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J Biol Chem, Vol. 274, Issue 35, 25181-25186, August 27, 1999
From the Department of Biological Sciences, Rutgers, The State
University of New Jersey, Newark, New Jersey 07102
Tic22 previously was identified as a component of
the general import machinery that functions in the import of
nuclear-encoded proteins into the chloroplast. Tic22 is peripherally
associated with the outer face of the inner chloroplast envelope
membrane, making it the first known resident of the intermembrane space of the envelope. We have investigated the import of Tic22 into isolated
chloroplasts to define the requirements for targeting of proteins to
the intermembrane space. Tic22 is nuclear-endoded and synthesized as a
preprotein with a 50-amino acid N-terminal presequence. The analysis of
deletion mutants and chimerical proteins indicates that the precursor
of Tic22 (preTic22) presequence is necessary and sufficient for
targeting to the intermembrane space. Import of preTic22 was stimulated
by ATP and required the presence of protease-sensitive components on
the chloroplast surface. PreTic22 import was not competed by an excess
of an authentic stromal preprotein, indicating that targeting to the
intermembrane space does not involve the general import pathway
utilized by stromal preproteins. On the basis of these observations, we
conclude that preTic22 is targeted to the intermembrane space of
chloroplasts by a novel import pathway that is distinct from known
pathways that target proteins to other chloroplast subcompartments.
The chloroplast is a complex organelle that is subdivided into at
least six suborganellar compartments by a double-membrane envelope and
an internal thylakoid membrane. These three membranes segregate and
organize a number of essential biochemical processes, including the
light and dark reactions of photosynthesis and aspects of amino acid
and fatty acid synthesis. The biogenesis and maintenance of this
elaborate architecture requires the selective targeting of numerous
nuclear-encoded proteins from their site of synthesis on cytoplasmic
ribosomes to their proper suborganellar compartment. Targeting is
mediated by a set of hierarchical targeting signals that are intrinsic
to the nuclear-encoded protein. These signals act independently to
initiate translocation of the protein across one or more of the
chloroplast membranes en route to their proper destination (1).
Proteins destined for the stromal and thylakoid compartments are
translocated across the double membrane envelope through a common,
general import machinery as the first step in their import and assembly
pathways. Envelope translocation is mediated by the cleavable,
N-terminal transit sequence of the preprotein. The general import
machinery consists of two oligomeric membrane complexes, the outer
membrane Toc complex and the inner membrane Tic complex, that cooperate
to catalyze direct transport of preproteins from the cytoplasm to the
stroma at the expense of ATP and GTP (2). Targeting to the thylakoid is
mediated by secondary targeting signals that act subsequent to envelope
translocation. Translocation into the thylakoid lumen or integration
into the thylakoid membrane involves at least four distinct targeting
pathways (3). Analyses of the components involved in these pathways
suggest that each of these systems evolved from secretion systems
similar to those that exist in prokaryotes.
Targeting to the outer and inner membranes of the chloroplast envelope
involves at least two distinct pathways. Inner envelope membrane
proteins contain cleavable transit sequences, and translocation across
the envelope occurs with the assistance of the general import
machinery. Integration into the inner membrane occurs concomitant with
membrane translocation (4) or subsequent to translocation via a stromal
intermediate (5) and is directed by elements contained within one or
more of the transmembrane regions of these proteins.
In contrast to inner membrane proteins, most outer envelope membrane
proteins are synthesized in the cytoplasm as their mature forms. The
signals for insertion into the membrane reside within regions of the
proteins that encompass the transmembrane domains and adjacent
sequences (6-11). Outer membrane proteins integrate without the
assistance of the general import machinery or the requirement of an
obvious energy source, suggesting that they directly partition into the
lipid bilayer from the cytoplasm. Toc75, a component of the general
import machinery, is an exception among known outer membrane proteins
(12, 13). Toc75 is synthesized with a cleavable presequence similar to
stromal proteins. The N-terminal domain of the presequence functions as
a typical transit sequence to direct Toc75 to the general import
machinery during the initial stages of targeting. In contrast to
stromal proteins, the transit sequence is followed in tandem by a
sequence that blocks complete transport across the envelope and
triggers integration of Toc75 into the outer membrane.
The pathway of targeting to the intermembrane space of the envelope has
not been studied due to the lack of a known marker for this
subcompartment. We recently identified Tic22 as an intermembrane space
protein (14). Tic22 is a component of the general import machinery and
is peripherally bound to the outer face of the inner envelope membrane
(15). In this study, we used recombinant
preTic221 to investigate the
pathway of targeting to the intermembrane space. It is synthesized as a
preprotein containing an N-terminal extension with similarity to
transit sequences. This raises the possibility that preTic22 may
utilize the general import machinery en route to the intermembrane
space. We used chimerical proteins in which the preTic22 presequence
and the transit sequence of preSSU were reciprocally exchanged to
investigate the functional role of the Tic22 presequence and mature
sequence in targeting to the intermembrane space. Our experiments
suggest that the presequence of preTic22 does not function as a stromal
transit sequence but directs import into the intermembrane space by a
previously unidentified mechanism.
Chloroplast Isolation and Subfractionation--
Intact
chloroplasts were isolated from 14-day-old pea seedlings (Pisum
sativum variant Green Arrow) by homogenization and Percoll silica
gel gradient centrifugation as described previously (16). A protease
inhibitor mixture (P9599, Sigma) was included at all stages of
chloroplast isolation according to the manufacturer's recommendations.
Isolated chloroplasts were resuspended in 50 mM Hepes-KOH,
pH 7.7, 0.33 M sorbitol (HS buffer) to a concentration equivalent to 2-3 mg chlorophyll/ml. To separate the total membrane and soluble fractions, intact chloroplasts were lysed under hypertonic conditions (17), and total membrane and soluble fractions were separated by centrifugation at 40000 × g for 20 min in
a TLA100.3 rotor (Beckman Instruments, Palo Alto, CA).
Preparation of Fusion Proteins--
Plasmid pET21d-pTic22 for
the in vitro expression of preTic22 was constructed as
follows. The 759-base pair open reading frame of preTic22 cDNA (15)
including the stop codon was amplified by PCR to incorporate
NcoI and NotI sites at the 5' and 3' ends, respectively. The fragment was inserted into the NcoI and
NotI sites of pET21d (Novagen, Madison, WI). Plasmid
pET21d-Tic22MS, encoding the mature sequence of Tic22, was constructed
by amplifying the 608-base pair fragment of preTic22 cDNA from
nucleotide 163 to 771 by PCR to incorporate NheI and
NotI sites at the 5' and 3' ends, respectively. The fragment
was subcloned into the NheI and NotI sites of
pET21d (Novagen).
Plasmid pET8c-pTic22-SSU, encoding the presequence of preTic22 fused to
mature SSU, was constructed as follows. A region of pET21d-pTic22 was
amplified by PCR with a sense primer specific for the T7 promoter of
the vector and an antisense NcoI adaptor primer
(5'-CATGCCATGGCAGTCAAAGTAGCGGC-3') annealing to nucleotides 150-174 of
the preTic22 cDNA. The DNA fragment was digested and inserted into
the XbaI and NcoI sites of pET8c-S (18). The
resulting open reading frame consisted of the N-terminal 54 amino acids of preTic22 fused to the complete mature sequence of SSU.
Plasmid pET21d-pS-Tic22, encoding the transit sequence of preSSU fused
to the mature sequence of preTic22, was constructed as follows. A
region of pET8c-pS (18) was amplified by PCR with a sense primer
specific for the T7 promoter of the vector and an antisense
NheI adaptor primer (5'-GAATTCGCTAGCGCACTTTACTCTTCC-3') annealing to nucleotides 157-171 of the preSSU cDNA. The DNA
fragment was digested and inserted into the XbaI and
NheI sites of pET21d-Tic22MS. The resulting open reading
frame consisted of the N-terminal 57 amino acids of preSSU fused to the
complete mature sequence of preTic22.
In Vitro Translation and Protein Import Reactions--
All
in vitro synthesized substrates for the import assays
were generated in a coupled transcription-translation system containing reticulocyte lysate according to the supplier's recommendations (Promega Corp., Madison, WI) using T7 RNA polymerase in the presence of
[35S]methionine. The translation mixture was gel filtered
using Sephadex G-25 (Amersham Pharmacia Biotech) to remove free
nucleotides before use in the import reactions.
Chloroplasts (50 µg of chlorophyll) were incubated in 150 µl of
HS-buffer containing 50 mM KOAc, 4 mM MgOAc,
400 nM nigericin (import buffer) for 15 min at 26 °C in
the dark to deplete them of endogenous ATP. The energy-depleted
chloroplasts were incubated for an additional 5 min with 10 units/ml
apyrase or the indicated concentration of ATP, and the import reaction
was initiated subsequently by the addition of gel-filtered reticulocyte
lysate containing 3 × 105 to 4.5 × 105 cpm of 35S-labeled substrate. The reactions
were incubated for 30 min at 26 °C. After the import reaction, the
chloroplasts were reisolated through 40% Percoll silica gel (Amersham
Pharmacia Biotech) and analyzed by 12 or 14% SDS-PAGE and
phosphorimagery. Radioactive signals in dried gels were quantitated
using a PhosphorImager SI (Molecular Dynamics, Inc., Sunnyvale,
CA) with the Iplab Gel scientific image processing program, version
1.5c (Signal Analytics, Vienna, VA).
The protease treatment of chloroplasts was performed by incubating the
import reaction in the presence or absence of 200 µg/ml of
thermolysin or trypsin on ice for 30 min. After protease treatment, the
reactions were diluted 5 times with the import buffer containing 10 mM EDTA, 1 mM phenylmethylsulfonyl fluoride,
0.05 mg/ml 1-chloro-3-tosylamido-7-amino-2-heptanone, 0.1 mg/ml soybean
trypsin inhibitor, and 2 µg/ml aprotinin (19). The chloroplasts were
reisolated over 40% Percoll containing all inhibitors and washed two
times with the import buffer containing all inhibitors.
Newly Imported Tic22 Is Localized to the Outer Surface of the Inner
Membrane--
To investigate the import characteristics of Tic22, we
performed in vitro import experiments using
[35S]preTic22 synthesized in a reticulocyte lysate.
PreTic22 was incubated with isolated chloroplasts in the presence of 2 mM ATP in a standard import assay. To assess the
localization of imported preTic22, we treated samples of chloroplasts
from the import reactions with thermolysin or trypsin. Thermolysin is
an outer membrane impermeable protease that selectively degrades
proteins exposed to the cytoplasm (20), whereas trypsin is capable of
accessing the intermembrane space and degrading proteins that are
exposed to the intermembrane space (19, 20). We previously demonstrated that endogenous Tic22 was resistant to thermolysin but sensitive to
trypsin treatment, consistent with its localization in the intermembrane space (15).
In vitro synthesized preTic22 associated with intact
chloroplasts, and a portion of the preprotein was processed to its
mature form, as indicated by the appearance of a radioactive band with a mobility identical to Tic22 (Fig.
1A, lane 1). The mature Tic22 was resistant to thermolysin treatment (Fig. 1A, lane 2),
but 50% of Tic22 was digested by trypsin (Fig. 1A, lane 3).
The resistance of Tic22 to thermolysin was not due to inherent protease
resistance because disruption of the envelope with detergent prior to
thermolysin treatment resulted in complete degradation of the protein
(Fig. 1C). Furthermore, neither protease digested a newly
imported stromal protein, preSSU, eliminating the possibility that the
degradation of Tic22 resulted from inadequate inhibition of the enzymes
prior to disruption of the chloroplasts (Fig. 1B). The
pattern of protease sensitivity for newly imported Tic22 is
indistinguishable from that observed for endogenous Tic22 (15),
indicating that a portion of preTic22 was translocated into the
intermembrane space of the envelope and processed to its mature
form.
Remarkably, a significant portion of bound preTic22 was insensitive to
thermolysin digestion (Fig. 1A, lane 2). This observation suggests that the thermolysin-insensitive preTic22 might be
translocated across the outer membrane but incompletely processed to
mature Tic22. The insensitivity of bound preTic22 to thermolysin was not a kinetic effect because increasing the concentration of
thermolysin to 500 µg/ml did not increase the degradation of the
bound precursor (data not shown). To investigate the possibility that a
portion of bound preTic22 was imported to the intermembrane space, we performed a time course of preTic22 import. At various time points after the addition of preTic22 to a standard import reaction, samples
were removed from the reaction, and the chloroplasts were reisolated by
sedimentation through 40% Percoll silica gel. The chloroplasts from
each time point were split into two equal samples, and one sample was
treated with thermolysin to remove surface bound precursor. Fig.
2 demonstrates that preTic22 associated with the chloroplast and was processed to Tic22 in a
time-dependent manner. Quantitative analysis of the import
reaction indicates that the binding of preTic22 to chloroplasts reaches
a maximum of 16% of added translation product after 5 min of
incubation (Fig. 2, lane 6). Although the binding of
preTic22 reached a maximum at 5 min, the proportion of bound preTic22
that was insensitive to protease increased from 24% at 5 min to 56%
at 30 min (Fig. 2, compare lanes 7 and 13). These
data indicate that preTic22 binds to the chloroplast surface and is
translocated across the outer membrane in a time-dependent
manner. The accumulation of mature Tic22 increased up to 30 min of
incubation, reaching a maximum of 4% of the precursor added to the
import reaction (Fig. 2, compare lanes 2 and 12).
The processed form of the Tic22 showed a pattern of thermolysin
sensitivity similar to that of preTic22 over the course of the import
reaction. The processed form of Tic22 was almost completely degraded by
thermolysin at the early time points (Fig. 2, lanes 3 and
5); however, the sensitivity to protease decreased with
time, and 90% of mature Tic22 was resistant to protease by 30 min
(Fig. 2, lane 13). These data suggest that processing can
occur prior to complete translocation across the outer membrane.
Furthermore, the maturation of Tic22 does not appear to be tightly
coupled to translocation because the major fraction of preTic22 is
translocated to a thermolysin-sensitive compartment without being
processed to its mature form. We attempted to test whether the imported
preTic22 could be chased to the mature form by reisolating chloroplasts
from the 20 min time point and subjecting them to a second 20-min
incubation in the absence of added preTic22. No significant difference
between the relative pattern of bound preTic22 or processed Tic22
before or after the second incubation was observed (data not shown).
Therefore, the processing of preTic22 appears to be very slow.
Our import experiments suggest that the in vitro import
assay reproduced proper targeting and translocation of Tic22 to the intermembrane space. However, it was not clear whether newly imported preTic22 or Tic22 associated with the inner membrane in a manner similar to endogenous Tic22. To test whether both forms of newly imported Tic22 associated with the inner membrane, we examined their
distributions within various chloroplast subfractions. After a 30-min
in vitro import reaction with preTic22, intact chloroplasts were reisolated, lysed, and fractionated to yield stroma and fractions enriched in outer and inner envelope membranes. Analysis of the distribution of the newly imported protein demonstrates that the majority of preTic22 and Tic22 are associated with the fraction enriched in inner membranes (Fig. 3,
lane 1). Minor amounts of preTic22 and Tic22 were detected
in the outer membrane fraction (Fig. 3, lane 2), and no
significant amounts of either form were detected in the stromal
fraction (Fig. 3, lane 3). We conclude that both preTic22
and mature Tic22 associate with the inner membrane upon entering the
intermembrane space in a manner indistinguishable from endogenous
Tic22.
Energy Dependence of Tic22 Import--
Translocation of stromal or
integral inner membrane proteins across the envelope strictly requires
ATP hydrolysis, whereas the insertion of most integral outer membrane
proteins is not dependent on an energy source. We tested the effects of
added ATP on the import of preTic22 to determine the energy
requirements for translocation to the intermembrane space. To eliminate
the interference of nucleoside triphosphates in the reticulocyte
lysate, the preTic22 in vitro translation mixture was gel
filtered before addition to the import reaction. As an added
precaution, chloroplasts were preincubated in the presence of 400 nM nigericin and apyrase in the dark to deplete exogenous
and endogenous nucleoside triphosphates. As controls for the energy
state of chloroplasts in the import reaction, we monitored the binding
and import of preSSU. As expected, 100 µM ATP stimulated
the binding of preSSU to chloroplasts (Fig. 4B, lane 3). At 2 mM ATP, preSSU was imported into the chloroplast stroma and
processed to its mature form (Fig. 4B, lane 7). The results
shown in Fig. 4 demonstrate that preTic22 binds to isolated chloroplasts and is processed to Tic22 in the absence or presence of
the externally added ATP (Fig. 4A, compare lanes 2, 4, and 5). However, 50% of Tic22 is susceptible to
externally added thermolysin in the absence of ATP (Fig. 4A, lane
3), whereas Tic22 formed in the presence of low (100 µM) or high (2 mM) concentrations of ATP is
not degraded by thermolysin (Fig. 4A, lanes 6 and
7, asterisks). These data suggest that the
binding of preTic22 to chloroplasts does not require ATP hydrolysis.
However, complete translocation to the intermembrane space is
stimulated by ATP hydrolysis.
Two proteolytic degradation products of 24 and 18 kDa were observed in
chloroplasts incubated with thermolysin (Fig. 4A, lanes 6 and 7). These fragments appear to represent
protease-protected regions of preTic22 that result from partial
translocation of the preprotein across the outer membrane. The
degradation products are more pronounced when Tic22 is imported in the
presence of ATP, providing additional evidence that ATP stimulates
insertion of preTic22 across the outer membrane.
Import of PreTic22 Is Dependent on the Presence of Proteinaceous
Components in the Outer Membrane--
Import of nuclear-encoded
stromal preproteins requires the presence of thermolysin-sensitive
proteins in the outer membrane of the envelope (21). We wished to
investigate the role of surface exposed proteins in translocation to
the intermembrane space by testing whether proteolytic treatment of
isolated chloroplasts inhibited preTic22 import. Isolated chloroplasts
were treated with 100 µg/ml thermolysin for 30 min on ice prior to
the import assay. The results of the experiments demonstrate that
pretreatment of chloroplasts with thermolysin decreases binding and
import of both preTic22 and preSSU by 60% (Fig.
5, compare lanes 1 and 2 and lanes 4 and 5). The decrease in
Tic22 binding and import was not due to the degradation of preTic22 by
residual thermolysin because no significant proteolysis of the unbound
preTic22 from the import reaction was observed (data not shown). These
data suggest that import of preTic22 into chloroplasts is mediated by
proteinaceous components in the outer membrane.
The Tic22 Presequence Contains the Information for Targeting to the
Intermembrane Space--
The deduced amino acid sequence of the Tic22
cDNA indicates that the protein is synthesized as a precursor with
a 50-amino acid N-terminal presequence (15). The length and charge
distribution of the presequence are similar to those of typical transit
sequences. To determine the significance of the N-terminal presequence
in targeting, we tested the ability of in vitro synthesized
mature Tic22 to bind and import into isolated chloroplasts. Mature
Tic22 associated with isolated chloroplasts (Fig.
6, lane 2), but the bound
protein was completely degraded by externally added thermolysin (Fig.
6, lane 1). This result clearly demonstrates that the
N-terminal presequence of Tic22 is necessary for protein import into
isolated chloroplasts.
To test whether the preTic22 presequence was sufficient for targeting
to the intermembrane space, we engineered two chimerical import
substrates in which the presequences of preTic22 and preSSU were
reciprocally exchanged. The first construct, preTic22-SSU, consisted of
the presequence of preTic22 fused to the mature sequence of the small
subunit of rubisco. This construct was used to test the ability of the
preTic22 presequence to target a passenger protein to the intermembrane
space. The second construct, pSSU-Tic22, consisted of the transit
sequence of preSSU fused to mature Tic22. This chimera was used to test
the influence of the mature sequence of Tic22 on translocation across
the envelope membranes. The chimeras were incubated with isolated
chloroplasts in the standard protein import reaction, and the
topologies of the newly imported proteins were determined by their
sensitivity to exogenous thermolysin and trypsin. PreTic22-SSU bound to
chloroplasts, and 2% of the added protein was processed to its mature
size (Fig. 7A, lane 2). The
newly imported preTic22-SSU and SSU were exclusively associated with
the chloroplast membrane fraction in chloroplasts that were not treated
with protease (Fig. 7A, compare lanes 2 and
5). The membrane-associated SSU is insensitive to exogenous
thermolysin, whereas bound preTic22-SSU is partially digested by
thermolysin (Fig. 7A, lane 3). SSU is a soluble protein, and
it is unclear why it remains associated with the envelope membrane
after processing in the intermembrane space. The simplest explanation
is that SSU cannot properly fold in the intermembrane space and may
aggregate in an insoluble form that associates with the membrane. Only
25% of chloroplast bound preTic22-SSU remains intact following
thermolysin treatment, and the majority is associated with the
chloroplast membrane fraction (Fig. 7A, lane 3). However,
23% of protease-protected preTic22-SSU is observed in the soluble
fraction of lysed chloroplasts (Fig. 7A, lane 6). This
observation suggests that imported preTic22-SSU might remain associated
with the translocation machinery, but disruption of the machinery by
proteolysis releases the bound preprotein. Taken together, the
membrane-bound and soluble preTic22-SSU present after thermolysin
treatment correspond to 23% of the preTic22-SSU added to the import
reaction. The resistance of the mature and precursor forms to
thermolysin indicates that both forms were imported across the outer
membrane. Two partial degradation products of 16 and 12 kDa are
apparent after treatment of the import reaction with thermolysin (Fig.
7A, lane 3, asterisks), suggesting that a portion
of bound preTic22-SSU is partially inserted across the outer membrane
and thereby protected from complete digestion. These proteolytic
products are comparable to those observed in thermolysin treated
preTic22 import reactions (Fig. 4A). Both the processed and
precursor forms of the chimera were completely digested with trypsin
(Fig. 7A, lane 4), indicating that the
thermolysin-insensitive proteins were not translocated across the inner
envelope membrane. Based on these observations, we conclude that the
presequence of preTic22 is sufficient to target a passenger protein to
the intermembrane space of the envelope.
The preSSU-Tic22 chimera is imported into the chloroplast and
efficiently processed to mature SSU (Fig. 7B). The imported chimera is insensitive to both thermolysin (Fig. 7B, lane 3)
and trypsin (Fig. 7B, lane 4) in intact chloroplasts and is
found in the soluble fraction after chloroplast lysis (Fig.
7B, compare lanes 2-4 to lanes 5-7).
These data are consistent with import to the stroma. A small fraction
of preSSU-Tic22 remains bound to the chloroplast membrane fraction
(Fig. 7B, lane 5), but it is sensitive to thermolysin
digestion (Fig. 7B, lane 6), suggesting that it is bound to
the outer surface of the envelope. In addition, a small portion of the
imported mature protein is associated with the membrane fraction (Fig.
7B, lane 5). This mature Tic22 is not sensitive to either
protease in intact chloroplasts (Fig. 7B, lanes 6 and
7), suggesting that it represents newly imported stromal
Tic22 that contaminates the membrane fraction. The import properties of
preSSU-Tic22 are indistinguishable from authentic preSSU, indicating
that targeting is not detectably influenced by the mature sequences of Tic22.
PreTic22 Does Not Compete with preSSU for Import into Isolated
Chloroplasts--
The similarities between the import characteristics
and presequences of preTic22 and stromal preproteins raised the
possibility that targeting to the intermembrane space may involve
components of the general import machinery. To explore this
possibility, we performed a competition experiment in which import of
radioactively labeled preTic22 was carried out in the presence of
excess unlabeled preSSU. Unlabeled preSSU efficiently competed for the
import of radiolabeled preSSU (Fig.
8A). Inhibition of import was
dose-dependent, reaching a maximum of 70% inhibition at 2 µM unlabeled preSSU (Fig. 8B). An equivalent
concentration of mature SSU had no detectable effect on import. In
contrast, the same concentrations of unlabeled preSSU had no detectable
effect on the import and processing of preTic22 (Fig. 8, A, lane
6, and B). These results make it unlikely that the
import pathways of preTic22 and preSSU share common components.
In this report, we investigated the import of preTic22 into
isolated chloroplasts to define the signals and pathway for targeting to the chloroplast intermembrane space. PreTic22 was imported into
isolated chloroplasts, and the pattern of protease sensitivity of the
newly imported protein was similar to endogenous Tic22, indicating that
the protein was accurately targeted to the intermembrane space in
vitro. Our results show that the import of preTic22 is directed by
the N-terminal presequence of the protein. The presequence is required
for translocation across the outer membrane (Fig. 6), and it can target
a soluble passenger protein to the intermembrane space (Fig.
7A). Therefore, it appears to be necessary and sufficient for targeting to the intermembrane space. Recognition of the
presequence appears to be mediated by a proteinaceous receptor system
at the surface of the outer membrane because proteolytic treatment of chloroplasts significantly reduces preTic22 binding and translocation (Fig. 5).
Approximately 20% of radiolabeled preTic22 added to the import assay
was imported across the outer membrane, and the majority of the
imported product associated with the inner membrane. Although import of
the protein across the outer membrane was relatively efficient,
processing of imported preTic22 was inefficient. Only 20% of the total
imported protein was cleaved to mature Tic22 over the time course of
our import assays (Fig. 2). We were unable to detect a significant
conversion of imported preTic22 to mature Tic22 when chloroplasts from
a preTic22 import reaction were reisolated and subjected to a second
incubation under import conditions in the absence of added preprotein
(data not shown). Processing of imported preTic22 therefore appears to
be a slow event in the import process. The high proportion of imported
preTic22 that remains unprocessed and the ability of preTic22 to
associate with the inner membrane suggest that cleavage of the
presequence is not tightly coupled to translocation across the outer
membrane or binding to the inner membrane.
PreTic22 binds to chloroplasts and is processed with similar efficiency
in the presence or absence of ATP (Fig. 4). However, the amount of
imported protein that was insensitive to thermolysin degradation
doubled in the presence of ATP. Therefore, it appears that ATP promotes
efficient translocation across the outer membrane. It remains to be
established whether ATP is utilized for translocation or for
stabilizing Tic22 in the intermembrane space. The fact that a fraction
of preTic22 is imported to a protease-insensitive compartment in the
absence of exogenous ATP suggests that translocation can occur at
reduced levels without energy. This observation favors the hypothesis
that ATP may be necessary to stabilize Tic22 during or shortly after
translocation. This interpretation is supported by the observation that
thermolysin generates discrete proteolytic fragments of the
envelope-bound authentic preTic22 (Fig. 4A) and the
preTic22-SSU chimera (Fig. 7A) in import reactions
containing exogenous ATP. These fragments most likely represent
portions of the proteins that are partially translocated and therefore are protected from digestion by the outer membrane.
The characteristics of preTic22 import suggest that the pathway of
targeting to the intermembrane space is distinct from known chloroplast
targeting pathways that operate at the envelope membranes. The
integration of outer membrane proteins with typical *
This work was supported by National Science Foundation
Grants MCB-9722914 and BIR-94131980 and a Charles and Johanna Busch Memorial Fund Award.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
The abbreviations used are:
preTic22, precursor
of Tic22;
SSU, small subunit of ribulose-1,5-bisphosphate
carboxylase/oxygenase;
preSSU, precursor of SSU;
PCR, polymerase chain
reaction;
PAGE, polyacrylamide gel electrophoresis.
Tic22 Is Targeted to the Intermembrane Space of Chloroplasts
by a Novel Pathway*
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Import of preTic22 in into isolated
chloroplasts. Rabbit reticulocyte lysate containing in
vitro synthesized [35S]preTic22 or
[35S]preSSU was incubated with isolated chloroplasts (50 µg of chlorophyll) at 26 °C in a standard import reaction
containing 2 mM ATP. After the import reaction, the
chloroplasts were incubated in the absence ( 
) or presence (+) of 200 µg/ml thermolysin (T-lysin) or trypsin on ice for 30 min.
Chloroplasts were reisolated in the presence of protease inhibitors.
A, phosphorimage of SDS-PAGE-resolved chloroplast proteins
from [35S]preTic22 import. B, phosphorimage of
SDS-PAGE-resolved chloroplast proteins from [35S]preSSU
import. C, phosphorimage of SDS-PAGE-resolved chloroplast
proteins from [35S]preTic22 import in which chloroplasts
were incubated in the presence (+) or absence () of 200 µg/ml
thermolysin (T-lysin) and 1% w/v Triton X-100
(TX-100) after the import reaction. Tr,
of the translation product added to the import assay. The
positions of preTic22 (pTic22), Tic22, preSSU
(pSSU), and SSU are indicated to the left of each
panel.
View larger version (29K):
[in a new window]
Fig. 2.
Time-dependent import and
processing of preTic22. Rabbit reticulocyte lysate containing
in vitro synthesized [35S]preTic22 was
incubated with isolated chloroplasts at 26 °C in a standard import
reaction containing 2 mM ATP. At the times indicated,
samples from the import reaction (50 µg of chlorophyll) were removed
and incubated for 30 min on ice in the presence (+) or absence ( ) of
200 µg/ml thermolysin (T-lysin). Chloroplasts were
reisolated in the presence of protease inhibitors and analyzed by
SDS-PAGE and phosphorimaging. Tr, of the
translation product added to the import assay. The positions of
preTic22 (pTic22) and Tic22 are indicated to the
right of the figure.
View larger version (59K):
[in a new window]
Fig. 3.
Localization of the newly imported preTic22
to the inner envelope membrane. Chloroplasts (20 mg chlorophyll)
from a [35S]preTic22 import reaction were lysed, and the
total membrane fraction was separated from the stroma by differential
centrifugation. Outer and the inner envelope membranes were resolved
from the total membrane fraction by centrifugation on a linear gradient
of 20-45% w/v sucrose. Proteins from the stroma, outer envelope
membrane (OM), and inner envelope membrane (IM)
were resolved by SDS-PAGE and analyzed by phosphorimaging. Each lane
contains 30 µg of protein. The positions of preTic22
(pTic22) and Tic22 are indicated to the left of
the figure.
View larger version (60K):
[in a new window]
Fig. 4.
Energy dependence of preTic22 import.
Gel-filtered rabbit reticulocyte lysate containing in vitro
synthesized [35S]preTic22 or [35S]preSSU
was incubated with isolated chloroplasts at 26 °C in the presence
(+) or absence ( ) of apyrase or the indicated concentrations of ATP.
A, chloroplasts (100 µg of chlorophyll) from the
[35S]preTic22 import reactions were divided into equal
samples and treated in the presence or absence of 200 µg/ml
thermolysin on ice for 30 min. Chloroplasts were reisolated in the
presence of protease inhibitors and analyzed directly by SDS-PAGE and
phosporimaging. B, chloroplasts (50 µg of chlorophyll)
from the [35S]preSSU import reactions were lysed and
separated into stroma and membrane fractions by differential
centrifugation. Proteins from these fractions were resolved by SDS-PAGE
and analyzed by phosphorimaging. Tr, of the
translation product added to the import assay. The positions of
preTic22 (pTic22), Tic22, preSSU (pSSU), and SSU
are indicated to the right of each panel.
View larger version (80K):
[in a new window]
Fig. 5.
Import of preTic22 requires the presence of
the thermolysin-sensitive components in the chloroplast outer
membrane. Isolated chloroplasts were incubated in the presence (+)
or absence ( ) of 100 µg/ml thermolysin on ice for 30 min.
Chloroplasts were reisolated in the presence of protease inhibitors and
incubated with [35S]preTic22 or [35S]preSSU
in a standard import reaction containing 2 mM ATP at
26 °C for 30 min. Chloroplasts were reisolated and analyzed by
SDS-PAGE and phosphorimaging. Tr, of the
translation product added to the import assay. The positions of
preTic22 (pTic22) and Tic22 are indicated to the
left of the figure, and preSSU (pSSU) and SSU are
indicated to the right.
View larger version (56K):
[in a new window]
Fig. 6.
Import of mature Tic22 into isolated
chloroplasts. Rabbit reticulocyte lysate containing in
vitro synthesized [35S]Tic22 was incubated with
isolated chloroplasts (50 µg of chlorophyll) at 26 °C in a
standard import reaction containing 2 mM ATP. After the
import reaction, the chloroplasts were incubated in the absence ( ) or
presence (+) of 200 µg/ml thermolysin (T-lysin) on ice for
30 min. Chloroplasts were reisolated in the presence of protease
inhibitors and analyzed by SDS-PAGE and phosphorimaging. Tr,
of the translation product added to the import
reaction.
View larger version (58K):
[in a new window]
Fig. 7.
Import of preTic22-SSU and preSSU-Tic22
chimeras into isolated chloroplasts. Rabbit reticulocyte lysate
containing in vitro synthesized
[35S]preTic22-SSU or [35S]preSSU-Tic22 was
incubated with isolated chloroplasts (50 µg of chlorophyll) at
26 °C in a standard import reaction containing 2 mM ATP.
After the import reaction, the chloroplasts were incubated in the
absence ( ) or presence (+) of 200 µg/ml thermolysin
(T-lysin) or trypsin on ice for 30 min. Chloroplasts were
reisolated in the presence of protease inhibitors, lysed, and separated
into membrane and soluble fractions by differential centrifugation.
A, phosphorimage of SDS-PAGE-resolved proteins from soluble
and membrane fractions of chloroplasts from an
[35S]preTic22-SSU import reaction. B,
phosphorimage of SDS-PAGE-resolved proteins from soluble and membrane
fractions of chloroplasts from an [35S]preSSU-Tic22
import reaction. Tr, of the translation product
added to the import assay. The positions of preTic22-SSU
(pTic22-SSU), SSU, preSSU-Tic22 (pSSU-Tic22), and
Tic22 are indicated to the left of the each panel.
View larger version (39K):
[in a new window]
Fig. 8.
Competition of preTic22 import by
preSSU. Rabbit reticulocyte lysate containing in vitro
synthesized [35S]preTic22 or [35S]preSSU
was incubated with isolated chloroplasts (50 µg of chlorophyll) at
26 °C in a standard import reaction containing 2 mM ATP
and various concentrations of unlabeled preSSU (pSSU) or
SSU. After the import reaction, the chloroplasts were incubated in the
presence of 200 µg/ml thermolysin (T-lysin) on ice for 30 min. Chloroplasts were reisolated in the presence of protease
inhibitors. A, phosphorimage of SDS-PAGE-resolved
chloroplast proteins from [35S]preSSU and
[35S]preTic22 import. The positions of preSSU
(pSSU), SSU, preTic22 (pTic22), and Tic22 are
indicated to the right of the figure. Tr,
of translation product added to the import reaction.
B, quantitative analysis of A.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-helical transmembrane domains, such as Toc34 (9, 11), OEP14 (10), and OEP24
(8), does not appear to involve protease-sensitive components of the
outer membrane, nor does integration require ATP. Upon first
evaluation, the characteristics of preTic22 import appeared similar to
aspects of the targeting stromal and integral inner membrane proteins
on the general import pathway. For example, the energetics of preTic22
translocation across the outer membrane are similar to the requirements
for the early stages of translocation of stromal preproteins across the
envelope. At low levels of ATP (<0.1 mM), stromal
preproteins insert across the outer envelope membrane to form an early
import intermediate (18, 22). Translocation of preTic22 across the
outer membrane required similarly low levels of ATP. This observation,
coupled with the resemblance between the preTic22 presequence and the
transit sequences of stromal preproteins, suggested that preTic22
translocation across the outer membrane may involve components of the
general import machinery. However, the preTic22 presequence was unable
to direct import to the stroma (Fig. 7A), and the targeting
of preTic22 to the intermembrane space was not competed by a stromal
preprotein (Fig. 8). Therefore, the preTic22 presequence represents a
new type of targeting signal specific for the chloroplast intermembrane space, and preTic22 import represents a third, novel system for targeting to and across the chloroplast envelope. The nature of the
machinery involved in the pathway of targeting to the intermembrane space remains to be determined, but the identity of intermediates that
are partially inserted across the outer membrane should facilitate the
identification of the components of this pathway.
FOOTNOTES
To whom correspondence should be addressed: Dept. of Biological
Sciences, Rutgers, The State University of New Jersey,
101 Warren St., Newark, NJ 07102. Tel.: 973-353-1082; Fax:
973-353-1007; E-mail: schnell@andromeda.rutgers.edu.
ABBREVIATIONS
REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.
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