b). In plants, PEX1, PEX2, PEX4, PEX6,

PEX10, PEX12, PEX13 and PEX14 have been suggested to

be involved in the mechanism of peroxisomal import, based

on the analysis of transgenic Arabidopsis plants in which

PEX gene expression was suppressed by RNA interference

(Nito et al., 2007). However, little is known about the

mechanism underlying the targeting/recycling of PEX7 in

plant cells.

Here, we have used bimolecular fluorescence comple-

mentation (BiFC) and yeast two-hybrid assay, in combina-

tion with the overexpression of fluorescent protein-tagged

PEX7, in various Arabidopsis pex mutants to identify the

molecular components of PEX7 targeting to peroxisomes

in planta. We present direct evidence that the targeting of

PEX7 to peroxisomes requires at least four factors in plant

cells: a PTS2-containing protein, PEX14, PEX13 and PEX12.

Our data also demonstrates that GFP-PEX7 can be a

useful tool to study the mechanism of PEX7 targeting to

peroxisomes.

RESULTS

Effect of GFP-PEX7 on peroxisomal function

To analyze the molecular mechanism of PEX7 function, we

constructed a chimeric gene encoding GFP fused to Ara-

bidopsis PEX7 under the control of the cauliflower mosaic

virus 35S promoter (Figure S1). The gene was inserted into a

Ti vector, and then introduced into wild-type Arabidopsis

(ecotype Columbia) using Agrobacterium tumefaciens.

Transgenic Arabidopsis progeny expressing GFP-PEX7 were

selected and designated as AtGFP-PEX7. We first compared

the phenotypes of AtGFP-PEX7 and wild-type Arabidopsis

grown under normal atmospheric conditions. Previously, it

was shown that plants with a defect in leaf peroxisomal

function exhibit a dwarf phenotype with pale-green leaves

(Hayashi et al., 2000, 2005; Schumann et al., 2007). As

shown in Figure 1(a), the growth of AtGFP-PEX7 was similar

to that of wild-type plants, which indicated that the over-

expression of GFP-PEX7 does not affect leaf peroxisomal

function. It has been reported that plants lacking peroxi-

somal fatty acid b-oxidation become resistant to 2,4-dichlo-

rophenoxybutyric acid (2,4-DB), and require sucrose during

post-germinative growth (Hayashi et al., 1998; Zolman et al.,

2000; Bartel et al., 2001). To determine the status of fatty acid

b-oxidation in transgenic plants, we examined the effect of

Factors required for PEX7 targeting to peroxisomes 489

ª 2009 The AuthorsJournal compilation ª 2009 Blackwell Publishing Ltd, The Plant Journal, (2009), 60, 488–498

Page 3: Molecular components required for the targeting of PEX7 to peroxisomes in Arabidopsis thaliana

2,4-DB and sucrose on the growth of AtGFP-PEX7. When

AtGFP-PEX7 seedlings were grown on medium containing

0.3 lg ml)1 2,4-DB, they developed long roots, which indi-

cated that AtGFP-PEX7 was resistant to 2,4-DB (Figure 1b,

upper panel). In addition, AtGFP-PEX7 seedlings could not

germinate in the absence of sucrose (Figure 1b, middle

panel). It required sucrose for germination. These aspects of

the AtGFP-PEX7 phenotype were identical to the ped1

mutant, which has a defect in the peroxisomal fatty acid

b-oxidation enzyme 3-ketoacyl CoA thiolase (Hayashi et al.,

1998). These results indicated that the overexpression of

GFP-PEX7 in plants results in a defect in glyoxysomal fatty

acid b-oxidation.

Subcellular localization of fluorescent protein-tagged PEX7

To study the relationship between the glyoxysomal defect in

AtGFP-PEX7 and the subcellular localization of GFP-PEX7,

we analyzed AtGFP-PEX7 plants by confocal laser-scanning

microscopy. As shown in Figure 2(a), all of the cells exam-

ined showed punctate green fluorescence, which indicated

that the majority of GFP-PEX7 localizes to peroxisomes in

these cells. To confirm the peroxisomal localization of GFP-

PEX7, we constructed two chimeric cDNAs encoding N- and

C-terminal fusion proteins of Arabidopsis PEX7 and YFP

(YFP-PEX7 and PEX7-YFP, respectively; Figure S1). The chi-

meric cDNAs were placed under the control of the cauli-

flower mosaic virus 35S promoter, and were then introduced

into Allium ampelopresum (Japanese leek) epidermal cells

using the particle bombardment technique. A peroxisomal

marker protein composed of the red fluorescent protein

tdTomato (Shaner et al., 2004) and 10 C-terminal amino acid

residues from hydroxypyruvate reductase (Mano et al.,

2002) (tdTomato-PTS1) was also introduced into cells as a

marker for transformation. Cells were incubated for 18 h

after bombardment, and then the subcellular localization of

the transiently expressed fluorescent fusion proteins was

examined. Both YFP-PEX7 and PEX7-YFP were detected in

the peroxisome (Figure 2), and co-localized with tdTomato-

PTS1 (Figure 2d,g). The placement of YFP at either the N- or

C-terminus of PEX7 did not affect the transport of PEX7 to

peroxisomes. These results were consistent with the sub-

cellular localization of GFP-PEX7 in transgenic Arabidopsis

(Figure 2a), and provided additional evidence that fluo-

rescent fusion proteins of PEX7 are properly targeted to

peroxisomes.

We next analyzed whether fluorescent protein-tagged

PEX7 localized to the peroxisomal membrane or peroxi-

somal matrix (Figure 3). Total tissue homogenate was

prepared from 5-day-old etiolated cotyledons of AtGFP-

PEX7 and wild-type Arabidopsis. The crude homogenate

was then treated separately with low-salt buffer (Figure 3, L;

0.05 M NaCl), high-salt buffer (Figure 3, H; 0.5 M NaCl) or

alkaline solution (Figure 3, A; 0.1 M Na2CO3), and was

then fractionated into supernatant (S) and pellet (P) after

centrifugation. The protein fractions from the AtGFP-PEX7

and the wild-type plants were analyzed by immunoblot

using anti-GFP, anti-PEX7 and anti-PEX5 antibodies.

(a)

(b)

GFP-PEX7

GFP-PEX7 WT ped1

2.4-DB

- Sucrose

GM

WT

Figure 1. Phenotype of GFP-PEX7 transgenic Arabidopsis plants.

(a) Transgenic plants expressing GFP-PEX7 (left) and wild-type Arabidopsis

plants (Columbia, right) were grown in a normal atmosphere under constant

illumination for 3 weeks. The arrowheads indicate the tops of the inflores-

cence apices, indicating the normal growth of GFP-PEX7 similar to wild-type

Arabidopsis.

(b) Effect of 2,4-dichlorophenoxybutyric acid (2,4-DB) and sucrose on the

growth of AtGFP-PEX7, wild-type (WT) Arabidopsis and ped1 mutant seed-

lings. GFP-PEX7, WT and ped1 seedlings were grown for 10 days on growth

medium containing 0.3 lg ml)1 2,4-DB, or on growth medium without

sucrose ()sucrose), under constant illumination. As a control, seedlings were

grown on growth medium (GM) with sucrose. The seedlings were removed

from the media and rearranged on agar plates before being photographed.

Scale bar: 1.5 cm.

490 Tanuja Singh et al.

ª 2009 The AuthorsJournal compilation ª 2009 Blackwell Publishing Ltd, The Plant Journal, (2009), 60, 488–498

Page 4: Molecular components required for the targeting of PEX7 to peroxisomes in Arabidopsis thaliana

As shown in Figure 3(a), anti-GFP antibody (a-GFP) revealed

that a 69-kDa protein corresponding to GFP-PEX7 was

detected primarily in the pellet, even after treatment with

alkaline solution (Figure 3a, arrowhead in a-GFP). A small

quantity of protein was also detected in the supernatant.

These results indicated that GFP-PEX7 accumulates in the

peroxisomal membrane. In contrast, a 43-kDa protein corre-

sponding to endogenous PEX7 from AtGFP-PEX7 was

present in approximately equal quantities in the supernatant

and pellet, even after treatment with alkaline solution

(Figure 3a, arrowhead in a-PEX7), which suggested that

endogenous PEX7 is present in two subcellular compart-

ments: the cytosol and the peroxisomal membrane. These

results were consistent with previous findings that PEX7 is

targeted to peroxisomes, and is then recycled back to the

cytosol in mammals and yeast (Mukai et al., 2002; Nair et al.,

2004). As a control, 3-ketoacyl-CoA thiolase and peroxisomal

ascorbate peroxidase were analyzed as markers for the

peroxisomal matrix and the peroxisomal membrane,

respectively (Figure 3a, a-thiolase and a-APX). These results

suggested that GFP-PEX7 is properly targeted to peroxi-

somes, similar to endogenous PEX7, but that the addition of

GFP inhibits the last step of the peroxisomal protein import

pathway: the recycling of PEX7 from the peroxisomal

membrane to the cytosol. In 5-day-old etiolated cotyledons

of wild-type plants, 43-kDa PEX7 and 99-kDa PEX5 were

detected in both supernatant and pellet fractions (Figure 3b,

arrowheads in a-PEX7 and a-PEX5), whereas in AtGFP-PEX7,

(a)

(b) (c) (d)

(e) (f) (g)

Figure 2. Subcellular localization of fluorescence-conjugated PEX7.

(a) A 10-day-old transgenic plant expressing GFP-PEX7 in root cells was

grown on growth medium under constant illumination, and then imaged by

confocal laser scanning microscopy. The green color shows the location of

GFP-PEX7 in peroxisomes (indicated by an arrowhead). Scale bar: 5 lm.

(b–g) Transient expression of YFP-PEX7 and PEX7-YFP in leek epidermal cells.

YFP-PEX7 (b–d), or PEX7-YFP along with tdTomato-PTS1 (e–g), was intro-

duced into leek epidermal cells. The green color indicates YFP-PEX7 and

PEX7-YFP in the peroxisome (b, e); the red color indicates tdTomato-PTS1 in

the peroxisome (c, f); (d) the images in (b) and (c) were merged to generate a

composite image; (g) the images in (e) and (f) were merged to create a

composite image. Images were obtained by confocal laser scanning micros-

copy. Scale bar: 10 lm.

(a)

(b)

Figure 3. Subcellular localization of GFP-PEX7 and endogenous PEX7.

(a) The total homogenate prepared from 5-day-old etiolated cotyledons of

AtGFP-PEX7 was treated with low-salt buffer (L), high-salt buffer (H) or an

alkaline solution (A), and was then separated into supernatant (S) and pellet

(P). T represents the total fraction. An immunoblot analysis was performed

using antibodies raised against GFP (a-GFP), PEX7 (a-PEX7), PEX5 (a-PEX5), 3-

ketoacyl-CoA thiolase (a-thiolase) and peroxisomal ascorbate peroxidase (a-

APX). 3-Ketoacyl-CoA thiolase and peroxisomal ascorbate peroxidase were

used as markers of the peroxisomal matrix and peroxisomal membrane,

respectively.

(b) Total homogenate prepared from 5-day-old etiolated cotyledons of wild-

type Arabidopsis was treated with low-salt buffer (L), high-salt buffer (H) or an

alkaline solution (A), as above, and was then separated into supernatant (S)

and pellet (P). Immunoblot analysis was performed using antibodies raised

against PEX7 (a-PEX7) and PEX5 (a-PEX5).

Factors required for PEX7 targeting to peroxisomes 491

ª 2009 The AuthorsJournal compilation ª 2009 Blackwell Publishing Ltd, The Plant Journal, (2009), 60, 488–498

Page 5: Molecular components required for the targeting of PEX7 to peroxisomes in Arabidopsis thaliana

PEX5 was mostly detected in the pellet fractions, with only a

small quantity detected in the supernatant fractions

(Figure 3a, arrowhead in a-PEX5), indicating that PEX5 is

trapped on the membrane together with GFP-PEX7 in

AtGFP-PEX7.

Detection of complex formation between PEX7 and a

PTS2-containing protein in living plant cells using BiFC

Previously, the binding of Arabidopsis PEX7 to PTS2 was

demonstrated using the yeast two-hybrid system (Nito et al.,

2002). To determine the subcellular localization of protein

complexes composed of PEX7 and PTS2-containing

proteins in plant cells, we carried out a series of BiFC assays

using N- and C-terminal fragments of YFP (Bracha-Drori

et al., 2004; Walter et al., 2004; Citovsky et al., 2006; Ohad

et al., 2007). An interaction between two proteins that

are fused to two different YFP fragments results in the

reconstitution of a functional YFP molecule with yellow

fluorescence. We constructed two chimeric genes encoding

the N-terminal half of YFP fused to PEX7, and the N-terminal

48 amino acid residues of citrate synthase, which contains a

PTS2 (Kato et al., 1996), fused to the C-terminal half of

YFP. These chimeras were designated as nYFP-PEX7

and PTS2-cYFP, respectively (Figure S1). We transiently

expressed nYFP-PEX7 and PTS2-cYFP in leek epidermal cells

together with tdTomato-PTS1 using the particle bom-

bardment technique (Figure 4a–c). As shown in Figure 4(a),

YFP fluorescence (as indicated by the green pseudocolor)

was observed exclusively in peroxisomes, and the signal

co-localized perfectly with the red fluorescence of tdTomato-

PTS1 (Figure 4b,c). These results demonstrated that

fluorescent protein-tagged PEX7 is targeted to the peroxi-

some together with a PTS2-containing protein.

To confirm these results, we substituted a conserved

arginine (arginine 16) of the PTS2 sequence of PTS2-cYFP

with Glycine (R16G) to create PTS2*-cYFP. This mutation has

been shown to disrupt the peroxisomal targeting of PTS2

(Kato et al., 1996). When PTS2*-cYFP was co-expressed with

nYFP-PEX7 together with tdTomato (a cytosolic marker for

transformation; Figure 4e) or tdTomato-PTS1 (a peroxi-

somal marker for transformation; Figure 4h), weak YFP-

positive signals were detected in the cytosol (Figure 4d,g).

These results suggested that the peroxisomal targeting of

PEX7 requires an intact PTS2 sequence in the cargo protein.

We next examined whether there was a direct interaction

between PTS1- and PTS2-containing proteins during import

using BiFC (Figure 4j). We constructed a gene chimera

encoding nYFP and PTS1 (nYFP-PTS1). When nYFP-PTS1

and PTS2-cYFP were co-expressed together with tdTomato-

PTS1, YFP fluorescence was detected in the peroxisome

(Figure 4j), and co-localized with tdTomato-PTS1 (Fig-

ure 4k,l). These results were in agreement with the model

in which receptor–cargo complexes composed of PEX5,

PEX7, a PTS1-containing protein and a PTS2-containing

protein are targeted to peroxisomes during import. We

carefully examined control cells that expressed nYFP-PEX7

and cYFP (Figure 4m), nYFP and PTS2-cYFP (Figure 4p),

nYFP-PTS1 and cYFP, and PTS2*-cYFP and nYFP (data

not shown), and none of the cells exhibited significant levels

of YFP fluorescence, which indicated that the reconstitution

of YFP fluorescence was indeed the result of complex

formation between the two fusion proteins.

Furthermore, to show that the expression of protein is

occurring without the reconstitution of YFP fluorescence, we

transiently expressed nYFP fused to mRFP1 (red fluorescent

protein; nYFP-mRFP1) or cYFP fused to mRFP1 (cYFP-

mRFP1) in leek epidermal cells. As shown in Figure S2(a,b),

YFP fluorescence was then detected in cells with a signifi-

cant red fluorescent signal.

Subcellular localization of tdTomato-PEX7 in ped2, apm2

and pex12i mutants

To determine whether there were additional factors required

for PEX7 targeting to the peroxisomal membrane, we ana-

lyzed the subcellular localization of tdTomato-PEX7 in root

cells of several Arabidopsis pex mutants. It has been

reported that mutants with nucleotide substitutions in

PEX14 and PEX13 are defective in peroxisomal function.

These mutants are designated ped2 and apm2 (Hayashi

et al., 2000; Mano et al., 2006). Furthermore, Nito et al.

(2007) recently reported reduced PEX12 function in the RNAi

knock-down mutant pex12i. In this mutant, GFP-PTS1 fails to

localize properly, and accumulates in the cytosol (Nito et al.,

2007). We introduced tdTomato-PEX7 into the root cells of

wild-type Arabidopsis, and into pex14, pex13 and pex12i

mutants, by particle bombardment (Figure 5). tdTomato-

PEX7 localized to the peroxisomes in wild-type plants

(Figure 5, WT), consistent with the subcellular localization of

GFP-PEX7 and YFP-PEX7 (Figure 2). In contrast, tdTomato-

PEX7 localized to the cytosol in pex14, pex13 and pex12i

mutants (Figure 5). These results indicated that the loss of

PEX14, PEX13 or PEX12 prevents the targeting of PEX7 to

peroxisomes.

Detection of complex formation between PEX7 and PEX12

in living plant cells using BiFC

To determine whether PEX7 directly interacted with PEX12

in vivo, we carried out a BiFC assay using nYFP-PEX7, a

fusion protein of PEX12 and the C-terminal half of YFP

(cYFP-PEX12). Transient expression of nYFP-PEX7 and

cYFP-PEX12 in leek epidermal cells resulted in punctate

YFP fluorescence signals in the peroxisome (Figure 6a).

The YFP-specific spherical structures co-localized with

tdTomato-PTS1 (Figure 6b,c), which suggested that the

interaction of PEX7 and PEX12 occurred at the peroxisome.

The PEX7-PEX12 complex was most likely localized to the

peroxisomal membrane, as PEX12 is a peroxisomal mem-

brane protein (Mano et al., 2006). These results provide

492 Tanuja Singh et al.

ª 2009 The AuthorsJournal compilation ª 2009 Blackwell Publishing Ltd, The Plant Journal, (2009), 60, 488–498

Page 6: Molecular components required for the targeting of PEX7 to peroxisomes in Arabidopsis thaliana

experimental evidence of a novel, direct protein–protein

interaction between PEX7 and PEX12 in plant cells.

Detection of an interaction between PEX7 with PEX12 or

PTS2 using the yeast two-hybrid system

To confirm the interaction of PEX7 with PEX12 and PTS2, as

shown by BiFC, the yeast two-hybrid system was employed.

PEX12 contains two membrane-spanning domains, so we

thought that it was difficult to use the full-length PEX12 for

the two-hybrid assay, as was the case with PEX13 (Mano

et al., 2006). Accordingly, the truncated constructs with the

N-terminal 240 amino acids (PEX12N) or the C-terminal 102

amino acids (PEX12C) were generated. PEX7 interacted with

PEX12C, but did not interact with PEX12N (Figure 7a). With

regard to the interaction of PEX7 with PTS2, PEX7 was able

to interact with both the wild-type and mutated versions of

PTS2 (Figure 7b).

These data confirmed that PEX7 was capable of binding

with PEX12 and PTS2, and the interaction was detected by

BiFC (Figures 4 and 6). In addition, BiFC is a useful

(a) (b) (c)

(d) (e) (f)

(g) (h) (i)

(j) (k) (l)

(m) (n) (o)

(p) (q) (r)

Figure 4. Bimolecular fluorescence complemen-

tation (BiFC) analysis of the interaction between

PEX7 and a PTS2-containing protein.

(a) Epidermal cells expressing nYFP-PEX7 and

PTS2-cYFP. YFP fluorescence was present in the

peroxisome.

(d) Epidermal cells expressing nYFP-PEX7 and

PTS2(R16G)-cYFP. YFP fluorescence was present

exclusively in the cytosol.

(g) Epidermal cells expressing nYFP-PEX7 and

PTS2(R16G)-cYFP. YFP fluorescence was present

in the cytosol.

(j) Epidermal cells expressing nYFP-PTS1 and

PTS2-cYFP. YFP fluorescence was present in the

peroxisome.

(m) Epidermal cells expressing nYFP-PEX7 and

cYFP (negative control).

(p) Epidermal cells expressing nYFP and PTS2-

cYFP (negative control).

The indicated constructs were transiently ex-

pressed in leek epidermal cells using particle

bombardment. tdTomato-PTS1, a red fluores-

cent peroxisomal marker protein (b, h, k), and

tdTomato, a cytosolic marker protein (e, n, q),

were used as markers for transformation. (c, f, i, l,

o, r) Merged images. Images were obtained by

confocal laser-scanning microscopy. Scale bar:

10 lm.

Factors required for PEX7 targeting to peroxisomes 493

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Page 7: Molecular components required for the targeting of PEX7 to peroxisomes in Arabidopsis thaliana

technique for detecting the interaction between peroxi-

somal proteins.

DISCUSSION

In the current study, we established transgenic Arabidopsis

that expressed GFP-PEX7 (Figure 1). A detailed analysis of

the phenotype of AtGFP-PEX7 revealed that the overex-

pression of GFP-PEX7 causes a defect in glyoxysomal

function, including fatty acid b-oxidation, but not in leaf

peroxisomal photorespiration. This phenotype was identical

with that of a PEX7 knock-down mutant obtained through

RNA interference (Hayashi et al., 2005) and the pex5-1 mu-

tant (Woodward and Bartel, 2005). We previously demon-

strated that PEX7 knock-down mutants exhibit dysfunctional

PTS2-dependent protein import, and are defective in glyox-

ysomal function, but not in leaf peroxisomal function. Thus,

the overexpression of GFP-PEX7 appears to exert a domi-

nant-negative effect on glyoxysomal function.

To understand the mechanism underlying the dominant-

negative phenotype of AtGFP-PEX7 plants, we analyzed the

subcellular localization of several different fluorescent

protein-tagged PEX7 variants in plant cells (Figure 2). The

results indicated that the fusion proteins localized primarily

to the peroxisomal membrane, whereas endogenous PEX7

was detected in approximately equal quantities in the

cytosol and peroxisome (Figure 3). Similar results have

been observed in S. cerevisiae. Nair et al. (2004) reported a

bimodal distribution of both endogenous PEX7 and PEX7-

GFP. The authors proposed that endogenous PEX7 is

targeted to peroxisomes and then recycled back to the

cytosol, and that the proportion of PEX7-GFP detected in

peroxisomes increases as a result of the addition of a GFP

moiety to the C-terminus of PEX7. Our results were in

agreement with these previous results, and indicate that in

Arabidopsis PEX7 is targeted to peroxisomes, and is then

recycled back to the cytosol, similar to other organisms,

such as yeast and mammals. We also demonstrated that

fluorescent protein-tagged PEX7 can form the receptor–

cargo complex with a PTS2-containing protein, and is

also properly targeted to plant peroxisomal membranes

(Figures 4a and 7b). Thus, fluorescent protein-tagged PEX7

is a useful tool for studying the mechanism of PEX7

targeting to plant peroxisomes.

In addition, bimolecular fluorescence complementation

and yeast two hybrid analyses (Figures 4 and 7) indicated

that the mutated PTS2 could bind with PEX7, but that the

complex stayed in the cytosol. The result suggested that the

binding of a cargo to PEX7 is necessary but not sufficient for

the targeting of the complex. One possible reason could be

that the specific stereo structure of the receptor–cargo

complex is required during peroxisomal import, not just

the primary structure of each component. The addition of a

Figure 5. Subcellular localization of tdTomato-PEX7 in Arabidopsis pex

mutants.

Transient expression of tdTomato-PEX7 in the root cells of 10–12-day-old

wild-type Arabidopsis and pex14, pex13 and pex12i mutants, which are

defective in PEX14, PEX13 and PEX12, respectively. Images were obtained by

confocal laser-scanning microscopy after incubation in the dark for 18 h. The

red color indicates tdTomato-PEX7 fluorescence. Scale bar: 10 lm.

(a)

nYFP-PEX7cYFP-PEX12

tdTomato-PTS1

Merge

(b)

(c)

Figure 6. Bimolecular fluorescence complementation (BiFC) analysis of the

interaction between PEX7 and PEX12.

(a) Epidermal cells expressing nYFP-PEX7 and cYFP-PEX12. YFP fluorescence

(green color) was present in the peroxisome. The indicated constructs were

transiently expressed in leek epidermal cells together with tdTomato-PTS1

(b), using particle bombardment; (c) composite image obtained by merging

the images in (a) and (b). Images were obtained by confocal laser-scanning

microscopy. Scale bar: 10 lm.

494 Tanuja Singh et al.

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Page 8: Molecular components required for the targeting of PEX7 to peroxisomes in Arabidopsis thaliana

fluorescent tag to PEX7 and the mutation of PTS2 might

destroy the stereo structure of the receptor–cargo complex

that is necessary for the subsequent import process. The

addition of a fluorescent protein tag to PEX7 prevented the

later recycling step of the peroxisomal protein import

pathway, and promoted the irreversible binding of PEX7 to

the peroxisomal membranes. The adhesion of PEX7 to

peroxisomal membranes might prevent further import of

PTS2-containing proteins into peroxisomes, and may

explain in part the dominant-negative phenotype of

AtGFP-PEX7.

Using fluorescent protein-tagged PEX7 as a tool, we

demonstrated that at least three peroxisomal membrane

proteins, PEX14, PEX13 and PEX12, play a critical role in the

peroxisomal targeting of PEX7 in vivo. Our results clearly

indicated that all three of these proteins are essential for

peroxisomal targeting, as PEX7 remained in the cytosol and

failed to properly localize to peroxisomes in cells that were

deficient in PEX14, PEX13 or PEX12 (Figure 5, pex14, pex13

and pex12i, respectively). Previous studies in vitro using the

yeast two-hybrid system and co-immunoprecipitation anal-

ysis suggested that PEX7 forms a cytosolic receptor–cargo

complex composed of PEX7, PEX5, PTS2- and PTS1-con-

taining proteins, and that PEX7 and PEX5 bind directly to

PEX13 and PEX14, respectively (Hayashi et al., 2000, 2005;

Nito et al., 2002; Mano et al., 2006). These results have led to

a model in which PEX7 binds directly to PEX13 and indirectly

to PEX14 in the initial step of peroxisomal targeting of

receptor–cargo complexes (Zolman and Bartel, 2004; Zol-

man et al., 2005; Hayashi and Nishimura, 2006). Here, we

present direct experimental evidence that both PEX14 and

PEX13 play a critical role in the initial docking of PEX7 on

peroxisomal membranes in plant cells, and that both PEX14

and PEX13 are essential for the proper targeting of PEX7 to

plant peroxisomes.

Arabidopsis pex12 mutants exhibit defects in the perox-

isomal import of both PTS1- and PTS2-containing proteins

(Mano et al., 2006; Nito et al., 2007), which has led to the

suggestion that PEX12 is involved in the peroxisomal

protein import machinery in plant cells. Here, we demon-

strated that PEX12 is also essential for the peroxisomal

targeting of PEX7 (Figure 5, pex12i), and that PEX12 inter-

acts directly with PEX7 at the peroxisomal membrane

(Figures 6 and 7). Thus, the peroxisomal import defect of

pex12 mutants is most likely caused by the failure of the

PEX7-containing receptor–cargo complexes to be properly

targeted to the peroxisomal membrane. In mammalian cells,

PEX12 is believed to play a role in the translocation of cargo

after the docking of the receptor–cargo complex with the

PEX14/PEX13 protein complex (Chang et al., 1999; Miyata

and Fujiki, 2005). The results of the current study suggest

that in plant cells, receptor–cargo complexes are initially

captured by a PEX14/PEX13 complex on the peroxisomal

membrane, and are then transferred to PEX12.

We propose the following model for the targeting of PEX7

to peroxisomal membranes in plants (Figure 8): (i) PEX7 and

PEX5 form a cytosolic receptor complex; (ii) PTS1- and PTS2-

containing proteins are captured by the complex by binding

to PEX5 and PEX7, respectively; (iii) the receptor–cargo

complex docks onto peroxsiomal membranes through the

binding of PEX5 with PEX14 and PEX7 with PEX13; (iv) the

receptor–cargo complex is transferred to PEX12 through a

direct interaction with PEX7; (v) cargo proteins are released

into the peroxisomal matrix; and (vi) the PEX5/PEX7 receptor

complex is recycled back to the cytosol. In this model, the

addition of a fluorescent protein tag to PEX7 disrupts the

final recycling step. Although it has been suggested that

other PEX genes are involved in this process, the molecular

mechanism underlying the function of these additional PEX

genes in the peroxisomal targeting of PEX7 remains to be

clarified.

EXPERIMENTAL PROCEDURES

Plant materials

Arabidopsis thaliana ecotypes Columbia and Landsberg erecta,which were used as wild-type plants, and the mutants ped2, apm2and pex12i, expressing GFP-PTS1, were grown as describedpreviously (Hayashi et al., 2000; Mano et al., 2006; Nito et al., 2007).All seeds were surface sterilized in 2% NaClO, 0.02% Triton X-100,

(a)1

2

3

4

1

2

3

4

(b)

Figure 7. Detection of the interaction between PEX7 and PEX12 or PTS2 by

yeast two-hybrid assay.

(a) Interaction between PEX7 and PEX12. Lane 1, pGBD-C1 and pGAPEX12N;

lane 2, pGBD-C1 and pGAPEX12C; lane 3, pGBPEX7 and pGAPEX12N; lane 4,

pGBPEX7 and pGAPEX12C.

(b) Interaction between PEX7 and PTS2. Lane 1, pGBD-C1 and pGAPTS2wt;

lane 2, pGBD-C1 and pGAPTS2m; lane 3, pGBPEX7 and pGAPTS2wt; lane 4,

pGBPEX7 and pGAPTS2m. The two-hybrid interaction was detected by

growth in the absence of adenine. Serial dilutions were spotted on plates and

incubated at 30�C.

Factors required for PEX7 targeting to peroxisomes 495

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and were grown on germination media [2.3 mg ml)1 MS salts(Wako, http://www.wako-chem.co.jp/english), 1% sucrose,100 lg ml)1 myo-inositol, 1 lg ml)1 thiamine-HCl, 0.5 lg ml)1

pyridoxine, 0.5 lg ml)1 nicotinic acid, 0.5 mg ml)1 2-(N-morpholine)-ethanesulphonic acid (MES)-KOH (pH 5.7), 0.8% agar (INA, http://www.funakoshi.co.jp/export/product/NF060010.html)]. Where indi-cated, 0.3 lg ml)1 of 2,4-DB was added, or sucrose was removedfrom the growth medium. Germination was induced by incubationin the dark for 48 h at 4�C, followed by white light at 22�C. Seedlingsthat were grown for 7 days on growth medium were transferred to a1:1 mixture of perlite and vermiculite. Plants were grown underconstant illumination at 22�C.

Plasmid construction and Arabidopsis transformation

To generate the GFP-PEX7 chimera, a DNA fragment encoding theMet1–Ser317 region of PEX7 was amplified by PCR. The amplifiedfragment was inserted into pDONR221 (Invitrogen, http://www.invitrogen.com) and then transferred into the Ti vector pK7WGF2(VIB, Ghent University), using BP and LR clonases, according to themanufacturer’s instructions (Invitrogen). The resultant vector wasintroduced into A. tumefaciens strain C58C1RifR by electroporation,and the transformed bacteria were vacuum-infiltrated into A. thali-ana (ecotype Columbia) (Bechtold et al., 1993). Transgenic plantswere selected on germination media plates containing 50 lg ml)1 ofkanamycin.

Construction of chimeric genes

For the construction of YFP-PEX7, PEX7-YFP, nYFP-PTS1, PTS2-cYFP, nYFP-PEX7, cYFP-PEX12 and tdTomato-PEX7 fusion genes,cDNAs for the pair of interacting proteins [full-length PEX7 (Nitoet al., 2002) and PEX12 (Met1–Thr372) (Mano et al., 2006)], thePTS1-containing C-terminal 10 amino acids of Arabidopsishydroxypyruvate reductase (Mano et al., 2002), and the PTS2-con-taining N-terminal 48 amino acids of pumpkin citrate synthase (Katoet al., 1996) were amplified by PCR using specific sets of primers. Allof the constructs were amplified with attB1 and attB2 sites at their 5¢and 3¢ ends, respectively. The amplified fragments were cloned intothe entry vector pDONR221 (Invitrogen) using BP clonases,according to the manufacturer’s instructions (Gateway technology;Invitrogen). The PEX7 coding region in pDONR221 was then trans-ferred to the Gateway expression vectors pUGW41 and pUGW42(kindly provided by Dr T. Nakagawa, Shimane University) by LRrecombination (Gateway technology; Invitrogen) to generate plas-mids that encoded YFP-PEX7 and PEX7-YFP, respectively (Nakaga-wa et al., 2007). The PEX7, PEX12, PTS1 and PTS2 coding regions inpDONR221 and mRFP1 in pCR8/mRFP1 were transferred to the

attR1-attR2 sites of nYFP/pUGW0, cYFP/pUGW0 and cYFP/pUGW2,respectively, using the same procedure. PTS2 with a single base-pair substitution (CGC fi GGC, arginine 16 fi glycine) wasgenerated by site-directed mutagenesis using a Quick-change site-directed mutagenesis kit (Stratagene, http://www.stratagene.com).

SDS-PAGE and immunoblot analysis

Wild-type Arabidopsis (ecotype Columbia) and transgenic GFP-PEX7 seeds (3000; approximately 60 mg) were grown on growthmedium for 5 days in darkness at 22�C. The 5-day-old etiolatedcotyledons of AtGFP-PEX7 and wild-type Arabidopsis werehomogenized with 250 ll of homogenization buffer [150 mM

HEPES-KOH (pH 7.2), 1 mM EDTA and protease inhibitor cocktailtablet (Boerhinger, http://www.boehringer-ingelheim.com)]. Totalhomogenate was then treated with low-salt buffer, high-salt bufferor alkaline solution consisting of 150 mM HEPES-KOH (pH 7.2), with50 mM NaCl, 150 mM HEPES-KOH (pH 7.2) with 500 mM NaCl, or0.1 M Na2CO3, respectively. Samples were incubated for 1 h at 4�C,and were then subjected to centrifugation at 100 000 g for 30 min,and the supernatant and pellet fractions were collected. The pelletwas resuspended in 1· SDS sample buffer, incubated for 1 h at 4�Cand then subjected to centrifugation at 100 000 g for 30 min. Theprotein concentration of the extracts was estimated using the Bio-Rad protein assay (Bio-Rad Laboratories, http://www.bio-rad.com)and bovine gamma albumin as a standard.

Proteins were separated by SDS-PAGE and then transferred to anitrocellulose membrane (Schleicher and Schuell BioscienceGmbH, now part of Whatman, http://www.whatman.com) using asemidry electroblotting system. The membrane was probed withantibodies raised against AtPEX7 (diluted 1:1000; Nito et al., 2002),GFP (diluted 1:5000; Mitsuhashi et al., 2000), AtPEX5 (diluted1:5000; Nito et al., 2002), 3-ketoacyl-CoA thiolase (diluted 1:10 000;Kato et al., 1996) and peroxisomal ascorbate peroxidase (pAPX)(diluted 1:1000), as indicated, followed by horseradish peroxidase(HRP)-conjugated anti-rabbit secondary antibody. Immunoreactiveproteins were detected using an ECL detection kit (GE Healthcare,http://www.gehealthcare.com).

Transient expression assays using particle bombardment

Transient expression assays to assess the subcellular localization ofYFP fusion proteins in Japanese leek epidermal cells were carriedout using the particle bombardment technique. Plasmids encodingnYFP and cYFP fusion proteins were mixed at a 1:1 (w/w) ratio, andwere adsorbed onto gold particles of 1.0 lm in diameter, accordingto the manufacturer’s instructions (Bio-Rad). Inner epidermal peels

Figure 8. Model for the targeting of PEX7 to

peroxisomal membranes in plants.

(i) PEX5 and PEX7 form a cytosolic receptor

complex; (ii) PTS1- and PTS2-containing proteins

are captured by the PEX5/PEX7 complex; (iii) the

receptor–cargo complex docks onto peroxsiomal

membranes through the binding of PEX5 and

PEX7 to PEX14 and PEX13, respectively; (iv) the

receptor–cargo complex is then transferred to

PEX12 through binding to PEX7; (v) cargo pro-

teins are released into the peroxisomal matrix;

(vi) the PEX5/PEX7 receptor complex is recycled

back to the cytosol (indicated by dotted arrow);

and (vii) the addition of a GFP epitope tag

prevents receptor recycling. Abbreviations: 5,

PEX5; 7, PEX7; 14, PEX14; 13, PEX13; 12, PEX12.

496 Tanuja Singh et al.

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of leek (1-cm square), with the inner side oriented upwards, wereplaced on a 0.8% (w/v) agar plate containing 2.3 mg ml)1 MS saltsand 0.5 mg ml)1 MES-KOH (pH 5.7). For each bombardment, 9 ll ofDNA-coated gold particles were placed on the macrocarrier. Leekepidermal cells were bombarded using the PDS-1000/He BiolisticParticle Delivery System (Bio-Rad) at 7590 kPa under a vacuum of84.7 kPa. Following bombardment, the cells were incubated in thesame petri dish for 18 h at 22�C in the dark before observation. Asa marker of transformation, we also analyzed the expression oftdTomato (a gift from Dr R.Y. Tsien, University of California) usingthe expression vector ptdGW and tdTomto-PTS1.

tdTomato-PEX7 was introduced into root cells of wild-typeArabidopsis and the following mutants: ped2 (defective in PEX14;Hayashi et al., 2000), apm2 (defective in PEX13; Mano et al., 2006)and pex12i (defective in PEX12; Nito et al., 2007) under the sameconditions described above.

Confocal microscopy

Leek epidermal cells were examined under an LSM510 confocallaser-scanning microscope equipped with Argon and He–Ne lasers,and with a set of filters capable of distinguishing between yellow(YFP) and red (RFP) fluorescence. The 488-nm argon ion laser wasused to excite GFP, whereas the 543-nm He line was used to exciteRFP. Fluorescence signals from YFP and RFP were excited by 514-and 543-nm light wavelengths, respectively, and emissions wereobserved with a 565–615-nm band-pass filter and a LP560 long-passfilter, respectively (Carl Zeiss, http://www.zeiss.com). Images werecollected in multi-channel mode.

Two-hybrid analysis

The two-hybrid analysis was carried out according to a methoddescribed previously (Fields and Song, 1989). The two vectors,pGBD-C1 and pGAD-C1, which contain the Gal4p DNA-bindingdomain or -activation domain, respectively, were modified todestination vectors to contain the Gateway cassette in the ClaI andBglII sites using the Gateway Vector Conversion System (Invitro-gen). They were designated as pGBD-C1GW3 and pGAC-C1GW3,respectively. To make the two truncated constructs of PEX12 withthe N-terminal 240 amino acids (PEX12N) or the C-terminal 102amino acids (PEX12C), DNA fragments conjugated at the attB1 andattB2 sites at the 5¢ and 3¢ ends, respectively, were amplified by PCRand purified. The PCR-amplified DNA fragments were transferred tothe donor vector, pDONR221, by a BP recombination reactionaccording to the Gateway technology (Invitrogen). After sequencingto confirm that no mutation was introduced, the insert was trans-ferred to pGAD-C1GW3 or pGBD-C1GW3 by an LR recombinationreaction, according to the Gateway technology (Invitrogen). Thevectors were designated as pGAPEX12N, pGBPEX12N, pGAPEX12Cand pGBPEX12C. Two entry vectors containing wild-type ormutated PTS2 fragments, which were described above, were usedto make four kinds of two-hybrid vectors, pGAPTS2wt, pGBPTS2wt,pGAPTS2m and pGBPTS2m. These two-hybrid vectors wereco-transformed into tester strain PJ69-4A (James et al., 1996)according to an established protocol (Gietz et al., 1995). Yeasttransformants were selected and grown on complete synthesizedmedia containing 2% glucose, 0.67% yeast nitrogen base withoutamino acid (DIFCO) and amino acids as needed. Two-hybrid inter-actions were assayed using His3 and LacZ reporters.

ACKNOWLEDGEMENTS

We thank Dr Tsuyoshi Nakagawa (Shimane University) for provid-ing pUGW vectors, Dr Roger Tsien (University of California, San

Diego) for providing tdTomato, and the Japan Society for the Pro-motion of Science (JSPS) for the financial support to TS. This workwas supported in part by the Japanese Ministry of Education,Sports, Culture, Science and Technology to MN [Grant-in-Aid forScientific Research on Priority Areas (no. 1685209) and Grant-in-Aidfor Scientific Research (B) (no. 20370024)] to MH [Grant-in-Aid forScientific Research (C) (no. 21570053)] and to MS [Grant-in-Aid forScientific Research (C) (no. 20570045)], and The Graduate Universityfor Advanced Studies (Sokendai).

SUPPORTING INFORMATION

Additional Supporting Information may be found in the onlineversion of this article:Figure S1. Schematic representation of bimolecular fluorescencecomplementation (BiFC) fusion proteins.Figure S2. Bimolecular fluorescence complementation (BiFC) anal-ysis of nYFP- and cYFP-fusion proteins.Please note: Wiley-Blackwell are not responsible for the content orfunctionality of any supporting materials supplied by the authors.Any queries (other than missing material) should be directed to thecorresponding author for the article.

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