A 1-Megadalton Translocation Complex Containing Tic20 andTic21 Mediates Chloroplast Protein Import at the InnerEnvelope Membrane W

Shingo Kikuchi,a Maya Oishi,a Yoshino Hirabayashi,a Dong Wook Lee,b Inhwan Hwang,b and Masato Nakaia,1

a Institute for Protein Research, Osaka University, Suita, Osaka 565-0871, Japanb Division of Integrative Biosciences and Biotechnology and Division of Molecular and Life Sciences, POSTECH, Pohang

790-784, Korea

Chloroplast protein import is mediated by two hetero-oligomeric protein complexes, the Tic and Toc translocons, which are

located in the inner and outer envelope membranes. At the inner membrane, many Tic components have been identified and

characterized, but it remains unclear how these Tic proteins are organized to form a protein-conducting channel or whether

a stable Tic core complex that binds a translocating preproteins exists. Here, we report the identification of a 1-megadalton

(MD) translocation complex as an intermediate during protein translocation across the inner membrane in Arabidopsis

thaliana and pea (Pisum sativum). This complex can be detected by blue native PAGE using the mild detergent digitonin

without any chemical cross-linkers. The preprotein arrested in the 1-MD complex can be chased into its fully translocated

form after a subsequent incubation. While Tic20 and Tic21 appear to be involved in the 1-MD complex, Tic110, a well-

characterized Tic component, exists as a distinct entity from the complex. Several lines of evidence suggest that the 1-MD

complex functions in between the Toc and Tic110-containing complexes, most likely as a protein-conducting channel at the

inner envelope.

INTRODUCTION

Nuclear-encoded chloroplast proteins are synthesized in the

cytosol as preproteins with N-terminal targeting signals, called

transit peptides. These proteins are then posttranslationally

imported across the double envelope membranes of chloro-

plasts. The chloroplastic outer and inner envelope membranes

contain multisubunit machinery for the import of preproteins,

termed the Toc (translocon at the outer envelope membrane of

chloroplasts) and the Tic (translocon at the inner envelope

membrane of chloroplasts) complexes, respectively (for reviews,

see Soll and Schleiff, 2004; Bedard and Jarvis, 2005; Jarvis,

2008; Kessler and Schnell, 2009). During import, the Toc and Tic

complexes are thought to come together at contact sites where

the outer and inner membranes are in proximity, allowing the

preprotein to pass through both membranes simultaneously

(Schnell and Blobel, 1993). During or shortly after import, the

transit peptide is removed by a stromal processing peptidase,

and the mature protein is then folded or targeted to one of the

internal compartments.

Protein import into chloroplasts requires ATP hydrolysis. In the

presence of low concentrations of ATP (<100 mM), irreversible

binding of preproteins to the translocon components occurs.

Although it is not clear where the ATP-utilizing component may

reside, previous studies have shown that a stable association of

preproteins with the translocon components, the so-called early

import intermediate, was generated under low ATP concentra-

tions (Waegemann and Soll, 1991; Perry and Keegstra, 1994;

Schnell et al., 1994; Ma et al., 1996; Akita et al., 1997; Kouranov

and Schnell, 1997; Nielsen et al., 1997; Young et al., 1999; Inoue

and Akita, 2008). In the presence of higher concentrations of ATP

(>1 mM), complete translocation of preproteins across the dou-

ble envelope of chloroplasts occurs. This high ATP level is

probably required by stromal molecular chaperones believed to

provide the driving force for unidirectional translocation of pre-

proteins.

At the outer membrane, Toc75, Toc159, and Toc34 form a

stable complex and mediate the transfer of preproteins through

the outer membrane (Schleiff et al., 2003; Kikuchi et al., 2006). At

the inner membrane, eight proteins (Tic110, Tic40, Tic20, Tic21,

Tic22, Tic55, Tic62, and Tic32) are reported to be involved in the

import process (Soll and Schleiff, 2004; Bedard and Jarvis, 2005;

Teng et al., 2006; Jarvis, 2008; Kessler and Schnell, 2009). Tic20

has been proposed to function as the protein-conducting chan-

nel of the inner membrane. Tic20 was identified by chemical

cross-linking to translocating preproteins in pea (Pisum sativum)

chloroplasts (Kouranov et al., 1998). Tic20 is an integral mem-

brane protein and is predicted to have three or four transmem-

brane helices. The reduction of Tic20 levels in Arabidopsis

thaliana antisense plants produced a specific defect in protein

translocation across the inner membrane (Chen et al., 2002).

Tic110 is predicted to have two transmembrane helices at its N

terminus and a large hydrophilic C-terminal domain, which was

1Address correspondence to nakai@protein.osaka-u.ac.jp.The author responsible for distribution of materials integral to thefindings presented in this article in accordance with the policy describedin the Instructions for Authors (www.plantcell.org) is: Masato Nakai(nakai@protein.osaka-u.ac.jp).WOnline version contains Web-only data.www.plantcell.org/cgi/doi/10.1105/tpc.108.063552

This article is a Plant Cell Advance Online Publication. The date of its first appearance online is the official date of publication. The article has been

edited and the authors have corrected proofs, but minor changes could be made before the final version is published. Posting this version online

reduces the time to publication by several weeks.

The Plant Cell Preview, www.aspb.org ã 2009 American Society of Plant Biologists 1 of 17

shown to be exposed to the stromal compartment (Jackson

et al., 1998). The stromal domain of Tic110 has been proposed to

function as a molecular scaffold by binding the preprotein and

recruiting the stromal chaperone Hsp93 with the assistance of

the putative cochaperone Tic40 (Akita et al., 1997; Nielsen et al.,

1997; Chou et al., 2003; Chou et al., 2006). These three proteins

(Tic110, Tic40, and Hsp93) are thought to drive protein import

into the stroma through repeated cycles of binding and release.

Although an alternative model for the topology and function of

Tic110 has also been proposed, in which Tic110 is a polytopic

membrane protein that functions as a protein-conducting channel

(Heins et al., 2002; Balsera et al., 2009), a truncated version of

Tic110 lacking the N-terminal transmembrane helices was shown

to exist as a soluble protein when expressed in Escherichia coli or

in the stroma of transgenic Arabidopsis (Inaba et al., 2003).

However, the existence of a stable Tic complex containing a

protein-conducting channel remains unclear. Here, we report the

identification of a 1-MD translocation complex as an intermedi-

ate during protein translocation across the inner membrane. This

complex can be detected by blue native PAGE (BN-PAGE) using

the mild detergent digitonin without any chemical cross-linkers.

The preprotein arrested in the 1-MD translocation complex can

be chased into its fully translocated form after a subsequent

incubation. Antibody-shift BN-PAGE, immunodepletion, and im-

munoprecipitation assays suggest that Tic20 and Tic21 are

involved in the 1-MD translocation complex but that Tic110 is

not involved in this complex.

RESULTS

A Translocation Intermediate Complex Was Observed

by BN-PAGE

BN-PAGE allows the separation of membrane protein com-

plexes under nondenaturing conditions (Schagger and von

Jagow, 1991; Schagger et al., 1994). We examined whether

BN-PAGE is applicable for the analysis of preproteins in the

process of translocation across the double envelope mem-

branes of chloroplasts. The precursor of the small subunit of

ribulose-1,5-bisphosphate carboxylase/oxygenase (pSSU) was

used as a model protein. pSSU was synthesized in vitro in the

presence of [35S]Met. In vitro import reactions were performed in

the presence of different concentrations of ATP using pea

Figure 1. ATP-Dependent Formation of a Translocation Intermediate Complex.

(A) Energy-depleted pea chloroplasts were mixed with [35S]pSSU in HS buffer containing the indicated concentrations of ATP, 5 mMMgCl2, 5 mMDTT,

3 mM Met, 3 mM Cys, and 5 mL/mL protease inhibitor cocktail. The reactions were incubated for 10 min at 258C in the dark. Reisolated chloroplasts

were solubilized in BN-PAGE sample buffer (containing 1% digitonin) to a final concentration of 0.5 mg chlorophyll/mL for 10 min on ice. After

ultracentrifugation, the supernatant was divided into two aliquots, one of which was mixed with Coomassie blue solution and subjected to 4 to 14%

BN-PAGE (top). The other was mixed with 10% SDS and 2-mercaptoethanol to final concentrations of 3.3 and 5%, respectively, denatured by heating

at 958C for 2 min, and subjected to 15% SDS-PAGE (bottom).

(B) A gel strip corresponding to lane 3 (0.5 mM ATP) of (A) was subjected to SDS-PAGE as a second dimension (2D-BN/SDS-PAGE).

(C) A gel strip corresponding to lane 5 (5 mM ATP) of (A) was analyzed as in (B). Radioactive signals in dried gels were detected by digital

autoradiography. Molecular mass markers are ferritin (880 and 440 kD) and BSA (66 kD). TP, 10% of the [35S]pSSU translation product added to each

reaction. The positions of the 1-MD translocation intermediate complex, free pSSU, and unassembled mSSUmigrating in the lowmolecular mass range

are indicated by brackets.

2 of 17 The Plant Cell

chloroplasts. Chloroplasts were reisolated, solubilized with 1%

digitonin, and subjected to BN-PAGE and autoradiography (Fig-

ure 1A, top). Radioactive signals were found at, approximately,

the 1-MD area and at a low molecular mass (<66 kD). By SDS-

PAGE, the precursor form of SSU was observed at all tested

concentrations of ATP, and the mature form of SSU (mSSU) was

observed at relatively high concentrations of ATP (>1mM) (Figure

1A, bottom).

Figure 1B shows two-dimensional (2D)-BN/SDS-PAGE anal-

ysis of lane 3 (0.5 mM ATP) from Figure 1A. pSSU was found in

two peaks: one was in the 1-MD area, demonstrating that pSSU

was arrested in the 1-MD translocation complex, and the other

was in the low molecular mass range, probably representing

pSSU dissociated from the translocation machinery in the pres-

ence of detergent. Figure 1C shows 2D-BN/SDS-PAGE analysis

of lane 5 (5mMATP) from Figure 1A. pSSUwas also found in this

1-MD area. In addition,mSSUwas observed in the lowmolecular

mass range. Formation of the 1-MD translocation intermediate

complex wasmaximal at 0.5mMATP (Figure 1A, top). Therefore,

we decided to use this concentration to generate the transloca-

tion intermediate complex for subsequent experiments.

The 1-MD translocation complex was observed only when the

mild detergent digitonin was used. All tested detergents except

digitonin failed to preserve the 1-MD complex (i.e., 1% Triton

X-100, 1% Nonidet P-40, 1% dodecyl maltoside, 1% decyl

maltoside, or 1% octyl glucoside; data not shown). Moreover,

addition of either dodecyl maltoside or Triton X-100 to the

digitonin-solubilized BN-PAGE sample caused dissociation of

pSSU from the 1-MD complex (Figure 2A). These observations

suggest that pSSU is loosely associated with the 1-MD complex.

The possibility that the association of pSSU occurred artificially

after solubilization of the chloroplasts can be excluded because

pSSU was efficiently associated with the 1-MD complex in the

presence of ATP only when incubated with intact chloroplasts

but not with solubilized chloroplast extract (see Supplemental

Figure 1 online). A similar 1-MD translocation complex was also

observed when the precursor of ferredoxin:NADP+ oxidoreduc-

tase was used (see Supplemental Figure 2 online).

Formationof the1-MDTranslocationComplex IsBlockedby

Slowly Hydrolyzable Analogs of GTP and ATP

It is well known that Toc34 and Toc159 have GTP binding motifs,

and the binding of preproteins to the Toc complex is inhibited by

non- or slowly hydrolyzable analogs of GTP (Young et al., 1999;

Soll and Schleiff, 2004; Inoue and Akita, 2008; Jarvis, 2008;

Kessler and Schnell, 2009). To test whether the 1-MD translo-

cation intermediate complex is formed after translocation

Figure 2. Characteristic Features of the 1-MD Translocation Complex.

(A) After pea chloroplasts carrying [35S]pSSU were solubilized with 1% digitonin (Dig), either dodecyl maltoside (DDM, lane 2) or Triton X-100 (lane 3)

was added to give a final concentration of 1%. After incubation for 30 min on ice, samples were subjected to BN-PAGE.

(B) After pea chloroplasts were preincubated with the indicated concentrations of GTP-g-S or ATP-g-S for 10 min at 258C in the dark, [35S]pSSU and

ATP (final 0.5 mM) were added and incubated for another 10 min at 258C in the dark. Reisolated chloroplasts were solubilized and subjected to

BN-PAGE (top) and SDS-PAGE (bottom).

The 1-MDa Tic complex of chloroplasts 3 of 17

through the Toc complex via the general import pathway, we

examined the effect of a slowly hydrolyzable analog of GTP

(GTP-g-S). Chloroplasts were preincubatedwith GTP-g-S before

addition of pSSU and ATP. GTP-g-S drastically reduced the

formation of the 1-MD complex (Figure 2B), suggesting that

the 1-MD complex is formed after translocation through the

well-defined Toc complex. In addition, by preincubating chloro-

plasts with ATP-g-S, formation of the 1-MD complex was also

drastically reduced (Figure 2B). This result shows that hydrolysis

of ATP is also essential for the formation of the 1-MD complex.

ThePreproteinArrested in the1-MDTranslocationComplex

Can Be Chased into Its Fully Translocated Mature Form

Chase experiments indicated that, in the absence of ATP, almost

no import of pSSU, which had been arrested in the 1-MD

translocation complex, was observed (Figure 3, top). In the

presence of ATP (0.5 and 5 mM), the radiolabel in the 1-MD

complex gradually decreased during the chase period, while

radiolabeled ribulose-1,5-bis-phosphate carboxylase/oxygen-

ase (Rubisco) holoenzyme found at the 520-kDposition gradually

increased (Figure 3, top). SDS-PAGE analysis also showed that

pSSU gradually decreased, while fully imported mature SSU

gradually increased (Figure 3, bottom). This means that pSSU

arrested in the 1-MD complex was translocated, processed into

its mature form, and further assembled into the 520-kD Rubisco

holoenzyme in organello in the presence of higher concentra-

tions of ATP. Sincemature SSU contains only three of the sixMet

residues that are present in precursor SSU, the percentage of

bound pSSU that was chased into mSSU under 5 mM ATP

conditions for 30 min was estimated as 90%. The possibility that

pSSU is associated with a stromal chaperonin can be excluded

because addition of higher concentrations of ATP after solubi-

lization of chloroplasts did not affect the signal intensity of the

1-MD complex (see Supplemental Figure 1 online). Therefore, we

conclude that the radioactive 1-MD signal represents a chase-

able genuine translocation intermediate complex.

Protease Treatments Revealed That the Translocation

Intermediate Complex Is Thermolysin Resistant and

Partially Sensitive to Trypsin

To determine where the 1-MD translocation complex was accu-

mulated in chloroplasts, we performed a selective proteolysis

using exogenous thermolysin and trypsin. Thermolysin is an

outer envelope–impermeable protease that selectively digests

chloroplast surface-exposed proteins, whereas a certain con-

centration of trypsin is able to destroy the membrane integrity of

the outer envelope and partially digest proteins within the inter-

membrane space while leaving the stromal proteins undigested

(Kouranov et al., 1998; Hirohashi et al., 2001). Toc159, which has

a large cytosolically exposed domain, was easily degraded by

thermolysin and trypsin, as demonstrated by the resultant 52-kD

protease-resistant fragment (Figure 4B). Toc34, which also has a

cytosolically exposed domain, was resistant to up to 10mg/mL of

thermolysin (Chen et al., 2000) but was degraded by 100 mg/mL

of thermolysin and trypsin. Toc75, an integral membrane protein,

was largely resistant to thermolysin and trypsin. Tic22, which is

known to reside in the intermembrane space (Kouranov et al.,

1998), was resistant to thermolysin but sensitive to trypsin.

Stromal Hsp70 and Tic110, an inner envelopemembrane protein

that is oriented toward the stroma (Jackson et al., 1998; Inaba

et al., 2003), were completely resistant to both thermolysin and

trypsin (Figure 4B).

The 1-MD complex was largely resistant to exogenous ther-

molysin (Figure 4A, top, lanes 2 and 3). In SDS-PAGE, degrada-

tion products of SSU (SSU-DPs) were observed (Figure 4A,

bottom, lanes 2 and 3), indicating that theC-terminal tail of pSSU,

which was exposed to the surface of chloroplasts, was digested

by exogenous thermolysin, while the N-terminal part of pSSU,

which was probably deeply buried in a translocation channel,

was resistant to proteolysis. Furthermore, 2D-BN/SDS-PAGE of

lane 3 fromFigure 4A confirmed that the radioactive signals in the

1-MD area remaining after thermolysin treatment were derived

mostly from SSU-DP (Figure 4D). When chloroplasts carrying

pSSU were treated with 100 mg/mL trypsin, the radiolabeled

Figure 3. The Preprotein Arrested in the Translocation Intermediate

Complex Can Be Chased into Its Fully Imported Mature Form under High

ATP Concentrations.

The translocation intermediate was generated under 0.5-mM ATP as

described in Methods. Reisolated pea chloroplasts carrying [35S]pSSU

were resuspended in HS buffer containing the indicated concentrations

of ATP, 5 mM MgCl2, 5 mM DTT, 3 mM Met, 3 mM Cys, and 5 mL/mL

protease inhibitor cocktail on ice. Translocation (import) was resumed by

transferring the reaction tubes to a water bath (258C). The reactions were

terminated by chilling and centrifuging the chloroplasts at the indicated

time points. These samples were then solubilized and subjected to BN-

PAGE (top) and SDS-PAGE (bottom). As a control, chloroplasts carrying

preproteins were directly solubilized without the subsequent chase

reaction (lane 1). The positions of the 1-MD translocation intermediate

complex, the 520-kD Rubisco holoenzyme, free pSSU, and unassem-

bled mSSU are indicated by brackets.

4 of 17 The Plant Cell

Figure 4. Protease Treatments of the Chloroplast Surface Reveal That the 1-MD Translocation Complex Is Resistant to Thermolysin but Partially

Sensitive to Trypsin.

(A) to (E) The translocation intermediate was generated under 0.5-mM ATP as described in Methods. The pea chloroplast suspension was divided into

six aliquots (lanes 1 to 6) and washed twice with HS buffer in the absence of protease inhibitor cocktail. Chloroplasts carrying [35S]pSSU were treated

with the indicated concentrations of thermolysin (Thl) or trypsin (Trp) for 20 min on ice. After inactivation of the proteases, the chloroplast pellet was

solubilized and subjected to BN-PAGE ([A], top) and SDS-PAGE ([A], bottom). When using trypsin, trypsin inhibitor was added to BN-PAGE sample

buffer.

(B) Aliquots of the same samples in (A) were subjected to SDS-PAGE and immunoblotting with antibodies against indicated proteins. Arrows indicate

intact Toc159, 86- and 52-kD fragments of Toc159, and Tic22. An asterisk denotes a nonspecific cross-reacting band.

(C) A gel strip corresponding to lane 1 (without protease) of (A) was subjected to 2D-BN/SDS-PAGE.

(D) A gel strip corresponding to lane 3 (with thermolysin) of (A) was analyzed as in (C).

The 1-MDa Tic complex of chloroplasts 5 of 17

complex shifted slightly to a lower molecular mass on BN-PAGE

(Figure 4A, top, lane 6), indicating partial degradation of the 1-MD

complex. SSU-DPs were also found in the trypsin-treated sam-

ples by SDS-PAGE (Figure 4A, bottom, lanes 5 and 6). Figure 4E

shows 2D-BN/SDS-PAGE of lane 6 from Figure 4A, confirming

that the radioactive signals in the shifted complex were also

mostly derived from SSU-DP.

As we have shown previously, the intact Toc complex is 800 to

1000 kD on BN-PAGE (Kikuchi et al., 2006). As a result, we

initially suspected that the observed radiolabeled 1-MD complex

corresponded to the Toc complex itself due to their almost

identical sizes. Contrary to our expectations, the 1-MD complex

shown here was resistant to thermolysin. This observation was

inconsistent with the protease accessibilities of the Toc complex.

When chloroplasts were isolated in the absence of protease

inhibitor cocktail, the Toc complex became 350 to 500 kD in size

because the cytosolically exposed A-domain of Toc159 was

degraded by endogenous proteases during isolation of chloro-

plasts (Kikuchi et al., 2006; see Supplemental Figure 3 online,

lane 5). Irrespective of the presence or absence of protease

inhibitor cocktail during the isolation procedure, the radiolabeled

1-MD translocation complex was generated at nearly the same

levels (see Supplemental Figure 3 online, top, lanes 1 and 3).

Furthermore, as described above, while the 1-MD complex

remained resistant by thermolysin treatment, the Toc complex

was degraded to a 250- to 350-kD subcomplex in the same

samples (lanes 6 and 8). Therefore, we conclude that the 1-MD

complex does not correspond to the Toc complex itself. More

importantly, the observed partial degradation of the transloca-

tion complex after trypsin treatment suggests that the 1-MD

complex has an intermembrane space-facing domain.

TheTranslocation IntermediateAppears toBeAccumulated

in the Inner Envelope Membrane

To determine where [35S]pSSU was accumulated in chloro-

plasts, we separated envelope membrane vesicles into fractions

enriched in outer envelope membrane vesicles and inner enve-

lope membrane vesicles using sucrose density gradient centrif-

ugation (see Supplemental Figure 4 online). pSSU was found

mainly in the mixed outer-inner membrane fraction (fractions 4

and 5). A very minor portion of pSSU found in the outer mem-

brane fraction (fraction 8) may represent pSSU bound to Toc

complex that was not associated with contact sites. Thermoly-

sin-resistant SSU-DP was found exclusively in the mixed outer-

inner membrane fraction but not in the outer membrane fraction.

These observations suggest that the 1-MD complex in which the

N-terminal region of pSSU is stably associated resides in the

inner membrane rather than in the outer membrane.

Tic21 Is Involved in the 1-MD Translocation Complex

To identify which components are involved in the 1-MD complex,

we screened antisera against known Toc and Tic proteins by

antibody-shift BN-PAGE. In antibody-shift BN-PAGE, chloro-

plasts carrying pSSUwere first solubilized in digitonin-containing

buffer, followed by the addition of antibodies. The specific

binding of antibodies to a subunit(s) within the complex was

predicted to result in a shift of the complex to a higher molecular

mass on BN-PAGE. This assay was used in several reports (e.g.,

Johnston et al., 2002; Truscott et al., 2002). Arabidopsis chloro-

plasts were used in addition to pea chloroplasts for the assay

because some of the antibodies used were raised against

Arabidopsis antigens. We confirmed that incubation of Arabi-

dopsis chloroplasts with pSSU and ATP resulted in a virtually

identical translocation intermediate complex to that of the pea

complex (see Supplemental Figure 5 online). By thorough

screening of antisera, we found that an antibody against Arabi-

dopsis Tic21 caused a significant shift in this assay when

Arabidopsis chloroplasts were used (Figure 5A, lane 2). Tic21

was recently identified by Arabidopsis genetics (Teng et al.,

2006). When antibodies against pea Tic110 and SPA-820 mono-

clonal antibody (StressGen), which recognizes an as yet uniden-

tified intermembrane space Hsp70 (Schnell et al., 1994; Becker

et al., 2004), were added, the mobility of the 1-MD complex on

BN-PAGE was not affected (Figure 5A). All other antibodies,

which we tested, against known translocon components failed

to shift the 1-MD complex by antibody-shift BN-PAGE (i.e.,

antibodies against Toc159, Toc34, Tic40, Tic22, Cpn60a,

Cpn60b, and stromal Hsp70) (see Supplemental Figure 6 online).

These results indicate that at least Tic21 is involved in the 1-MD

complex.

The inability of anti-Tic110 antibody to shift the 1-MD trans-

location complex suggests that Tic110 is not involved in the

1-MD complex. To establish the absence of Tic110 in the 1-MD

complex, we removed Tic110 proteins from the solubilized

Arabidopsis chloroplast extract containing the translocation

intermediate by immunodepletion with anti-Tic110 antibody

bound to rProtein A-Sepharose. BN-PAGE followed by immu-

noblotting of the protein complexes remaining in the supernatant

showed almost complete depletion of a 200- to 300-kD Tic110

complex (Figure 5B, lane 4). By contrast, the amount of radio-

labeled 1-MD complex was not affected (Figure 5B, compare

lanes 1 and 2). The size of the Tic110 complex that migrated in

the 200- to 300-kD range is well consistent with previous reports

(Caliebe et al., 1997; Kuchler et al., 2002; Kikuchi et al., 2006).

Based on observations of antibody-shift BN-PAGE and immu-

nodepletion together with the substantial size difference be-

tween the 1-MD complex and the 200- to 300-kD Tic110

Figure 4. (continued).

(E) A gel strip corresponding to lane 6 (with trypsin) of (A)was analyzed as in (C). The three excised first-dimension BN-PAGE gel strips used in (C) to (E)

were derived from the same first-dimension BN-PAGE gel. The positions of the 1-MD translocation intermediate complex, a partially degraded

translocation intermediate complex, free pSSU, unassembled mSSU, and dissociated SSU-DP are indicated by brackets. mSSU assembled into the

520-kD Rubisco holoenzyme is indicated by arrows ([C] to [E]).

6 of 17 The Plant Cell

complex, we conclude that Tic110 is not involved in the 1-MD

translocation complex.

We also performed immunodepletion with anti-Tic40, -Hsp93,

and -Toc159 antibodies. Immunoblot analyses showed almost

complete depletion of these proteins from digitonin-solubilized

extract (see Supplemental Figure 7 online), demonstrating the

binding ability of these antibodies to respective solubilized

proteins. Nevertheless, these antibodies were unable to shift

the 1-MD complex. Therefore, we think that not only Tic110 but

also Tic40, Hsp93, and Toc159 are not involved in the 1-MD

translocation complex. We were unable to conclude, at this

stage, whether Tic20 is involved in the 1-MD complex either by

antibody-shift BN-PAGE or by immunodepletion, since all avail-

able Tic20 antisera appear not to recognize solubilized native

Tic20 proteins (see below).

Tic20 and a Minor Population of Tic21 Migrated at 1 MD

on BN-PAGE

We analyzed the size of native Tic21-containing complex in

Arabidopsis chloroplasts by BN-PAGE. Immunoblotting with

anti-Tic21 antibody identified several populations of the Tic21

complexes (Figure 6A, top). The majority of Tic21 was found in

the 100-kD area and thus is not present in the 1-MD complex.

However, a minor population of Tic21 was found in the 1 MD

area, which supports the hypothesis that Tic21 is involved in the

1-MD translocation complex.

We next analyzed the size of native Tic20-containing complex

since Tic20 is proposed to form the inner membrane transloca-

tion channel. Almost all Tic20 proteins were found in the 1-MD

area (Figure 6A, second). Although the Tic20 protein itself was

largely resistant to both thermolysin and trypsin, the Tic20

complex was resistant to thermolysin but partially sensitive to

trypsin (Figure 6A, bottom two panels), suggesting that some

other subunit(s) in the Tic20 complex has an intermembrane

space-facing domain. The BN-PAGE profile of the 1-MD Tic20

complex and its protease accessibility were very similar to those

of the 1-MD translocation complex shown in Figure 4. We also

analyzed the pea Tic20 complex by the same procedure and

obtained almost identical results (Figure 6F). In addition, by size

exclusion chromatography on a Superose 6 column, Tic20 was

found at, approximately, the 1-MD position, whereas Tic110 was

not cofractionated with Tic20 (Figure 6C), consistent with the

above observations using BN-PAGE.

Figure 5. Antibody-Shift BN-PAGE and Immunodepletion Experiments.

The translocation intermediate was generated using Arabidopsis chloroplasts by the same method described in Methods.

(A) The digitonin-solubilized chloroplastic extract containing [35S]translocation intermediate complex was mixed with purified anti-At Tic21 (8 mg), anti-

Ps Tic110 (5 mg), or monoclonal SPA-820 (5 mg) antibodies and incubated for 30 min on ice. Samples were subjected to BN-PAGE and digital

autoradiography. As a control, the solubilized chloroplastic extract without any antibody addition was applied (lane 1). The positions of a shifted

complex, the 1-MD translocation intermediate complex, free pSSU, and unassembled mSSU are indicated by brackets.

(B) The digitonin-solubilized chloroplastic extract containing [35S]translocation intermediate complex was immunodepleted with anti-Ps Tic110

antibody that had been bound to rProtein A-Sepharose. The immunodepleted fractions were subjected to BN-PAGE and autoradiography (lanes 1 and

2). The same samples were subjected to BN-PAGE and immunoblotting with anti-Ps Tic110 antibody (lanes 3 and 4). The native Tic110 complex is

indicated. An asterisk denotes a nonspecific cross-reacting band corresponding to the native Rubisco complex, which migrates in large quantities at

this position.

The 1-MDa Tic complex of chloroplasts 7 of 17

Immunodepletion Experiments Suggest That Tic20 Is

Associated with Tic21

Wenext investigated if there are direct interactions among Tic21,

Tic20, and Tic110. In Tic21-depleted extract, the majority of

Tic20 was depleted, while the amount of Tic110 was not affected

(Figure 6D). By contrast, neither Tic21 nor Tic20 was reduced in

Tic110-depleted extract. These results suggest that Tic21 and

Tic20 are associated and form the 1-MD complex under steady

state conditions but that Tic110 does not form any detectable

complex with Tic20 or Tic21 after solubilization with digitonin.

Tic20 Is a Core Component of the 1-MD Complex, whereas

Tic21 Appears to Be Loosely Associated with the Complex

Next, we looked for conditions in which Tic21 protein(s) are

dissociated from the Tic20 complex. After the Tic21-containing

Figure 6. Two-Dimensional BN/SDS-PAGE Analyses and Size Exclusion Chromatography of Tic Proteins.

(A) Intact Arabidopsis chloroplasts were treated with 100 mg/mL thermolysin (Thl), 100 mg/mL trypsin (Trp), or without proteases (Prot-) for 20 min on ice.

Reisolated chloroplasts were solubilized in BN-PAGE sample buffer (containing 1% digitonin) and subjected to 2D-BN/SDS-PAGE followed by

immunoblotting with anti-At Tic21 or anti-At Tic20 ES antibodies.

(B) Arabidopsis chloroplasts were solubilized in 1 M NaCl-containing BN-PAGE sample buffer (1% [w/v] water-soluble digitonin, 50 mM BisTris-HCl, pH

7.0, 500 mM 6-amino-n-caproic acid, 1 M NaCl, 10% [w/v] glycerol, and 10 mL/mL protease inhibitor cocktail) and subjected to 2D-BN/SDS-PAGE.

Immunoblotting was performed as in (A).

(C) Arabidopsis chloroplasts were solubilized with 1% digitonin and subjected to size exclusion chromatography on a Superose 6 column equilibrated

with 0.1% digitonin, 50 mMHEPES-KOH, pH 7.5, and 150mMNaCl. Immunoblotting was performed with anti-At Tic20 ES or anti-Ps Tic110 antibodies.

Molecular mass markers are ferritin (880 and 440 kD) and BSA (132 and 66 kD).

(D) The digitonin (1%)-solubilized Arabidopsis chloroplastic extract was immunodepleted with anti-At Tic21 or anti-Ps Tic110 antibodies that had been

bound to rProtein A-Sepharose. The immunodepleted fractions were subjected to SDS-PAGE and immunoblotting with anti-At Tic21, -At Tic20 ES, or

-Ps Tic110 antibodies.

(E) The dodecyl maltoside (1%)-solubilized Arabidopsis chloroplastic extract was immunoprecipitated with anti-At Tic21 antibody that had been cross-

linked to rProtein A-Sepharose by dimethyl pimelimidate. After the beads were washed with 0.5% Triton X-100 in TBS (25 mM Tris-HCl, pH 7.5, 150 mM

NaCl, and 10% [w/v] glycerol), the beads were further washed with various stringent conditions (2% Triton X-100 [TX], 0.2% SDS, 1%SDS, 1%CHAPS,

0.5 M NaCl, or 1 M NaCl; all reagents were in TBS). The proteins remained associated with the beads were eluted with 0.1 M glycine, pH 2.5, and 0.5%

Triton X-100 and subjected to SDS-PAGE followed by immunoblotting with anti-At Tic21 or -At Tic20 ES antibodies.

(F) Experiments were performed as in (A), except that pea chloroplasts were used.

8 of 17 The Plant Cell

Tic20 complex was immunoprecipitated with anti-At Tic21 anti-

body-immobilized (cross-linked) beads, the beads were incu-

bated under various severe conditions to disrupt the interaction

between Tic21 and the Tic20 complex. Incubation of this

immunoprecipitated complex under high salt conditions resulted

in a significant loss of Tic20 from the complex, while Tic21

remained associated with the beads (Figure 6E, lanes 6 and 7).

Similar results were obtained when the immunoprecipitated

complex was incubated with SDS (lanes 3 and 4). By contrast,

in the presence of stringent detergent conditions of 2% Triton

X-100 and 1% 3-[(3-cholamidopropyl)dimethylammonio]-1-

propanesulfonate (CHAPS), approximately half of the Tic20 sub-

complex remained associated with the beads via Tic21 (lanes 2

and 5).

Then, we asked how the 1-MD complex containing Tic20 and

Tic21 was influenced by treatment with high salt. To this end, we

performed 2D-BN/SDS-PAGE analysis of Arabidopsis chloro-

plasts after solubilization with digitonin in the presence of 1 M

NaCl. Almost all Tic21 was recovered in the 100-kD area, and a

minor population of Tic21 found in the 1-MD area in the absence

of NaCl disappeared (cf. Figures 6A with 6B, top panels). Cor-

respondingly, the size of the Tic20 complex appeared to be

slightly shifted to a lower molecular mass range (cf. Figures 6A,

second panel, with 6B, bottom panel). Notably, Tic20 proteins

that appeared as a smeared band around 1 MD and larger in the

absence of NaCl migrated as an intense spot in the presence of

1 M NaCl. This observation suggests that Tic21 was dissociated

from the Tic20 complex under high salt conditions, and the size

of the Tic20 complex was decreased slightly because of the

removal of Tic21 protein(s) and other potential unidentified

protein(s). More importantly, this observation suggests that

Tic20 exists as a core component of the 1-MD complex, whereas

Tic21 is loosely associated with the 1-MD complex.

Both the tic20 and tic21Mutants Were Defective in Plastid

Protein Import

There are two conflicting reports about the function of Tic21.

Teng et al. (2006) identified Tic21 as a preprotein translocon at

the inner envelope membrane, whereas Duy et al. (2007) inde-

pendently identified the same protein as an iron transporter

(PERMEASE IN CHLOROPLASTS1 [PIC1]). To clarify these con-

troversial findings, we employed two independent experimental

approaches. First, we performed transient expression and tar-

geting of preproteins using Arabidopsis knockout mutants. Be-

cause both homozygous tic21/pic1 and tic20 mutants show

severe albino phenotypes (Teng et al., 2006), standard import

experiments using isolated plastids are difficult. Therefore, we

used transient expression system in protoplasts (Jin et al., 2001;

Lee et al., 2008). In this system, plasmids encoding a fusion

protein consisting of the N-terminal transit peptide (79 amino

acids) of the small subunit of Rubisco and green fluorescent

protein (RbcS-nt:GFP) or a fusion protein consisting of the

N-terminal transit peptide (80 amino acids) of the pyruvate

dehydrogenase E1a subunit and GFP (E1a-nt:GFP) were intro-

duced into protoplasts by polyethylene glycol–mediated trans-

formation (Jin et al., 2001). RbcS-nt:GFP was used as a model

for photosynthetic proteins, whereas E1a-nt:GFP was used as a

model for nonphotosynthetic proteins (Smith et al., 2004). Protein

import into plastids was assessed by immunoblotting with anti-

GFP antibody using protein extracts prepared from transformed

protoplasts (Lee et al., 2008).

In the wild-type protoplasts, the majority of RbcS-nt:GFP and

E1a-nt:GFP were found in processed forms (Figure 7, lanes

1 and 2), indicating that the transiently expressed preproteins

were efficiently imported into wild-type plastids. By contrast,

approximately half of RbcS-nt:GFP remained unprocessed in

tic21/pic1 and tic20 mutants (lanes 3 and 5), indicating the

significant impairments in the protein import of photosynthetic

proteins in these mutants. On the other hand, the majority of

E1a-nt:GFP was imported into plastids in the tic21/pic1 and

tic20 mutants, although slightly less efficient than the wild type

(lanes 4 and 6). This observation suggests that some nonphoto-

synthetic preproteins are normally imported into plastids in spite

of the lack of Tic21/PIC1 and Tic20. Indeed, stromal Hsp70 and

thylakoidal Albino3, which are nonphotosynthetic and house-

keeping proteins, were accumulated normally in the tic21/pic1

and tic20 mutants, while accumulation of photosynthetic pro-

teins were severely affected (Teng et al., 2006; Duy et al., 2007;

see Supplemental Figure 8 online). As a control experiment,

another albino mutant, albino3, was used for the transient ex-

pression assay. Albino3 is a thylakoidal membrane protein and

unrelated to protein transport across the envelope (Sundberg

et al., 1997; Asakura et al., 2008). In the albino3 protoplasts, both

RbcS-nt:GFP and E1a-nt:GFP were found in their processed

form (see Supplemental Figure 9 online, lanes 3 and 4), suggest-

ing that the observed impairments of the import of RbcS-nt:GFP

are specific to the tic21/pic1 and tic20mutants but not common

in other albinomutants. From these results, we propose that both

Tic21/PIC1 and Tic20 play critical roles in import of photosyn-

thetic proteins into chloroplasts (see Discussion for details).

Figure 7. Protein Import into Plastids Analyzed by Transient Expression

in Protoplasts.

Protoplasts isolated from Arabidopsis wild-type, homozygous tic21/

pic1-1 (SALK_104852), and tic20 (SALK_039676) mutants were trans-

formed with RbcS-nt:GFP or E1a-nt:GFP. After 8 h of incubation at 228C,

proteins were extracted from transformed protoplasts and subjected

to SDS-PAGE followed by immunoblotting with anti-GFP antibody.

RbcS-nt, the N-terminal transit peptide of the small subunit of Rubisco;

E1a-nt, the N-terminal transit peptide of the pyruvate dehydrogenase

E1a subunit.

The 1-MDa Tic complex of chloroplasts 9 of 17

Upregulation of Iron Homeostasis Proteins Are Common in

Some Albino Mutants

In a second approach, we examined expression levels of iron

homeostasis proteins in the tic21/pic1 mutant and other albino

mutants. One reason that Duy et al. (2007) termed Tic21/PIC1 as

an iron transporter is based on the observation that genes related

to iron homeostasis are upregulated in the tic21/pic1 mutant.

Supplemental Figure 8 online shows immunoblot analyses of

several plastid-localized proteins in the tic21/pic1, tic20, and

albino3 mutants, all of which show albino phenotypes. As

reported by Duy et al. (2007), in the tic21/pic1 mutant, upregu-

lation of ferritin and copper superoxide dismutase 1 (CSD1) and

CSD2, which are related to iron homeostasis in plastids, was

reproduced. However, this upregulation was also observed in

other albino mutants, tic20 and albino3. This means that the

upregulation of these proteins is not specific to the tic21/pic1

mutant but is seen in other albino mutants.

Chemical Cross-Linking of the Preprotein to Tic20

To obtain direct evidence that Tic20 associates with the prepro-

tein in the translocation intermediate, chemical cross-linking of

the preprotein to translocon components was performed. In this

experiment, a fusion preprotein pSSU-DHFR, which consists of

full-length pSSU and dihydrofolate reductase (DHFR), was used

because it produced a much higher amount of cross-linked

products than pSSU. We then selectedm-maleimidobenzoyl-N-

hydroxysuccinimide ester (MBS) as a noncleavable cross-linker

after screening of more than a dozen reagents. Arabidopsis

chloroplasts were incubated with pSSU-DHFR under either low

or high ATP conditions. After reisolation, chloroplasts were

cross-linked with MBS followed by SDS-PAGE and autoradiog-

raphy. Several cross-linked products were observed, and the

relative amounts of these products varied between low and high

ATP conditions (e.g., a 48-kD and a 100-kDbandwere prominent

under low ATP conditions, whereas a 140-kD band was prom-

inent under high ATP conditions) (Figure 8A, compare lanes 2 and

3). This indicates dynamic changes of association partners in the

translocation event.

To examine whether Tic20 is cross-linked with the preprotein,

immunoprecipitation was performed after denaturation with

SDS. All four anti-At Tic20 antisera which we prepared and

raised in different rabbits can recognize denatured At Tic20

antigens, but are unable to immunoprecipitate or immunode-

plete native Tic20 proteins. The most probable explanation for

this is that Tic20 is deeply embedded within its large complex

and does not have surface-exposed epitopes. Therefore, cross-

linked products were denatured with 2% SDS to dissociate the

1-MD Tic20 complexes and allow Tic20 to be immunoprecipi-

tated. Anti-At Tic20 antibody immunoprecipitated the single

48-kD cross-linked band (Figure 8B, lane 3), demonstrating a

direct interaction between the preprotein and Tic20. Please note

that a slight shift of this 48-kD band to a lower molecular mass

was caused by an overlap of IgG that migrated in large quantities

at this position (cf. lanes 1 and 3).

We also performed immunoprecipitations using several Toc

and Tic antibodies. Immunoprecipitation with anti-Toc75 anti-

body gave a strong band at 100 kD, which most likely represents

a 1:1 cross-linked product with Toc75, and smeared bands of

>120 kD, which probably represent products including at least

one molecule of Toc75 (lane 4). Immunoprecipitation with anti-At

Tic21 antibody did not detect any specific band (data not shown),

probably because Tic21 is not positioned close to the prepro-

tein-translocating channel. Also, immunoprecipitation with anti-

Tic110 antibody after MBS cross-linking did not detect any

specific band (see Supplemental Figure 10 online). Almost all

major cross-linked bands except a 55-kD band observed under

low ATP conditions were immunoprecipitated with either anti-

Toc75 or -Tic20 antibodies, strongly suggesting that Toc75 and

Tic20 are the proteins in the closest contact with the trans-

locating preprotein.

Moreover, we used the cleavable cross-linker dithiobis(succi-

nimidyl propionate) (DSP), which has been successfully used to

cross-link preproteins to Toc and Tic proteins (Akita et al., 1997;

Chou et al., 2003). Consistent with the results of MBS cross-

linking, anti-Toc75 and -Tic20 antibodies immunoprecipitated

pSSU-DHFR more efficiently under low ATP conditions than

under high ATP conditions (Figure 8C, lanes 3, 4, 7, and 8). In

addition, immunoprecipitation with anti-Tic110 antibody was

able to capture a small amount of pSSU-DHFR under low ATP

conditions (lane 5) and both pSSU-DHFR and mSSU-DHFR

under high ATP conditions (lane 9), which are consistent with the

results of Chou et al. (2003).

DISCUSSION

We report the identification of a 1-MD translocation complex as

an intermediate during preprotein import into chloroplasts. Char-

acteristic features of the 1-MD translocation complex are sum-

marized as follows. (1) This complex is formed under limited ATP

conditions and can be detected by BN-PAGE with the mild

detergent digitonin. (2) Preproteins arrested in this complex can

be chased under high ATP conditions. (3) Protease accessibility

assays and sucrose density gradient centrifugation revealed that

this complex resides in the inner membrane. (4) Tic20 forms a

1-MD complex with a minor population of Tic21 at the inner

membrane under steady state conditions, and this complexmost

likely corresponds to the 1-MD translocation complex.

All tested detergents except digitonin failed to preserve the

1-MD translocation complex (Figure 2A; data not shown), sug-

gesting a loose association between the preprotein and the

translocation complex. This feature may have impeded previous

identification of the 1-MD translocation complex.

Although the 1-MD translocation complex described in this

study was derived from the inner membrane, protease treat-

ments shown in Figure 4 revealed that the C-terminal tail of the

preprotein was exposed to the surface of chloroplasts, suggest-

ing that the arrested preprotein spans both Toc and Tic com-

plexes. Indeed, in the presence of cross-linkers, the preprotein

was cross-linked with Toc75 and Tic20 (Figure 8). On BN-PAGE,

high concentrations of Coomassie blue (0.125%), which has an

anionic feature, was added to the detergent-solubilized protein

complexes, potentially causing dissociation of loosely associ-

ated proteins (Schagger and Pfeiffer, 2000; Gavin et al., 2003;

10 of 17 The Plant Cell

Figure 8. Chemical Cross-Linking of a Preprotein to Toc/Tic Proteins.

(A) Energy-depleted Arabidopsis chloroplasts were incubated with [35S]pSSU-DHFR under either low ATP concentrations (100 mM) or high ATP

concentrations (4 mM) in HS buffer containing 5 mM MgCl2, 5 mM DTT, 3 mM Met, 3 mM Cys, 5 mL/mL protease inhibitor cocktail, and 20 mM

methotrexate (MTX) for 10 min at 258C in the dark. The DHFR fusion protein, pSSU-DHFR, was used in this experiment instead of pSSU. Reisolated

chloroplasts carrying [35S]pSSU-DHFR, at 0.24 mg chlorophyll/mL in HS buffer in the presence of 20 mM MTX, were treated with (lanes 2 and 3) or

without (lane 1) 0.2 mM MBS. Noncleavable cross-linked products were subjected to 7.5% SDS-PAGE and autoradiography. Single asterisks indicate

cross-linked products of pSSU-DHFR with Toc75 or Tic20. The double asterisk indicates a cross-linked product with unidentified protein.

(B) The translocation intermediate was generated under low ATP concentrations and cross-linked with 0.2 mM MBS as in (A). Chloroplasts carrying

cross-linked products were solubilized under denaturing conditions containing 2% SDS. The resulting extract was diluted 18.5-fold with 0.5% Triton

X-100 in TBS followed by immunoprecipitation with anti-At Tic20 FL (lane 3) or anti-Ps Toc75 (lane 4) antibodies or with preimmune serum (lane 2). The

eluates were subjected to 7.5% SDS-PAGE and autoradiography. Input represents 30% of the material used for immunoprecipitation (lane 1). The

region marked with a diamond probably represents cross-linked products containing at least one Toc75 molecule.

(C) The translocation intermediate was generated under either low ATP concentrations (lanes 1 to 5) or high ATP concentrations (lanes 6 to 9) as in (A).

Cross-linking was performed with 0.2 mM DSP, which is cleavable with reducing agents. Chloroplasts carrying cross-linked products were solubilized

under denaturing conditions containing 2% SDS. The resulting extract was diluted 18.5-fold with 0.5% Triton X-100 in TBS followed by

immunoprecipitation with anti-At Tic20 FL (lanes 3 and 7), anti-Ps Toc75 (lanes 4 and 8), or anti-Ps Tic110 (lanes 5 and 9) antibodies or with

preimmune serum (lane 2). Bound proteins were eluted by boiling in 23 Laemmli sample buffer containing 10% 2-mercaptoethanol and subjected to

12.5% SDS-PAGE and autoradiography. Input represents 30% of the material used for immunoprecipitation (lanes 1 and 6).

The 1-MDa Tic complex of chloroplasts 11 of 17

Wittig and Schagger, 2005). It is highly probable that the Toc

complex was dissociated from the preprotein under the condi-

tions of BN-PAGE, whereas the Tic complex remained associ-

atedwith the preprotein during BN-PAGE andwas observable as

the 1-MD complex. Considering unidirectional protein transport

from the outer membrane to the inner membrane, it seems

reasonable that the translocating preprotein at this stage binds

more tightly to the Tic complex than to the Toc complex. When

chemical cross-linkingwasperformed to stabilize the associations

of Toc and Tic proteins prior to solubilization, the 1-MD translo-

cation complex was shifted to a higher molecular mass range

around 1.4 to 1.8MDonBN-PAGE,whichmost likely corresponds

to a Toc-Tic supercomplex (see Supplemental Figure 11 online).

The 1-MD translocation complex characterized in this study is

neither the Toc complex, to which preproteins initially bind, nor

the Tic110-containing complex, which should mediate a later

translocation step on the stromal side. We propose that the

1-MD translocation complex functions in between the Toc- and

Tic110-containing complexes. Tic110 andHsp93migrated in the

range of 200 to 300 kD on BN-PAGE (see Supplemental Figure 7

online; Caliebe et al., 1997; Kuchler et al., 2002), and Tic22

migrated at a low molecular mass (<66 kD) (data not shown),

supporting the idea that these Tic components are not involved in

the 1-MD translocation complex. Figure 8C shows that Toc75 is

preferentially associated with the precursor form of the prepro-

tein, whereas Tic110 is associated with both the precursor and

mature forms of the preprotein, consistent with the results of

Chou et al. (2003). Meanwhile, Tic20 is preferentially associated

with the precursor form of the preprotein, supporting the idea

that the Tic20-containing 1-MD complex functions in between

the Toc- and Tic110-containing complexes. The preprotein

arrested in the 1-MD complex would be transferred to the

Tic110/Hsp93 complex in a subsequent step, where the transit

peptide would be processed.

By analogy with mitochondrial import machinery, it is often

stated that Tic110 is an analogous component to Tim44 (Bedard

and Jarvis, 2005), which serves as a binding site for matrix Hsp70.

The situation that the 1-MD complex containing Tic20 and Tic21

holds preproteins in the absence of Tic110 is very similar to that of

the mitochondrial import since a Tim core complex containing

Tim23 and Tim17 can hold preproteins in the absence of Tim44 in

digitonin extracts (Dekker et al., 1997). It should be noted that,

despite the absence of any significant sequence similarities, Tic20

and Tic21, and mitochondrial Tim23 and Tim17, all have similar

molecular weights and contain three or four predicted transmem-

brane domains, suggesting functional similarity.

We have observed the protease-resistant fragment SSU-DPs

arrested in the 1-MD complex (Figure 4). Earlier studies have

depicted similar protease-resistant fragments (Friedman and

Keegstra, 1989; Waegemann and Soll, 1991, 1993; Chigri et al.,

2005; Inoue and Akita, 2008). Protease-resistant fragments are

called deg and classified into deg1, deg2, deg3, or deg4 based

on different molecular sizes, from the largest (deg1) to the

smallest (deg4). deg1 and deg2 were shown to cofractionate

with the outer membrane, whereas deg3 and deg4, which would

correspond to SSU-DPs in this study, were shown to cofrac-

tionate with the inner membrane (Waegemann and Soll, 1993;

Soll and Tien, 1998), supporting our observations.

Akita et al. (1997) have shown that a translocation intermediate

complex of ;600 kD could be isolated using chemical cross-

linkers. They generated the intermediate under incubation con-

ditions on ice for 20 min in the presence of 75 mM ATP, whereas

we incubated at 258C for 10 min in the presence of 0.5 mM ATP.

Under their conditions, an early intermediate is predicted to be

formed; therefore, the 600-kD complex most likely corresponds

to the Toc subcomplex (II) in our previous study (Kikuchi et al.,

2006). In addition, Chen and Li (2007) have recently shown that

two intermediate complexes of;880 and 1320 kD, which were

referred to as C1 and C2, respectively, were observed by BN-

PAGE. They concluded that both C1 and C2 contained the Toc

complex, while C2 additionally contained Tic110, Hsp93, and the

intermembrane space Hsp70. There are significant experimental

differences between their study and ours. After the import

reaction at 208C for 20 min, they first performed chemical

cross-linking to preserve the translocation intermediate com-

plexes. Then, isolated total membranes were solubilized with the

more stringent detergent decyl maltoside and fractionated by

sucrose density gradient centrifugation. C1 most likely corre-

sponds to the intact Toc complex characterized in our previous

study (Kikuchi et al., 2006). C2 likely corresponds to the Toc-Tic

supercomplex shown in Supplemental Figure 11 online. How-

ever, the involvement of Tic21 and/or Tic20 in formation of C2

was not analyzed. Moreover, since there are significant exper-

imental differences, some Tic proteins may be added to or

removed from the C2 complex.

Kouranov and Schnell (1997) have shown that the association

of the preprotein with Tic20 was increased in the presence of

high ATP (2 mM) compared with low ATP (0.1 mM). By contrast,

the cross-linked product between the preprotein and Tic20 was

more prominent in low ATP (0.1 mM) than in high ATP (4 mM) in

this study (Figure 8A). This difference can be explained by the

following reasons. In the study by Kouranov and Schnell (1997),

they used a urea-denatured preprotein that had been overex-

pressed in E. coli and purified from inclusion bodies. By

contrast, we used in vitro–translated soluble preproteins. In

addition, the preprotein used by Kouranov and Schnell con-

tained an IgG binding domain of Protein A at the C terminus.

Perhaps this are why the preprotein they used seems to require

more ATP to be unfolded and reach the Tic20-containing

complex than that required for the in vitro–synthesized prepro-

tein used in this study.

There are two conflicting reports about the function of Tic21,

which has been reported to be a component of the protein import

machinery at the inner envelope membrane (Teng et al., 2006) or

an iron transporter (Duy et al., 2007). To clarify the function of

Tic21, we performed transient expression and targeting of pre-

proteins in mutant protoplasts and compared expression levels

of metal homeostasis proteins in mutants. Figure 7 shows a

defect in protein import of photosynthetic proteins in the tic21/

pic1 mutant, which is comparable to that in the tic20 mutant.

Supplemental Figure 8 online shows that upregulation of ferritin,

CSD1, and CSD2, all of which are iron homeostasis-related

proteins, is not specific to the tic21/pic1 mutant but is seen in

other albino mutants. These observations support the proposal

by Teng et al. (2006) that Tic21/PIC1 functions in chloroplast

protein import.

12 of 17 The Plant Cell

The results shown in Figures 6A and 6B suggest that Tic21 is

not a central component of the 1-MD translocation complex but

loosely associated component of the complex. Nevertheless, the

lack of Tic21 causes severe defects in chloroplast protein import,

similar to those observed in the tic20 mutant. Preliminary anal-

ysis of the Tic20 complex in the tic21 mutant by 2D-BN/SDS-

PAGE showed that the Tic20 complex of the tic21mutant did not

migrate at the same position as that of the wild type but

accumulated at the top of the separation gel in BN-PAGE (S.

Kikuchi and M. Nakai, unpublished data), suggesting that an

improper assembly or an aggregation of the Tic20 complex

probably occurs in the tic21 mutant. Tic21 may function in the

proper assembly of the Tic20 complex. This hypothesis can

explain similar severe albino phenotypes of the tic20 and tic21

mutants (Teng et al., 2006).

Although the tic20 and tic21 mutants show severe albino

phenotypes and are seedling lethal, they are able to produce

albino leaves and occasionally inflorescence tissues on synthetic

media supplemented with sucrose. This suggests residual im-

port capacities in the tic20 and tic21mutants. As shown in Figure

7, import defects in the tic20 and tic21 mutants were clearly

observed using photosynthetic preprotein (RbcS-nt:GFP). How-

ever, interestingly, less severe import defects were observed

using nonphotosynthetic preprotein (E1a-nt:GFP). Indeed, stro-

mal Hsp70, ferritin, and thylakoidal Albino3, which are nonpho-

tosynthetic and housekeeping proteins, accumulated normally in

the plastids of the tic20 and tic21 mutants (see Supplemental

Figure 8 online). From these observations, we propose that Tic20

and Tic21 have substrate specificity for photosynthetic prepro-

teins. The phenotypes of the tic20 and tic21 mutants are very

similar to that of an Arabidopsis ppi2mutant in which the Toc159

gene is disrupted (Bauer et al., 2000; Teng et al., 2006). Many

photosynthetic proteins are deficient in theppi2mutant, whereas

nonphotosynthetic proteins seem to accumulate normally. In the

ppi2 mutant, Toc132 and Toc120, which are homologs of

Toc159, compensate for the absence of Toc159, at least to

some extent. The Tic20 family consists of four genes in Arabi-

dopsis: Tic20-I, Tic20-IV, Tic20-II, and Tic20-V (Jarvis, 2008).

Tic20-I characterized in this study is the most abundant isoform

among four proteins and is the closest homolog to the biochemi-

cally identified pea Tic20 (Kouranov et al., 1998). The other three

Tic20 isoforms in Arabidopsis may be responsible for the import

of nonphotosynthetic and housekeeping proteins. Preliminary

experiments indicated that certain double knockout mutations

introduced into the fourArabidopsis Tic20 genes resulted inmore

severe embryo lethal phenotype (S. Kikuchi, Y. Hirabayashi, and

M. Nakai, unpublished data), suggesting that the residual import

capacities observed in the tic20 and tic21 mutants may be due

to the presence of another Tic20 isoform-containing channel

that probably has different substrate specificity. These issues are

currently under investigation.

To date, the Tic110/Tic40/Hsp93 complex, which mediates

stromal side events, has received considerable attention with

regard to translocation across the inner membrane. However, a

Tic core complex that functions in between the Toc complex and

the Tic110/Tic40/Hsp93 complex has not yet been reported. We

believe that the 1-MD translocation complex characterized in

this study corresponds to this Tic core complex, which should

contain a protein-conducting channel. While Tic20 and Tic21

would play a crucial role in the 1-MD complex, we can easily

speculate that most constituents of the 1-MD complex have not

yet been identified. Therefore, further work will be required to

identify new components.

METHODS

Plant Material and Growth Conditions

Pea (Pisum sativum var Alaska) seedlings were grown on vermiculite in a

growth chamber under 14 h light at 258C/10 h dark at 238Ccycles for 12 to

13 d. The Arabidopsis thaliana mutants tic20 (SALK_039676) and tic21/

pic1-1 (SALK_104852) carrying T-DNA insertion(s) were kindly provided

by the Salk Institute Genomic Analysis Laboratory (Alonso et al., 2003).

Arabidopsis ecotype Columbia was used as the wild type. Arabidopsis

(wild type and mutants) were grown on MS plates (13 Murashige and

Skoog salts [Sigma-Aldrich], 13 Gamborg’s B5 vitamin [Sigma-Aldrich],

2% sucrose, pH 5.8, and 0.3% phytagel [Sigma-Aldrich]) in a growth

chamber under 16 h light at 238C /8 h dark at 218C cycles for 18 to 21 d

(Yabe et al., 2004; Asakura et al., 2008).

Isolation of Chloroplasts

Chloroplast isolation frompea leaveswasdescribed in our previous report

(Kikuchi et al., 2006). Arabidopsis chloroplasts were isolated by direct

homogenization method as described (Bruce et al., 1994; Aronsson and

Jarvis, 2002; Schulz et al., 2004) with the following modifications. Ap-

proximately 20 g of aerial parts were homogenized in 400 mL of blending

buffer (50 mM HEPES-KOH, pH 7.8, 330 mM sorbitol, 2 mM EDTA, 1 mM

MnCl2, 1 mM MgCl2, and 50 mM sodium ascorbate [freshly added in

powder]) with or without 5 mL/mL protease inhibitor cocktail (for plant

extracts, P-9599; Sigma-Aldrich), in five 2-s pulses in a kitchen blender

equipped with disposable razor blades. The homogenate was filtered

through four layers of Miracloth (Calbiochem) and then centrifuged at

4000g for 3 min. The crude chloroplast pellet was resuspended in HS

buffer (50 mM HEPES-KOH, pH 7.8, and 330 mM sorbitol) and overlaid

onto 30% (v/v) Percoll in HSbuffer. After centrifugation at 1350g for 15min

in a swinging bucket rotor, the pellet was washed twice with HS buffer.

Purified intactchloroplastswere kepton ice in thedarkandusedwithin3h.

In Vitro Transcription and Translation of Preproteins

Plasmid pGEM-4Z-pSSU for the in vitro expression of a precursor protein

of SSUwas constructedby inserting the coding sequencederived from its

cDNA prepared from pea into pGEM-4Z vector (Promega) at a XbaI/SalI

site. ThemRNAofpSSUwassynthesized from linearizedpGEM-4Z-pSSU

construct usingRiboMAX in vitro transcription system (Promega)withSP6

RNA polymerase. The resulting mRNA was translated in a wheat germ

extract (Promega) at 258C for 2 h in the presence of [35S]Met. A plasmid for

the in vitro expression of pSSU-DHFR, which consists of the full-length

precursor to SSU fused to the entire mouse dihydrofolate reductase, was

constructed by inserting the pSSU coding sequence lacking the stop

codon into pPC-DHFR/SP (Endo et al., 1994) at a PstI/BamHI site. The

fusion protein pSSU-DHFR was synthesized using TNT SP6-coupled

reticulocyte lysate system (Promega) at 308C for 90min in the presence of

[35S]Met. The translation mixtures were kept on ice and used within 3 h.

Formation of a Translocation Intermediate, Protease Treatments,

and Gel Electrophoresis

Isolated intact chloroplasts in HS buffer containing 5 mL/mL protease

inhibitor cocktail were preincubated for 10 min at 258C in the dark to

The 1-MDa Tic complex of chloroplasts 13 of 17

deplete endogenous ATP. Energy-depleted chloroplasts weremixedwith

in vitro–translated preproteins in HS buffer containing 0.5 mMMg-ATP, 5

mM MgCl2, 5 mM DTT, 3 mM Met, and 3 mM Cys. The reactions were

incubated for 10 min at 258C in the dark to form a translocation interme-

diate. Each reaction contained chloroplasts equivalent to 100 mg chlo-

rophyll in a reaction volume of 400 mL. The reactions were terminated by

reisolation of chloroplasts by centrifugation at 1500g for 1 min at 48C, and

chloroplasts were washed once with HS buffer containing 5 mL/mL

protease inhibitor cocktail. When chase experiments or protease treat-

ments were performed, the above reactions were performed as a batch

reaction corresponding to the number of various conditions. Before

protease accessibility assays, chloroplasts carrying preproteins were

washed twice with HS buffer in the absence of protease inhibitor cocktail.

For thermolysin treatment, chloroplasts in HS buffer were incubated with

10 to 100 mg/mL of thermolysin (Sigma-Aldrich) and 1 mM CaCl2 for 20

min on ice. Thermolysin was inactivated by the addition of 10 mM EDTA.

For trypsin treatment, chloroplasts in HS buffer were incubated with 10 to

100 mg/mL of trypsin (Sigma-Aldrich) for 20 min on ice. Trypsin was

inactivated by the addition of a fivefold excess of soybean trypsin inhibitor

(0.5 mg/mL; Sigma-Aldrich). Proteolyzed chloroplasts were pelleted and

washed twice with HS buffer containing either 5 mL/mL protease inhibitor

cocktail or 0.5 mg/mL trypsin inhibitor.

Samples prepared by the above methods were analyzed by two

different electrophoresis methods: BN-PAGE and SDS-PAGE. The

BN-PAGE method was described in detail in our previous report (Kikuchi

et al., 2006). The chloroplast pellet was solubilized in freshly prepared

BN-PAGE sample buffer (1% [w/v] water-soluble digitonin, 50 mM

BisTris-HCl, pH 7.0, 500 mM 6-amino-n-caproic acid, and 10% [w/v]

glycerol) containing 10 mL/mL protease inhibitor cocktail to a final

concentration of 0.5 mg chlorophyll/mL for 10 min on ice. Water-soluble

digitonin was prepared as described (Mori et al., 1999). When using

trypsin as a protease, trypsin inhibitor was added to a final concentration

of 0.5mg/mL toBN-PAGE sample buffer. Insolublematerial was removed

by ultracentrifugation at 100,000g for 10 min at 48C. The supernatant was

divided into two aliquots. One of which (40 mL) was mixed with

Coomassie Brilliant Blue G 250 solution (5% [w/v] Serva blue G, 50 mM

BisTris-HCl, pH 7.0, and 500 mM 6-amino-n-caproic acid) to give a

detergent:Coomassie ratio of 8:1 (w:w) and subjected to 4 to 14%

BN-PAGE. The other was mixed with 10% SDS and 2-mercaptoethanol

to final concentrations of 3.3 and 5%, respectively. The SDS-denatured

sample was heated at 958C for 2 min and subjected to SDS-PAGE. The

radioactive signals in dried gels were detected using a BAS-2000II image

analysis system (FujiFilm) and quantified using Image Gauge version 3.45

software (FujiFilm).

Antibody Preparation, Purification, and Immunoblotting

Two overlapping but different antigens of Arabidopsis Tic20 were

overexpressed as N-terminal hexahistidine-tagged fusion proteins in

Escherichia coli using recently developed cold shock–inducible system

(Qing et al., 2004). An antigen ofArabidopsis Tic21 was overexpressed as

a fusion protein with N-terminal 260 amino acids of T7 gene10 protein in

E. coli. An antigen of pea Tic22 was overexpressed as a C-terminal

hexahistidine-tagged fusion protein in E. coli. Regions of antigens used

were as follows: At Tic20 FL (full-length sequence not including transit

peptide), At Tic20 ES (C-terminal half part, amino acids 157 to 274), At

Tic21 (C-terminal part, amino acids 210 to 296), and Ps Tic22 (full-length

sequence including transit peptide). Recombinant At Tic20 FL, At Tic20

ES, and Ps Tic22 proteins were purified from inclusion bodies with nickel

chelate chromatography under denaturing conditions. Recombinant At

Tic21 protein was purified from inclusion bodies using SDS-PAGE and

subsequent electroelution. Purified recombinant At Tic20 FL, At Tic20 ES,

At Tic21, and Ps Tic22 proteins were injected into rabbits using Freund’s

complete adjuvant and (for subsequent boosts) Freund’s incomplete

adjuvant (Harlow and Lane, 1988). Preparation of antibodies against Ps

Toc75, Ps Toc159, Ps Toc34, Ps Tic110, So cpHsp70 (Spinacia oleracea

stromal Hsp70), So Cpn60a, and So Cpn60b was described in our

previous reports (Nishio et al., 1999; Asakura et al., 2004; Kikuchi et al.,

2006). Anti-At Tic21, -Ps Toc75, -Ps Toc159, and -Ps Toc34 antibodies

were affinity purified using the purified antigens as described previously

(Kikuchi et al., 2006). Anti-Ps Tic110 antibody was purified using rProtein

A-Sepharose (GE Healthcare). Anti-At Tic20 ES antibody was purified by

blot affinity purification (Tang, 1993).

Immunoblotting was performed as described previously (Kikuchi et al.,

2006). Toc75, Toc159, Toc34, Tic110, Tic22, and cpHsp70were detected

by an enhanced chemiluminescence system (GE Healthcare). Tic20 and

Tic21 were detected by the ECL plus system (GE Healthcare). We found

that At Tic21 protein band was lost in SDS-PAGE/immunoblotting after

standard denaturation by heating at 958C in Laemmli sample buffer;

therefore, when detecting Tic21 and distantly related Tic20, samples

were incubated at 378C for 30 min.

Antibody-Shift BN-PAGE

Antibody-shift BN-PAGE is used in several reports (e.g., Johnston et al.,

2002; Truscott et al., 2002). Chloroplasts carrying [35S]translocation

intermediate were solubilized in BN-PAGE sample buffer containing

10 mL/mL protease inhibitor as described above. After ultracentrifugation

at 100,000g for 10 min at 48C, 35 mL of the supernatant was mixed with

each purified antibody of known concentrations (0 to 10 mg) and incu-

bated for 30 min on ice with occasional mixing. After a clarifying spin, the

supernatant was mixed with Coomassie Brilliant Blue G 250 solution as

described above and subjected to BN-PAGE and autoradiography.

Immunodepletion

Chloroplasts carrying [35S]translocation intermediate were solubilized in

BN-PAGE sample buffer containing 10 mL/mL protease inhibitor as

described above. Insoluble material was removed by ultracentrifugation

at 100,000g for 10 min at 48C. Fifty to 100 mL of the supernatant was

incubated twice with 10 mL packed volume of rProtein A-Sepharose that

had been coupled with IgG for 1 h in cold room (68C) with rotational

mixing. The unbound fraction (supernatant) was recovered by centrifu-

gation and was filtered through a 0.2-mm membrane filter by centrifuga-

tion to remove residual beads.

Cross-Linking and Immunoprecipitation under

Denaturing Conditions

Chloroplasts carrying [35S]pSSU-DHFR, at 0.24 mg chlorophyll/mL in HS

buffer in the presence of 20 mM MTX and in the absence of protease

inhibitor cocktail, were treated with 0.2 mMMBS (Pierce) or 0.2 mM DSP

(Pierce) for 15 min on ice. The MBS cross-linking was quenched by

adding glycine and 2-mercaptoethanol to final concentrations of 20 mM

and 2%, respectively, and incubating on ice for another 15 min. The DSP

cross-linking was quenched by adding glycine to a final concentrations of

20 mM and incubating on ice for another 15 min. Chloroplasts were

recovered by centrifugation, washed with HS buffer containing 20 mM

MTX and 5 mL/mL protease inhibitor cocktail. Immunoprecipitation under

denaturing conditions was performed as described (Cline andMori, 2001)

with the following modifications. Chloroplasts carrying cross-linked pre-

proteins were solubilized under denaturing conditions (2% SDS, 0.5%

Triton X-100, 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, and 10 mL/mL

protease inhibitor cocktail) at 258C for 5 min at 1 mg chlorophyll/mL.

Insoluble material was removed by centrifugation, and the supernatant

was diluted 18.5-fold with 0.5% Triton X-100 in TBS (50 mM Tris-HCl, pH

7.5, and 150 mM NaCl) to enable immunoprecipitations. The diluted

sample (370 mL) was mixed with 10 mL packed volume of rProtein

14 of 17 The Plant Cell

A-Sepharose that had been coupled with IgG and incubated for 2 h at

258Cwith rotational mixing. After the beadswerewashed twicewith 0.5%

Triton X-100 in TBS, bound proteins were eluted by boiling 2 min in 23

Laemmli sample buffer. When using DSP as the cross-linker, bound

proteins were eluted by boiling 5 min in 23 Laemmli sample buffer

containing 10% 2-mercaptoethanol to cleave the cross-linker. The elu-

ates were subjected to SDS-PAGE and autoradiography.

Transient Expression in Protoplasts

Arabidopsis (wild type and mutants) were grown on MS plates. Homozy-

gous tic20 and tic21mutantswere selected frompools of plants based on

the visible albino phenotypes. Protoplasts were prepared as described

previously (Jin et al., 2001). The GFP fusion constructs were introduced

into protoplasts by polyethylene glycol–mediated transformation (Jin

et al., 2001). Protein import into wild-type and mutant plastids was

analyzedby immunoblottingwithanti-GFPantibodyusingprotein extracts

from transformed protoplasts as described previously (Lee et al., 2008).

Miscellaneous

2D-BN/SDS-PAGE was performed as described previously (Kikuchi

et al., 2006) except when detecting At Tic21, At Tic20, and Ps Tic20,

where excised first-dimension native gels were incubated at 378C for 30

min. Size exclusion chromatography was also performed as described

previously (Kikuchi et al., 2006) except that 1% water-soluble digitonin

was used for solubilization and 0.1% water-soluble digitonin was used in

equilibration buffer instead of dodecyl maltoside. Water-soluble digitonin

was prepared as described (Mori et al., 1999). Monoclonal antibody

SPA-820 against Hsp70/Hsc70 was purchased from StressGen.

Accession Numbers

Sequence data from this article can be found in the Arabidopsis Genome

Initiative or GenBank/EMBL databases under the following accession

numbers: Tic20 (At1g04940), Tic21 (At2g15290), pea Tic22 (AF095284),

and pea pSSU (X00806).

Supplemental Data

The following materials are available in the online version of this article.

Supplemental Figure 1. The 1-MD Translocation Complex Is Not a

Stromal Chaperonin Complex.

Supplemental Figure 2. The 1-MD Translocation Complex Was

Generated Using Ferredoxin:NADP+ Oxidoreductase as a Preprotein.

Supplemental Figure 3. Partial Degradation of the Toc Complex,

Which Had Occurred during the Isolation of Chloroplasts, Did Not

Affect the Formation of the 1-MD Translocation Complex.

Supplemental Figure 4. Sucrose Density Gradient Separation of

Outer and Inner Envelope Membrane Vesicles.

Supplemental Figure 5. The Arabidopsis 1-MD Translocation Com-

plex Has Identical Protease Accessibility Properties to That of the Pea

Complex.

Supplemental Figure 6. Antibody-Shift BN-PAGE.

Supplemental Figure 7. Tests for the Ability of Anti-Tic and -Toc

Antibodies to Native Proteins by Immunodepletion.

Supplemental Figure 8. Immunoblot Analyses of Several Proteins in

Albino Mutants.

Supplemental Figure 9. Protein Import into Plastids in a Transient

Expression System Using Protoplasts from the albino3 Mutant.

Supplemental Figure 10. Immunoprecipitation after Chemical Cross-

Linking with MBS.

Supplemental Figure 11. Chemical Cross-Linking of the Transloca-

tion Intermediate.

ACKNOWLEDGMENTS

We thank Toshiharu Hase, Yoko Kimata-Ariga, Guy T. Hanke, Yukari

Asakura, Toshiki Yabe, Jocelyn Bedard, and coworkers of our labora-

tory for valuable discussions, Tazu Uchida for technical assistance, and

Rieko Nakanishi for secretarial assistance. We also thank the Salk

Institute Genomic Analysis Laboratory for providing the sequence-

indexed Arabidopsis T-DNA insertion mutants tic20 and tic21/pic1,

Hsou-min Li for the gift of anti-At Tic40 antibody, Brigitte Touraine for

the gift of anti-ferritin antibody, Marinus Pilon for the gift of anti-CSD2

antibody, Toshiya Hirohashi for the preparation of anti-Ps Tic22 anti-

body, Kazuaki Nishio for the preparation of anti-stromal So Hsp70, So

Cpn60a, and So Cpn60b antibodies, and Tetsuya Nohara for the gift of

pea first-strand cDNA. This work was supported by Grants-in-Aid for

Scientific Research on Priority Areas (17028034 and 17051020) from

MEXT of Japan to M.N. This work was also supported in part by Korea

Science and Engineering Foundation through the Creative Research

Initiatives Program (20090063529) to I.H. S.K. was supported by a

research fellowship from the Japan Society for the Promotion of

Science.

ReceivedOctober 3, 2008; revisedMay 22, 2009; accepted June 1, 2009;

published June 16, 2009.

REFERENCES

Akita, M., Nielsen, E., and Keegstra, K. (1997). Identification of protein

transport complexes in the chloroplastic envelope membranes via

chemical cross-linking. J. Cell Biol. 136: 983–994.

Alonso, J.M., Stepanova, A.N., Leisse, T.J., Kim, C.J., Chen, H.,

Shinn, P., Stevenson, D.K., Zimmerman, J., Barajas, P., Cheuk, R.,

Gadrinab, C., and Heller, C. (2003). Genome-wide insertional muta-

genesis of Arabidopsis thaliana. Science 301: 653–657.

Aronsson, H., and Jarvis, P. (2002). A simple method for isolating

import-competent Arabidopsis chloroplasts. FEBS Lett. 529:

215–220.

Asakura, Y., Hirohashi, T., Kikuchi, S., Belcher, S., Osborne, E.,

Yano, S., Terashima, I., Barkan, A., and Nakai, M. (2004). Maize

mutants lacking chloroplast FtsY exhibit pleiotropic defects in the

biogenesis of thylakoid membranes. Plant Cell 16: 201–214.

Asakura, Y., Kikuchi, S., and Nakai, M. (2008). Non-identical contri-

butions of two membrane-bound cpSRP components, cpFtsY and

Alb3, to thylakoid biogenesis. Plant J. 56: 1007–1017.

Balsera, M., Goetze, T.A., Kovacs-Bogdan, E., Schurmann, P.,

Wagner, R., Buchanan, B.B., Soll, J., and Bolter, B. (2009). Char-

acterization of Tic110, a channel-forming protein at the inner envelope

membrane of chloroplasts, unveils a response to Ca2+ and a stromal

regulatory disulfide bridge. J. Biol. Chem. 284: 2603–2616.

Bauer, J., Chen, K., Hiltbunner, A., Wehrli, E., Eugster, M., Schnell,

D.J., and Kessler, F. (2000). The major protein import receptor of

plastids is essential for chloroplast biogenesis. Nature 403: 203–207.

Becker, T., Hritz, J., Vogel, M., Caliebe, A., Bukau, B., Soll, J., and

Schleiff, E. (2004). Toc12, a novel subunit of the intermembrane

space preprotein translocon of chloroplasts. Mol. Biol. Cell 15: 5130–

5144.

The 1-MDa Tic complex of chloroplasts 15 of 17

Bedard, J., and Jarvis, P. (2005). Recognition and envelope translo-

cation of chloroplast preproteins. J. Exp. Bot. 56: 2287–2320.

Bruce, B.D., Perry, S., Froehlich, J., and Keegstra, K. (1994). In vitro

import of protein into chloroplasts. In Plant Molecular Biology Manual,

S.B. Gelvin and R.A. Schilperoot, eds (Belgium: Kluwer Academic

Publishers), pp. 1–15.

Caliebe, A., Grimm, R., Kaiser, G., Lubeck, J., Soll, J., and Heins, L.

(1997). The chloroplastic protein import machinery contains a Rieske-

type iron-sulfur cluster and a mononuclear iron-binding protein.

EMBO J. 16: 7342–7350.

Chen, K., Chen, X., and Schnell, D.J. (2000). Initial binding of prepro-

teins involving the Toc159 receptor can be bypassed during protein

import into chloroplasts. Plant Physiol. 122: 813–822.

Chen, K.-Y., and Li, H.-m. (2007). Precursor binding to an 880-kDa Toc

complex as an early step during active import of protein into chloro-

plasts. Plant J. 49: 149–158.

Chen, X., Smith, M.D., Fitzpatrick, L., and Schnell, D.J. (2002). In vivo

analysis of the role of atTic20 in protein import into chloroplasts. Plant

Cell 14: 641–654.

Chigri, F., Soll, J., and Vothknecht, U.C. (2005). Calcium regulation of

chloroplast protein import. Plant J. 42: 821–831.

Chou, M.-L., Fitzpatrick, L.M., Tu, S.-L., Budziszewski, G., Potter-

Lewis, S., Akita, M., Levin, J.Z., Keegstra, K., and Li, H.-m. (2003).

Tic40, a membrane-anchored co-chaperone homolog in the chloro-

plast protein translocon. EMBO J. 22: 2970–2980.

Chou, M.-L., Chu, C.-C., Chen, L.-J., Akira, M., and Li, H.-M. (2006).

Stimulation of transit-peptide release and ATP hydrolysis by a

cochaperone during protein import into chloroplasts. J. Cell Biol.

175: 893–900.

Cline, K., and Mori, H. (2001). Thylakoid DpH-dependent precursor

proteins bind to a cpTatC-Hcf106 complex before Tha4-dependent

transport. J. Cell Biol. 154: 719–729.

Dekker, P.J.T., Martin, F., Maarse, A.C., Bomer, U., Muller, H.,

Guiard, B., Meijer, M., Rassow, J., and Pfanner, N. (1997). The Tim

core complex defines the number of mitochondrial translocation

contact sites and can hold arrested preproteins in the absence of

matrix Hsp70-Tim44. EMBO J. 16: 5408–5419.

Duy, D., Wanner, G., Meda, A.R., von Wiren, N., Soll, J., and

Philippar, K. (2007). PIC1, an ancient permease in Arabidopsis

chloroplasts, mediates iron transport. Plant Cell 19: 986–1006.

Endo, T., Kawakami, M., Goto, A., America, T., Weisbeek, P., and

Nakai, M. (1994). Chloroplast protein import. Chloroplast envelopes

and thylakoids have different abilities to unfold proteins. Eur. J.

Biochem. 225: 403–409.

Friedman, A.L., and Keegstra, K. (1989). Chloroplast protein import

quantitative analysis of precursor binding. Plant Physiol. 89: 993–999.

Gavin, P.D., Devenish, R.J., and Prescott, M. (2003). FRET reveals

changes in the F1-stator stalk interaction during activity of F1F0-ATP

synthase. Biochim. Biophys. Acta 1607: 167–179.

Harlow, E., and Lane, D. (1988). Antibodies: A Laboratory Manual.

(Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press).

Heins, L., Mehrle, A., Hemmler, R., Wagner, R., Kuchler, M.,

Hormann, F., Sveshnikov, D., and Soll, J. (2002). The preprotein

conducting channel at the inner envelope membrane of plastids.

EMBO J. 21: 2616–2625.

Hirohashi, T., Hase, T., and Nakai, M. (2001). Maize non-photosyn-

thetic ferredoxin precursor is mis-sorted to the intermembrane space

of chloroplasts in the presence of light. Plant Physiol. 125: 2154–2163.

Inaba, T., Li, M., Alvarez-Huerta, M., Kessler, F., and Schnell, D.J.

(2003). atTic110 functions as a scaffold for coordinating the stromal

events of protein import into chloroplasts. J. Biol. Chem. 278: 38617–

38627.

Inoue, H., and Akita, M. (2008). Three sets of translocation intermedi-

ates are formed during the early stage of protein import into chloro-

plasts. J. Biol. Chem. 283: 7491–7502.

Jackson, D.T., Froehlich, J.E., and Keegstra, K. (1998). The hydro-

philic domain of Tic110, an inner envelope membrane component of

the chloroplastic protein translocation apparatus, faces the stromal

compartment. J. Biol. Chem. 273: 16583–16588.

Jarvis, P. (2008). Targeting of nucleus-encoded proteins to chloroplasts

in plants. New Phytol. 179: 257–285.

Jin, J.B., Kim, Y.A., Kim, S.J., Lee, S.H., Kim, D.H., Cheong, G.-W.,

and Hwang, I. (2001). A new dynamin-like protein, ADL6, is involved

in trafficking from the trans-Golgi network to the central vacuole in

Arabidopsis. Plant Cell 13: 1511–1526.

Johnston, A.J., Hoogenraad, J., Dougan, D.A., Truscott, K.N., Yano,

M., Mori, M., Hoogenraad, N.J., and Ryan, M.T. (2002). Insertion

and assembly of human Tom7 into the preprotein translocase com-

plex of the outer mitochondrial membrane. J. Biol. Chem. 277: 42197–

42204.

Kessler, F., and Schnell, D. (2009). Chloroplast biogenesis: Diversity

and regulation of the protein import apparatus. Curr. Opin. Cell Biol.

21: in press.

Kikuchi, S., Hirohashi, T., and Nakai, M. (2006). Characterization of

the preprotein translocon at the outer envelope membrane of chlo-

roplasts by blue native PAGE. Plant Cell Physiol. 47: 363–371.

Kouranov, A., Chen, X., Fuks, B., and Schnell, D.J. (1998). Tic20 and

Tic22 are new components of the protein import apparatus at the

chloroplast inner envelope membrane. J. Cell Biol. 143: 991–1002.

Kouranov, A., and Schnell, D.J. (1997). Analysis of the interactions of

preproteins with the import machinery over the course of protein

import into chloroplasts. J. Cell Biol. 139: 1677–1685.

Kuchler, M., Decker, S., Hormann, F., Soll, J., and Heins, L. (2002).

Protein import into chloroplasts involves redox-regulated proteins.

EMBO J. 21: 6136–6145.

Lee, D.W., Kim, J.K., Lee, S., Choi, S., Kim, S., and Hwang, I. (2008).

Arabidopsis nuclear-encoded plastid transit peptides contain multiple

sequence subgroups with distinctive chloroplast-targeting sequence

motifs. Plant Cell 20: 1603–1622.

Ma, Y., Kouranov, A., LaSala, S.E., and Schnell, D.J. (1996). Two

components of the chloroplast protein import apparatus, IAP86 and

IAP75, interact with the transit sequence during the recognition and

translocation of precursor proteins at the outer envelope. J. Cell Biol.

134: 315–327.

Mori, H., Summer, E.J., Ma, X., and Cline, K. (1999). Component

specificity for the thylakoidal sec and delta pH-dependent protein

transport pathways. J. Cell Biol. 146: 45–55.

Nielsen, E., Akita, M., Davila-Aponte, J., and Keegstra, K. (1997).

Stable association of chloroplastic precursors with protein transloca-

tion complexes that contain proteins from both envelope membranes

and a stromal Hsp100 molecular chaperone. EMBO J. 16: 935–946.

Nishio, K., Hirohashi, T., and Nakai, M. (1999). Chloroplast chaper-

onins: Evidence for heterogeneous assembly of alpha and beta Cpn60

polypeptides into a chaperonin oligomer. Biochem. Biophys. Res.

Commun. 266: 584–587.

Perry, S.E., and Keegstra, K. (1994). Envelope membrane proteins that

interact with chloroplastic precursor proteins. Plant Cell 6: 93–105.

Qing, G., et al. (2004). Cold-shock induced high-yield protein produc-

tion in Escherichia coli. Nat. Biotechnol. 22: 877–882.

Schagger, H., Cramer, W.A., and von Jagow, G. (1994). Analysis of

molecular masses and oligomeric states of protein complexes by blue

native electrophoresis and isolation of membrane protein complexes by

two-dimensional native electrophoresis. Anal. Biochem. 217: 220–230.

Schagger, H., and Pfeiffer, K. (2000). Supercomplexes in the respira-

tory chains of yeast and mammalian mitochondria. EMBO J. 19:

1777–1783.

16 of 17 The Plant Cell

Schagger, H., and von Jagow, G. (1991). Blue native electrophoresis

for isolation of membrane protein complexes in enzymatically active

form. Anal. Biochem. 199: 223–231.

Schleiff, E., Soll, J., Kuchler, M., Kuhlbrandt, W., and Harrer, R.

(2003). Characterization of the translocon of the outer envelope of

chloroplasts. J. Cell Biol. 160: 541–551.

Schnell, D.J., and Blobel, G. (1993). Identification of intermediates in

the pathway of protein import into chloroplasts and their localization

to envelope contact sites. J. Cell Biol. 120: 103–115.

Schnell, D.J., Kessler, F., and Blobel, G. (1994). Isolation of compo-

nents of the chloroplast protein import machinery. Science 266: 1007–

1012.

Schulz, A., Knoetzel, J., Scheller, H.V., and Mant, A. (2004). Uptake of

a fluorescent dye as a swift and simple indicator of organelle intact-

ness: Import-competent chloroplasts from soil-grown Arabidopsis. J.

Histochem. Cytochem. 52: 701–704.

Smith, M.D., Rounds, C.M., Wang, F., Chen, K., Afitlhile, M., and

Schnell, D.J. (2004). atToc159 is a selective transit peptide receptor

for the import of nucleus-encoded chloroplast proteins. J. Cell Biol.

165: 323–334.

Soll, J., and Schleiff, E. (2004). Protein import into chloroplasts. Nat.

Rev. Mol. Cell Biol. 5: 198–208.

Soll, J., and Tien, R. (1998). Protein translocation into and across the

chloroplastic envelope membranes. Plant Mol. Biol. 38: 191–207.

Sundberg, E., Slagter, J.G., Fridborg, I., Cleary, S.P., Robinson, C.,

and Coupland, G. (1997). ALBINO3, an Arabidopsis nuclear gene

essential for chloroplast differentiation, encodes a chloroplast protein

that shows homology to proteins present in bacterial membranes and

yeast mitochondria. Plant Cell 9: 717–730.

Tang, W.-J.Y. (1993). Blot-affinity purification of antibodies. Methods

Cell Biol. 37: 95–104.

Teng, Y.-S., Su, Y.-s., Chen, L.-J., Lee, Y.J., Hwang, I., and Li, H.-m.

(2006). Tic21 is an essential translocon component for protein trans-

location across the chloroplast inner envelope membrane. Plant Cell

18: 2247–2257.

Truscott, K.N., Wiedemann, N., Rehling, P., Muller, H., Meisinger, C.,

Pfanner, N., and Guiard, B. (2002). Mitochondrial import of the ADP/

ATP carrier: the essential TIM complex of the intermembrane space is

required for precursor release from the TOM complex. Mol. Cell. Biol.

22: 7780–7789.

Waegemann, K., and Soll, J. (1991). Characterization of the protein

import apparatus in isolated outer envelopes of chloroplasts. Plant J.

1: 149–158.

Waegemann, K., and Soll, J. (1993). Isolation and characterization of a

functionally active protein translocation apparatus from chloroplast

envelopes. In Molecular Mechanisms of Membrane Traffic, NATO ASI

Ser. H74, D.J. Morre, K.E. Howell, and J.J.M. Bergeron, eds (Berlin/

Heidelberg: Springer-Verlag), pp. 101–104.

Wittig, I., and Schagger, H. (2005). Advantages and limitations of clear-

native PAGE. Proteomics 5: 4338–4346.

Yabe, T., Morimoto, K., Kikuchi, S., Nishio, K., Terashima, I., and

Nakai, M. (2004). The Arabidopsis chloroplastic NifU-like protein CnfU,

which can act as an iron-sulfur cluster scaffold protein, is required for

biogenesis of ferredoxin and photosystem I. Plant Cell 16: 993–1007.

Young, M.E., Keegstra, K., and Froehlich, J.E. (1999). GTP promotes

the formation of early-import intermediates but is not required during

the translocation step of protein import into chloroplasts. Plant

Physiol. 121: 237–243.

The 1-MDa Tic complex of chloroplasts 17 of 17

DOI 10.1105/tpc.108.063552; originally published online June 16, 2009;Plant Cell

Shingo Kikuchi, Maya Oishi, Yoshino Hirabayashi, Dong Wook Lee, Inhwan Hwang and Masato NakaiProtein Import at the Inner Envelope Membrane

A 1-Megadalton Translocation Complex Containing Tic20 and Tic21 Mediates Chloroplast

 This information is current as of June 20, 2018

 

Supplemental Data /content/suppl/2009/06/09/tpc.108.063552.DC1.html

Permissions https://www.copyright.com/ccc/openurl.do?sid=pd_hw1532298X&issn=1532298X&WT.mc_id=pd_hw1532298X

eTOCs http://www.plantcell.org/cgi/alerts/ctmain

Sign up for eTOCs at:

CiteTrack Alerts http://www.plantcell.org/cgi/alerts/ctmain

Sign up for CiteTrack Alerts at:

Subscription Information http://www.aspb.org/publications/subscriptions.cfm

is available at:Plant Physiology and The Plant CellSubscription Information for

ADVANCING THE SCIENCE OF PLANT BIOLOGY © American Society of Plant Biologists