toc64 is not required for import of proteins into chloroplasts in the moss physcomitrella patens

Toc64 is not required for import of proteins into chloroplastsin the moss Physcomitrella patens

Nancy Rosenbaum Hofmann and Steven M. Theg*

Section of Plant Biology, University of California at Davis, Davis, CA 95616, USA

Received 11 March 2005; revised 25 May 2005; accepted 6 June 2005.*For correspondence (fax þ530 752 5410; e-mail [email protected]).

Summary

Toc64 has been suggested to be part of the chloroplast import machinery in Pisum sativum. A role for Toc64 in

protein transport has not been established, however. To address this, we generated knockout mutants in the

moss Physcomitrella patens using the moss’s ability to perform homologous recombination with nuclear DNA.

Physcomitrella patens contains two genes that encode Toc64-like proteins. Both of those proteins appear to be

localized in the chloroplast. The double-mutant plants were lacking Toc64 protein in the chloroplasts but

showed no growth phenotype. In addition, these plants accumulated other plastid proteins at wild-type levels

and showed no difference from wild type in in vitro protein import assays. These plants did have a slightly

altered chloroplast shape in some tissues, however. The evidence therefore indicates that Toc64 proteins are

not required for import of proteins in Physcomitrella, but may point to involvement in the determination of

plastid shape.

Keywords: chloroplast, moss, OEP64, Physcomitrella patens, protein transport, Toc64.

Introduction

Over 90% of chloroplast proteins are encoded in the nuclear

genome and post-translationally targeted to the plastid

(Abdallah et al., 2000). Most of these proteins are synthe-

sized as precursors with NH2-terminal transit peptides that

contain the targeting information for transport into the

chloroplast. Precursor proteins cross the envelope mem-

branes in a process driven by nucleotide hydrolysis and

have their transit peptides cleaved upon entry to the stroma

(Chen et al., 2000; Keegstra and Cline, 1999; Soll and

Schleiff, 2004). Nuclear-encoded thylakoid lumen proteins

contain bipartite transit peptides, in which the second half of

the transit peptide is cleaved upon transport to the lumen.

The transport of precursor proteins across the envelope is

mediated by proteins in each membrane of the envelope and

by stromal proteins, such as chaperones and the processing

protease. The transporter proteins stably associate into

complexes referred to as the Toc and Tic complexes (for

Translocon at the Outer, and Inner, membrane of the

Chloroplast envelope, respectively).

Toc75 is a b-barrel protein of the Toc complex that

appears to form the channel through which precursor

proteins cross the outer membrane (Hinnah et al., 1997;

Reumann et al., 1999; Schnell et al., 1994). Toc159 is a

cytoplasm-facing guanosine triphosphatase (GTPase) that

interacts with precursor proteins during transport, per-

haps acting as a receptor (Hirsch et al., 1994; Kouranov

and Schnell, 1997). It behaves as an integral membrane

protein, but a cytoplasmic pool of Toc159 has also been

reported (Bauer et al., 2002; Hiltbrunner et al., 2001).

Toc34 is a related GTPase that is anchored in the outer

membrane with the bulk of the protein in the cytoplasm

(Kessler et al., 1994; Seedorf et al., 1995). It interacts with

precursors early in the transport process and has been

suggested by some groups to be a receptor (Kouranov

and Schnell, 1997; Soll and Schleiff, 2004). It also seems

to play a role in targeting Toc159 to the Toc complex

(Bauer et al., 2002; Smith et al., 2002; Wallas et al., 2003).

Another protein, called Toc64, was reported to co-purify

with the Toc complex from pea (Pisum sativum) chloroplasts

(Sohrt and Soll, 2000). This protein consists of a short

NH2-terminal hydrophobic domain, a region with homology

to amidases and three tetratricopeptide repeats (TPRs) at the

COOH-terminus. It is an outer membrane protein with the

COOH-terminus in the cytoplasm. Upon cross-linking, Toc64

was found in a complex containing Toc and Tic proteins as

well as precursor proteins (Sohrt and Soll, 2000). In addition,

ª 2005 Blackwell Publishing Ltd 675

The Plant Journal (2005) 43, 675–687 doi: 10.1111/j.1365-313X.2005.02483.x

antibodies to Toc64 co-immunoprecipitated Toc and Tic

proteins from outer envelope vesicles (Becker et al., 2004).

Arabidopsis thaliana has three genes that encode proteins

related to Toc64 based on sequence similarity (Jackson-

Constan and Keegstra, 2001) (Figure 1). Two of these

proteins have all three domains and, like the pea protein,

lack the putative catalytic residues (Patricelli and Cravatt,

2000) in the amidase region. A third protein consists of the

Figure 1. Toc64 sequences from Physcomitrella patens and other plants.

Two Toc64-like proteins from P. patens (PpToc64-1: AAS47584 and PpToc64-2: AAS47585) are aligned with related sequences from pea (PsToc64: AAF62870),

Arabidopsis (AtToc64-III: BAB02718, AtmtOM64: CAC0546 and AtToc64-I: NP_563831) and rice (Oryza sativa, OsToc64: AAK50116). Conserved residues are

represented with dark backgrounds. The NH2-terminal hydrophobic domain, the putative amidase catalytic residues and the starts of the three 34-amino-acid

tetratricopeptide repeats (TPRs) are labeled above the alignment. This alignment was previously published as supplemental material (Hofmann and Theg, 2003).

676 Nancy Rosenbaum Hofmann and Steven M. Theg

ª Blackwell Publishing Ltd, The Plant Journal, (2005), 43, 675–687

amidase region only and does have the catalytic residues.

One of the two proteins with all three domains, AtToc64-III,

has been shown to be localized to the chloroplast in vivo

(Lee et al., 2004). In addition, the NH2-terminal transmem-

brane domain together with a few flanking residues was

found to be sufficient to target a passenger protein to

chloroplasts (Lee et al., 2004). The other Arabidopsis protein

with all three domains was found to be a mitochondrial

protein and is now referred to as AtmtOM64, though its

function is not known (Chew et al., 2004).

The role that Toc64 might play in chloroplast protein

targeting is not clear. In fact, Toc64 was not required to

reconstitute Toc function in liposomes. When the Toc ‘core’

complex of Toc75, Toc159 and Toc34 was present, guano-

sine triphosphate (GTP)-dependent translocation of precur-

sor proteins was observed (Schleiff et al., 2003). It was

originally suggested that Toc64 might play a role in chloro-

plast protein transport as a docking factor for incoming

precursor proteins in a guidance complex (Sohrt and Soll,

2000). This was based on the fact that the TPRs, which are

usually protein–protein interaction domains, were facing the

cytoplasm. More recently, a role has been proposed for

Toc64 in interaction with transport proteins in the inter-

membrane space (Becker et al., 2004). However, the trans-

membrane domain of Toc64 is within 10–15 amino acid

residues of the NH2-terminus and the rest of the protein

appears to reside in the cytoplasm. Thus, it is not clear that

enough Toc64 extends into the intermembrane space to

facilitate the proposed interaction.

We sought to verify a role for Toc64 in chloroplast protein

targeting by generating knockout mutants using the moss

Physcomitrella patens as our model plant. This moss has

been used in cell biology for many years and has been

shown to undergo homologous recombination in the nuc-

lear genome at high rates, unlike any other land plant

characterized so far (Kammerer and Cove, 1996; Schaefer,

2002; Schaefer and Zryd, 1997). We recently showed that

P. patens is a good model organism for studying chloroplast

protein transport (Hofmann and Theg, 2003). We identified

known individual translocation machinery components in

the large expressed sequence tag databases available for

moss. We also showed that P. patens is amenable to

transport assays using isolated plastids. These findings,

coupled with the ability to perform targeted gene replace-

ments in moss, mean that it is possible to readily combine

molecular genetic and biochemical techniques in the same

model organism when using P. patens.

Results

Toc64 is present in moss

We previously cloned two genes encoding Toc64 proteins in

P. patens (Hofmann and Theg, 2003). Both proteins have the

NH2-terminal hydrophobic domain, an amidase-like region

and TPRs that are hallmarks of Toc64-like proteins (Figure 1)

(Hofmann and Theg, 2003). Unlike the Toc64s from vascular

plants, however, these proteins do have the putative cata-

lytic residues in the amidase region. PpToc64-1 and

PpToc64-2 are 72% identical and 85% similar to each other.

In addition, they are more similar to chloroplastic Toc64

proteins from pea and Arabidopsis than to the mitochondrial

AtmtOM64 (Table 1).

To confirm that Toc64 protein was located in moss

chloroplasts, we generated an antibody that should react

with both moss proteins (see Experimental procedures).

This antibody recognized an appropriately sized band when

reacted against P. patens chloroplasts (Figure 2a). We

compared the signal from chloroplasts with that obtained

from protoplasts to see whether any Toc64 protein was

found outside the chloroplasts in the cell (Figure 2a). A

second gel was loaded identically and stained to show all

proteins (Figure 2b). When corrections were made to

account for loading differences, there was no difference

between the amount of Toc64 protein in chloroplasts and

that in the entire cell (Figure 2c). This indicates that both

PpToc64-1 and PpToc64-2 are in the chloroplast, since all of

the protein that our antibody recognizes is associated with

chloroplasts.

Generation of Toc64 single mutants

To test whether Toc64 plays a role in chloroplast protein

targeting, we generated knockout mutants in P. patens.

PpToc64-2 was targeted using a construct designed to

interrupt the locus with a 35S-driven nptII gene for resist-

ance to G418 (Figure 3a). This resistance cassette was pre-

ceded by about 1 kb of genomic sequence from the

PpToc64-2 gene. Genomic DNA from transformants was

screened by PCR to amplify the PpToc64 loci. In the

PpToc64-2::nptII plant only the PpToc64-1 locus could be

amplified, while PpToc64-2 could not (Figure 3a, left panel).

This is presumably because the PpToc64-2 locus now

included the resistance gene and was too large to be

amplified. In wild-type plants, both loci could be amplified

(Figure 3a, left panel). To confirm targeting, primers that

annealed to PpToc64-2 outside the region included in the

transformation cassette (primers 2S and 2A, Figure 3a) were

Table 1 Percentage similarity among Toc64-like proteins. Proteinsare the same as those in Figure 1

PsToc64 AtToc64-III AtmtOM64

PpToc64-1 72 69 66PpToc64-2 71 70 64OsToc64 73 75 65PsToc64 81 66AtToc64-III 65

Toc64 in chloroplast protein targeting 677


used for PCR in addition to primers binding to the resistance

gene (primers RS and RA, Figure 3a). Primers 2S and RA

were able to amplify a product from PpToc64-2::nptII but not

from wild type, as were primers RS and 2A (Figure 3a, right

panel). These products correspond to the 5¢ and 3¢ junctions

of the transformation cassette in the PpToc64-2 locus,

respectively, and confirm the correct targeting of the cas-

sette in the locus.

To test whether the insertion of a resistance gene into the

PpToc64-2 locus disrupted the expression of the gene, we

performed reverse transcriptase (RT)-PCR. The message for

both Toc64s was present in wild-type RNA, but the PpToc64-2

message was absent in RNA from PpToc64-2::nptII (Fig-

ure 3b). Despite this, PpToc64-2::nptII plants were indistin-

guishable from wild type (Figure 3c). Since it seemed likely

that both Toc64 proteins in P. patens were chloroplast

proteins, we checked the level of Toc64 protein in the

knockout plant. Western blots revealed that a significant

amount of Toc64 protein remained in PpToc64-2::nptII

chloroplasts (Figure 3d). In the absence of PpToc64-2,

PpToc64-1 could account for the protein remaining in the

chloroplast.

Generation of a double-knockout plant

Because the single-knockout plant still had a significant

amount of Toc64 protein in its chloroplasts, we generated a

double knockout. PpToc64-2::nptII plants were transformed

with a construct containing a 35S-driven hygromycin

resistance gene flanked by about 1 kb of cDNA sequence

from the PpToc64-1 locus (Figure 4a). Again, PCR on

genomic DNA was performed to confirm the correct target-

ing (Figure 4a). Both loci could be amplified from wild-type

DNA, while neither targeting product could. In the double-

mutant plant neither full-length locus could be amplified, but

targeting products could be. Western blots confirmed that

Toc64 protein was no longer present in the chloroplasts of

the double mutant (Figure 4b). Other proteins continued to

accumulate at near wild-type levels, however. The levels of

the Tic translocon protein, Tic40, were unchanged in the

double mutant. In addition, the stromal proteins cpHsp93

(ClpC) and cpHsp70 were present, as were the thylakoid

proteins LHCP and OE33 (Figure 4b).

Consistent with this lack of change in protein accumula-

tion, the double-mutant plants did not look different from

wild type (Figure 5a). They were as green as wild type and

grew at similar rates. Light microscopy of chloronemal

tissue revealed no large phenotypic differences (Figure 5b,

panels i–iv). In caulonemal filaments, however, the chloro-

plasts of the double mutant seemed to be somewhat longer

and thinner than those of wild type (Figure 5b, panels v–vii).

Electron micrographs of wild-type and double-mutant chlo-

roplasts revealed dramatic differences in some tissues

(Figure 6). While not all chloroplasts appeared to be altered

in shape, some were unmistakably different (Figure 6,

panels a–d). Measurements of chloroplast length and width

in all tissues showed that the mutant chloroplasts were on

average slightly, but significantly, longer and thinner than

wild type (Table 2). Despite this change in shape there was

no difference between the number of chloroplasts in wild-

30--

45--

66--

97--

220--

1 32132kDa

chloroplasts protoplasts(a)

20.1--

30--

45--

66--

97--

220--

1 32132kDa

chloroplasts protoplasts(b)

*

(c)

0123456

0 1 2 3 4

Toc

64 p

rote

in (

arbi

trar

y un

its)

chloroplastsprotoplasts

Total protein (arbitrary units)

Figure 2. Physcomitrella patens chloroplasts contain Toc64 protein.

(a) Antibodies against Toc64 react with moss tissue. Increasing amounts of

moss chloroplasts and protoplasts were probed with antibodies raised

against Toc64. The arrow marks the Toc64 protein.

(b) Coomassie-stained SDS-PAGE gel of the samples probed in (a). The

asterisk marks bovine serum albumin from the buffer in the chloroplast

samples.

(c) All of the Toc64 protein in moss cells is in chloroplasts. Quantification of

Toc64 protein in (a) is shown after adjusting for differences in loading using

the gel in (b).



(a)

(b) (c)

(d)

PpToc64-2 locus RES

2S

2ARA

transformation cassette

RS*Non-specific products

64-1 64-2

mut

wtmut wtmut

wt

1--

2--

3--4----

6--

1.55--

kb

*1--

2--3--

4----6--

1.55--

kb

2S + RA 2S+2ARS+2Amut wt mutmut wtwt

*

mut = PpToc64-2::nptII

1--

2--4--kb

0.5--

C1 222 1 11 264 gene:+RT no RT +RT no RT

Wild type PpToc64-2::nptII

Wild

type

PpToc

64-2

::npt

II

220 --

97 --

66 --

45 --

30 --

kDa

Wild type PpToc64-2::nptII

Figure 3. Phenotype of the PpToc64-2::nptII knockout plant.

(a) PCR from wild-type (wt) or PpToc64-2::nptII (mut) genomic DNA using primers to amplify the PpToc64-1 or PpToc64-2 loci (left panel). The right panel shows PCR

from the same DNA using primers hybridizing to locations shown in the schematic diagram. In the schematic diagram the endogenous locus is shown as a line,

while the transformation cassette is represented as a rectangle. The transformation cassette contains a region homologous to the endogenous locus and a region

including a 35S-driven nptII gene (labeled RES). Primer binding sites are represented as arrows showing the direction of priming.

(b) RT-PCR from wild-type or PpToc64-2::nptII RNA to amplify the PpToc64-1 or PpToc64-2 message. No reverse transcriptase was added to the ‘no RT’ samples as a

control for genomic DNA contamination.

(c) Wild-type or PpToc64-2::nptII plants grown on agar for 2 months.

(d) Equal amounts of chloroplast proteins from wild-type or PpToc64-2::nptII plants were probed on a Western blot with antibodies against Toc64. Lanes shown are

non-adjacent lanes of the same gel. The arrow labels the Toc64 protein.



type or mutant chloronemal or caulonemal tissue (data not

shown). Within the chloroplasts, there were also no major

differences in ultrastructure. Thylakoid membranes were

clearly seen, though there may be a slight difference in

stacking (Figure 6, panels v and vi). The presence of

thylakoid membranes is consistent with the fact that thyla-

koid proteins continue to accumulate at wild-type levels in

the double mutant (Figure 4b).

In vitro import assays were then performed with chloro-

plasts isolated from wild-type or double-mutant tissue.

Figure 7 shows a representative experiment in which plast-

ids from either source imported moss plastocyanin (a

photosynthetic protein), two moss chloroplastic Hsp70

proteins (PpcpHsp70-1 and PpcpHsp70-2) or pea cpHsp93

(non-photosynthetic proteins) with similar efficiencies.

These import assays were repeated several times, and any

differences between the import rates of the plastids were

smaller than the variation between preparations of chloro-

plasts from plants of the same genotype (data not shown).

Thus, there was no statistically significant difference be-

tween the in vitro rates of protein import into isolated

chloroplasts from the mutant and wild-type plants, whether

photosynthetic or non-photosynthetic precursor proteins

were imported.

Discussion

As we reported previously, P. patens has two proteins rela-

ted to Toc64. Both have all three recognizable domains, al-

though they differ from Toc64s of flowering plants in that

they have the putative catalytic residues in the amidase

region (Hofmann and Theg, 2003). In Arabidopsis, a Toc64-

like protein of unknown function was found in the mito-

chondrion (Chew et al., 2004). In moss, however, all of the

Toc64 protein recognized by our antibody could be

accounted for by the Toc64 present in chloroplasts. In

addition, both moss Toc64s are more similar in sequence to

the chloroplastic pea and Arabidopsis Toc64s than to the

mitochondrial AtOM64. Both moss proteins are also capable

of inserting into the outer envelope membrane of isolated

chloroplasts (Hofmann and Theg, 2005), providing further

evidence that both proteins are normally found in the

chloroplast. While we cannot rule out the existence of an

additional Toc64 protein in moss, it does not seem likely.

The large expressed sequence tag databases which are

publicly and privately available are estimated to give nearly

complete coverage of the P. patens genome (Rensing et al.,

2002a,b). Searches of these databases have revealed only

the two Toc64 genes discussed in this work. Additionally,

when both Toc64 genes were knocked out, all of the Toc64

protein was gone. Together these suggest that if there are

other Toc64s in moss they are not closely enough related to

PpToc64-1 and PpToc64-2 to be recognized by the same

polyclonal antibody. The genome of P. patens is scheduled

to be sequenced, so it will soon be possible to learn with

certainty whether there are additional Toc64s in moss.

We used the ability of moss to undergo homologous

recombination in the nucleus to make knockout mutants of

both Toc64 genes. The single knockout in PpToc64-2 still had

a significant amount of Toc64 protein in its chloroplasts.

This suggests either that PpToc64-1 accounts for the major-

ity of Toc64 protein in protonemal chloroplasts or that the

(a)

(b)

Figure 4. The Toc64 double-knockout plant lacks Toc64 protein but not other

chloroplast proteins.

(a) PCR from wild-type or PpToc64-1::hyg PpToc64-2::nptII genomic DNA to

amplify the PpToc64-1 or PpToc64-2 loci (wt) or the 5¢ or 3¢ targeting products.

Different regions of the same gel are separated by a line. The schematic

diagram below shows the PpToc64-1 locus. The endogenous locus is shown

as a line, while the transformation cassette is represented as a rectangle. The

transformation cassette contains two regions homologous to the endogenous

locus and a region including a 35S-driven hygromycin resistance gene

(labeled RES). The locations of the primers used for PCR are represented as

arrows.

(b) Protein levels in wild-type or double-mutant chloroplasts. Equal amounts

of proteins from wild-type or PpToc64-1::hyg PpToc64-2::nptII chloroplasts

were probed with antibodies against Toc64, Tic40, cpHsp93 (ClpC), Hsp70,

OE33 and LHCP. The arrow marks the Toc64 protein. The asterisk marks a non-

specific band.



plants can compensate for the lack of one isoform by

upregulating the other. The latter possibility remains to be

explored. Although they had no PpToc64-2, there was no

obvious phenotype in the single-knockout plants.

The double-knockout plants lacked all Toc64 protein in

their chloroplasts. Despite this, the plants were not distin-

guishable from wild type. Both photosynthetic and non-

photosynthetic chloroplast proteins continued to accumu-

late at wild-type levels. In addition, isolated chloroplasts

from the Toc64-null mutant performed similarly to those

from wild-type plants in in vitro protein import assays.

Consistent with this, electron microscopy showed that

chloroplasts in the double mutant had similar amounts of

thylakoid membranes as found in the wild type. On the other

hand, chloroplast shape seemed to be altered in some

tissues. Chloroplasts from the double mutant were slightly,

but significantly, longer and narrower than those from wild-

type plants.

This phenotype in the double mutant stands in sharp

contrast to phenotypes seen for other Toc and Tic mutants.

In those cases, when all of a given protein was absent the

plants had severe growth defects and chloroplast develop-

ment was significantly altered. The Arabidopsis Toc159-null

mutant, ppi2, had an albino phenotype and could not

survive on soil (Bauer et al., 2000). The plastids in ppi2 had

no thylakoid membranes and did not accumulate photosyn-

thetic proteins at normal levels. Arabidopsis has two

proteins related to Toc34, AtToc33 and AtToc34. AtToc33

(a)

(b)

Wild type Toc64 double mutant

i

vii

vi

v

iviiiii

Figure 5. Phenotype of the Toc64 double knockout plant.

(a) Wild-type and PpToc64-1::hyg PpToc64-2::nptII plants grown on agar for 1 month.

(b) Light micrographs of wild-type (i, ii, v) and PpToc64-1::hyg PpToc64-2::nptII (iii, iv, vi, vii) tissue. Chloronemal cells are shown in (i–iv). Caulonemal cells are

shown in (v–vii). Bars represent 0.1 mm.



was absent from ppi1 plants, which were pale and had

smaller chloroplasts with fewer thylakoid membranes than

wild type (Jarvis et al., 1998). In addition, in vitro protein

import was impaired in ppi1. A similar effect was seen by

downregulating AtToc33, while reduction in AtToc34 did not

result in an obvious phenotypic difference (Gutensohn et al.,

2000). Recent work reported that an AtToc34 knockout alone

has little effect on plastid development, but the AtToc34

heterozygote in the ppi1 background (with only one func-

tional allele of AtToc34 and lacking AtToc33 altogether) had

a more severe phenotype than the original ppi1 plants

(Constan et al., 2004b). These plants were paler and had

even smaller chloroplasts with even fewer thylakoid mem-

branes. Furthermore, the complete lack of Toc34-like protein

from Arabidopsis is embryo lethal (Constan et al., 2004b).

Antisense plants showed that the less Tic20, a Tic complex

protein, a plant had, the more severe phenotype it showed

(Chen et al., 2002). Like the other translocon mutants, those

Wild type Toc64 double mutant

i

iviii

ii

viv

Figure 6. Electron microscopy of Toc64 double-knockout plants.

Scanning electron microscopy of tissue from wild-type (i, iii, v) or PpToc64-1::hyg PpToc64-2::nptII (ii, iv, vi) plants. Bars represent 5 lm in (i–iv) and 1 lm in (v) and

(vi).

Table 2 Measurements (mean � SE) of Toc64 double-mutant andwild-type chloroplasts from electron micrographs and t-test com-paring values

Chloroplastlength (lm)

Chloroplastwidth (lm) n

Wild type 6.03 � 0.27 1.51 � 0.08 76Toc64 double mutant 7.82 � 0.36 1.19 � 0.06 58P-value 0.0001 0.0028



plants were small and pale and had lower levels of plastid

proteins than wild type. The plastids themselves were also

smaller than wild type and had fewer and less stacked

thylakoid membranes. In addition, import of precursor

proteins was impaired. Likewise, mutants in the Tic-associ-

ated stromal chaperone, cpHsp93, have been recently des-

cribed (Constan et al., 2004a; Kovacheva et al., 2005;

Sjogren et al., 2004). There are two cpHsp93 proteins in

Arabidopsis. While the knockout of one had little effect, the

knockout in the other gave pale, small plants with fewer

thylakoid membranes and less stacking of the thylakoids.

The plastids in these plants were smaller than wild type and

in vitro import was impaired. As was observed for the other

translocon mutants, the complete lack of Hsp93 protein may

be lethal, since the double knockout could not be obtained

(Constan et al., 2004a). A similar phenotype was seen in an

Arabidopsis Tic110 mutant (Kovacheva et al., 2005). The

Tic110 mutation was lethal when homozygous, and even the

heterozygote was pale and had lowered rates of chloroplast

protein import. Finally, Arabidopsis mutants lacking the

apparent co-chaperone, Tic40, have been isolated (Chou

et al., 2003; Kovacheva et al., 2005). This is the first Toc or Tic

component not to be strictly required for chloroplast protein

import, but in the absence of Tic40 plants are pale, slower

growing and show lower rates of protein import than wild

type (Chou et al., 2003; Kovacheva et al., 2005).

Overall, the data to date point to severe consequences for

the plant when proteins known to be involved in chloroplast

protein transport are absent. The phenotypes of these plants

are remarkably similar. Small, pale plants are found, with

small plastids lacking normal amounts of thylakoid mem-

branes. When there is more than one similar protein, the

phenotype might be masked by the presence of the second

protein. Except for Tic40, the complete lack of a given

translocon protein appears to be lethal. The similarity of

phenotypes in these mutants is consistent with Toc159,

Toc34, Tic20, Tic40 and cpHsp93 all playing roles in the same

pathway.

The phenotypes of our mutants are not consistent with

Toc64 also being required for that pathway. Even in the

absence of any chloroplastic Toc64 protein, the plants

appeared healthy and normal. The double knockout was

not lethal, nor did it give rise to pale or slower-growing plants.

In addition, there was no appreciable difference in the

amount of thylakoid membrane in the plastids. We probed

both photosynthetic and non-photosynthetic plastid proteins

and saw no difference in the levels of either, suggesting that

Toc64 is not involved in targeting either group of proteins.

Finally, isolated plastids from the Toc64-null plants were

capable of importing proteins at rates similar to chloroplasts

from wild type. Our data show that Toc64 is not required for

chloroplast biogenesis in vivo or for protein import in vitro.

Physcomitrella patens has a relatively simple body plan

(Schaefer and Zryd, 2001), making it unlikely that Toc64 plays

a role in a cell or tissue type that was not examined. Indeed,

the removal of Toc64 from chloroplasts that normally contain

Toc64 protein did not appear to have any effect on protein

import.

Our mutants do suggest a role for Toc64 in determining

chloroplast morphology, however. The shape of the chloro-

plast in the double mutant was subtly altered and it is

possible that there was a tissue-specific effect. Whether this

phenotype is due to the lack of one or both moss Toc64

proteins remains to be examined. Since Toc64 has protein–

protein interaction domains in the cytoplasm, it is tempting to

speculate that it is a factor on the surface of the chloroplast

responsible for interacting with cytoplasmic factors deter-

mining shape. It is not known at present what determines

plastid shape, and this protein might begin to provide an

answer. While it is not clear what role the amidase-like region

might play, the TPRs in Toc64 resemble other chaperone-

binding TPRs (data not shown) and could function to organize

other proteins at the surface of the chloroplast.

In the light of our data it is not clear whether the reported

interactions between Toc64 and the Toc/Tic components are

physiologically relevant. It is possible that they are artifacts

due to the ability of Toc64 to interact with chaperone

Wild type

220--97--66--

45--

kDa 10%∆Toc64

- + thermolysin- +

mcpHsp70-1

kDa 10% - + thermolysin- +

20.1--

14.3--

30--

Wild type ∆Toc64

mPC

- + thermolysin- +Wild type ∆Toc64

mcpHsp70-2

- + thermolysin- +Wild type ∆Toc64

mcpHsp93

Figure 7. In vitro protein import into wild-type and double-mutant chloro-

plasts.

Moss plastocyanin (PC), two moss cpHsp70 proteins and a pea cpHsp93

protein were imported into wild-type or Toc64-null plastids as labeled.

Samples were treated with thermolysin as indicated. The positions of the

mature proteins are labeled with arrows.



proteins. It is also possible that some components of the

Toc/Tic machinery are involved in targeting Toc64 to the

chloroplast envelope (Hofmann and Theg, 2005) and that the

cross-linked protein was in the process of being targeted.

We thus propose renaming this protein with the more

general designation OEP64 (for Outer Envelope Protein of

64 kDa), to reflect the uncertainty about its function.

Experimental procedures

Alignments of proteins were generated with ClustalW (Thompsonet al., 1994) and edited in Genedoc (Nicholas et al., 1997). Genedocwas also used to determine per cent similarity and identity based onthe alignment. Chemicals were purchased from Sigma (St Louis,MO, USA), Fisher (Houston, TX, USA) or VWR (Brisbane, CA, USA)unless otherwise noted. ImageJ software was used to quantify gelsand blots (http://rsb.info.nih.gov/ij/).

Photographs of moss plants were acquired with a Nikon Coolpix(El Segundo, CA, USA) 950 digital camera. Light microscopy wasperformed with water as the medium at room temperature using aNikon Eclipse E600 microscope with a Nikon 40· objective that had anumerical aperture of 0.75. Images were captured with a Spot RTcamera and Spot software (Diagnostic Instruments, SterlingHeights, MI, USA). Adobe Photoshop was used for cropping andscaling images, as well as adjusting brightness and contrast and

Adobe Illustrator (Adobe Systems, San Jose, CA, USA) was used toassemble and label figures.

Antibody generation and Western blotting

The coding sequence for the PpToc64-1 soluble region was ampli-fied using the primers 641gateS and 641codeSA (Table 3) andinserted into the pEXP1-DEST vector (Invitrogen, Carlsbad, CA,USA) to enable the expression of PpToc64-1 protein without theNH2-terminal hydrophobic domain but with an NH2-terminal six-histidine tag. This protein was expressed in Escherichia coli BL21DE3 CodonPlus cells (Stratagene, La Jolla, CA, USA) purified on aNi-NTA (Qiagen, Valencia, CA, USA) column under denaturingconditions. The purified protein was sent to Zymed (South SanFrancisco, CA, USA) for antibody production in rabbits.

The Toc64 antibody was affinity purified by incubation with theantigen on blots as described (Olmsted, 1981). For Western blots,affinity-purified Toc64 antibodies were used at 1:100. a-LHCP (light-harvesting chlorophyll a/b-binding protein) antibodies (Payan andCline, 1991) were used at 1:10 000. Antibodies against stromalcpHsp70 (S78) and cpHsp93 (ClpC) (Akita et al., 1997; Constan et al.,2004a) were used at 1:2500 and 1:5000, respectively. Antibodiesagainst OE33 from pea were prepared in our laboratory (Ettingerand Theg, 1991) and used at 1:1000. Antibodies against Tic40 (Chouet al., 2003) were used at 1:5000. For all antibodies except OE33,goat a-rabbit secondary antibodies conjugated with horseradish

Table 3 Sequences of primers used in thisworkPrimer name Sequence (5¢ to 3¢)

641gateS CACCATGGATAATGGCGCCTTCATTGAa,c

641codeSA TATTATCTCGAGTTAACCGTACAGAAGCTTCTTCAGTb,d

642KO1 GGTCGATCTGAGTCCTGCTAGGTCTTa,e

642KO2 CGTAATCATGGTCATAGCTGTCTGCATAGCAGTTCGAAGGAGAGTAAb,f

642R1 TTACTCTCCTTCGAACTGCTATGCAGACAGCTATGACCATGATTACGa,f

642R2 ATGTCGAGAGCAGCTTGTACACGGGAGGGTTTTCCCAGTCACGACGb,f

641KO1 CGGGAAGGATATTGGCGCCTTCATTa,g

641KO2 CGTAATCATGGTCATAGCTGTCCAAAGCAGGTGCTAGATCAGGCTTTb,f

641KO3 CGTCGTGACTGGGAAAACCCTCTGGTACCTCTTCTCCAGAAGATCAa,f

641KO4 CCCATCCATTCTGACGACCTCGAAAb

641R1 AAAGCCTGATCTAGCACCTGCTTTGGACAGCTATGACCATGATTACGa,f

641R2 TGATCTTCTGGAGAAGAGGTACCAGAGGGTTTTCCCAGTCACGACGb,f

6411S ATGAGCCTGCTACACGCACa

641codeA TATTTACTCGAGACCGTACAGAAGCTTCTTCAb,d

PpToc641S CAGTGTTTAAGCCAGGAGGGGTTTa

PpToc641A CCCGCAGTCACACCTGCTTCAAGAb

PpToc642S CGTGCCGGGAGTGCCTGTTCCTTa

PpToc642A TACGCCGCTACGCTCAGCACGATb

R1LongRv TTGGCGTAATCATGGTCATAGCTGTb,h

R2LongFw CAACGTCGTGACTGGGAAAACCCTa,h

641codeS TATTATCCCGGGTATGCCGAACGATAAGATTTTGGCAa,i

642codeS TATTATCCCGGGCATGCCGAGCGACGCAGTGGTa,i

642codeSA TATTATCTCGAGTTAAATAAGAAGCTTCTTCAGTCb,d

aSense primer.bAntisense primer.cTOPO� directional cloning site (underlined) start codon in bold.dXhoI (underlined); stop in bold.eG to T mutation to generate stop codon (underlined).fpUC sequence underlined.gA to T mutation to destroy start codon (underlined).hHybridizes to pUC vector sequence.iSmaI (underlined); start in bold.



peroxidase (Bio-Rad, Hercules, CA, USA) were used. The signal wasvisualized with ECL substrate from either Amersham Biosciences(Piscataway, NJ, USA) (with 1:2500 secondary antibody) or PierceBiotechnology (Rockford, IL, USA: Supersignal West Pico Chemilu-minescent substrate with 1:50 000 secondary) on film or with acharge-coupled device (CCD) camera. For OE33, goat a-chickensecondary conjugated with alkaline phosphatase (Southern Bio-technology Associates, Birmingham, AL, USA) was used andvisualized on the Storm 860 Phosphor Imager (Molecular Dynamics,Sunnyvale, CA, USA) using ECF substrate (Amersham Biosciences).

Moss culture, chloroplast isolation and in vitro protein

import assays

Physcomitrella patens protonemal tissue was grown as reportedpreviously (Hofmann and Theg, 2003). Chloroplasts were isolatedfrom protoplasts as previously described (Hofmann and Theg,2003). Radio-labeled proteins were prepared by in vitro transcrip-tion and translation in rabbit reticulocyte lysate in the presence of[35-S]methionine (Promega, Madison, WI, USA). Physcomitrellapatens plastocyanin (accession no. AB026687) was prepared asdescribed (Hofmann and Theg, 2003). Two P. patens cpHsp70 pro-teins (Shi and ST, unpublished data) were transcribed from plas-mids containing cDNA. Pisum sativum cpHsp93 was transcribedfrom a plasmid containing the cDNA (accession no. L09547). Thisplasmid was a gift from Professor K. Inoue. In vitro import assaysand thermolysin treatments were carried out essentially as des-cribed, except that the import reactions were incubated in the lightfor only 5 min (Hofmann and Theg, 2003). Reactions that were nottreated with thermolysin were terminated by spinning throughsilicon oil into trichloroacetic acid as described (Theg et al., 1989),with the exception of the plastocyanin imports, which were ter-minated by 10-fold dilution. Proteins were visualized by autoradi-ography and, after exposure, phosphor screens were scanned usinga Storm 860 Phosphor Imager (Molecular Dynamics).

Transformation cassette preparation

Multiplex PCR was used to generate the transformation cassettes.For targeting the PpToc64-2 locus, a region of genomic DNA cor-responding to approximately the first half of the coding region wasamplified using the primers 642KO1 and 642KO2 (Table 3) andfused to the 35S:nptII gene (for G418 resistance) which was ampli-fied from the PBI426 plasmid (obtained from Dr C. Champagne) with642R1 and 642R2 (Table 3). The resultant product was gel extractedand used for transformation.

For targeting the PpToc64-1 locus, products corresponding to thefirst and second halves of the coding sequence were amplified fromcDNA using primers 641KO1 and 641KO2 (first half, Table 3) and641KO3 and 641KO4 (second half, Table 3). The 35S-driven hygro-mycin resistance gene was amplified from the plasmid 35S:hygpART7 (a gift from Dr C. Champagne) using the primers 641R1 and641R2 (Table 3). The final product had the resistance gene flankedby the PpToc64-1 sequence (see Figure 4). To generate largerquantities to use for transformation, this product was amplifiedusing the nested primers 6411S and 641codeA (Table 3). Fifteenreactions were pooled resulting in approximately 5 lg of product.

Generation of Toc64 knockouts

Physcomitrella patens knockout plants were generated by poly-ethylene glycol (PEG)-mediated transformation of protoplasts with

PCR products essentially as described (Cove and Knight, 1999;Grimsley et al., 1977). After 1 week, the regenerating plants weretransferred to media containing either 30 lg ml)1 hygromycin(Invitrogen) or 15 lg ml)1 G418 (Invitrogen). Plants were allowed toregenerate with selection for another 2 weeks and then werereleased from selection for 2 weeks. This was followed by anotherround of selection, and plants that survived were considered to bestable transformants and analyzed for targeting.

For generation of PpToc64-2 knockouts, wild-type plants weretransformed with the transformation cassette described in theprevious section. For generation of double mutants, PpToc64-2::nptII knockout plants were transformed with the PpToc64-1targeting construct described above.

Isolation of DNA and RNA from moss plants; PCR to check

targeting

Genomic DNA was isolated from moss plants as described (Coveand Knight, 1999). RNA was isolated from week-old protonemaltissue using the RNeasy Mini Kit (Qiagen). To check transformantsfor targeting, genomic DNA was used for PCR with various primerpairs. To amplify full-length PpToc64-1, PpToc641S and PpToc641A(Table 3) were used. For PpToc64-2, PpToc642S and PpToc642A(Table 3) were used. R1LongRv (Table 3) was used with PpToc641Sor PpToc642S for the 5¢ targeting products of PpToc64-1 orPpToc64-2, respectively. Similarly, for the 3¢ targeting products,R2LongFw (Table 3) was used with PpToc641A or PpToc642A.

For RT-PCR, cDNA was prepared from RNA using the Super-script First-Strand Synthesis System for RT-PCR (Gibco BRL,Rockville, MD, USA). To amplify PpToc64-1 message, PCR wasperformed with primers 641codeS and 641codeSA (Table 3). Toamplify PpToc64-2, primers 642codeS and 642codeSA (Table 3)were used.

Electron microscopy

Wild-type and double-mutant tissue was grown on cellophane-overlaid BCD minimal media plates (see Cove and Knight, 1999, fordetails) with 5 mM ammonium tartrate for 1 week. This tissue wasused for transmission electron microscopy (UC-Davis School ofMedicine Electron Microscopy Lab, Davis, CA, USA).

Acknowledgements

We would like to thank Professor Kentaro Inoue for gifts of clonesand antibodies and Dr Connie Champagne for advice and clones.Dr Lan-xin Shi also provided helpful comments and clones. Inaddition, we are grateful to John Perea for excellent technicalassistance and to Professor Anne Britt for critical reading of thismanuscript. This work was supported by an NSF Graduate ResearchFellowship to NRH and an NSF grant to SMT.

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toc64 is not required for import of proteins into chloroplasts in the moss physcomitrella patens

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