J Biol Chem, Vol. 274, Issue 45, 32351-32359, November 5, 1999
The C Terminus of a Chloroplast Precursor Modulates Its
Interaction with the Translocation Apparatus and PIRAC*
Carole
Dabney-Smith
,
Paul W. J.
van den Wijngaard§¶,
Yvonne
Treece
,
Wim J.
Vredenberg§, and
Barry D.
Bruce
**
From the Department of Biochemistry, Cellular, and
Molecular Biology, the Center for Legume Research,
University of Tennessee, Knoxville, Tennessee 37996 and
§ Laboratory of Plant Physiology, Wageningen
Agricultural University, Arboretumlaan 4, 6703 BD Wageningen, The
Netherlands
 |
ABSTRACT |
The import of proteins into chloroplasts involves
a cleavable, N-terminal targeting sequence known as the transit
peptide. Although the transit peptide is both necessary and sufficient to direct precursor import into chloroplasts, the mature domain of some
precursors has been shown to modulate targeting and translocation efficiency. To test the influence of the mature domain of the small
subunit of Rubisco during import in vitro, the precursor (prSSU), the mature domain (mSSU), the transit peptide (SS-tp), and
three C-terminal deletion mutants (
52, 67, and 74) of prSSU were expressed and purified from Escherichia coli. Activity
was then evaluated by competitive import of 35S-prSSU. Both
IC50 and Ki values consistently suggest that removal of C-terminal prSSU sequences inhibits its interaction with the translocation apparatus. Non-competitive import studies demonstrated that prSSU and 52 were properly processed and
accumulated within the chloroplast, whereas 67 and 74 were
rapidly degraded via a plastid-localized protease. The ability of
prSSU-derived proteins to induce inactivation of the
protein-import-related anion channel was also evaluated. Although the
C-terminal deletion mutants were less effective at inducing channel
closure upon import, they did not effect the mean duration of channel
closure. Possible mechanisms by which C-terminal residues of prSSU
modulate chloroplast targeting are discussed.
| |
INTRODUCTION |
Many chloroplast proteins are nuclear-encoded and produced in the
cytosol of the plant cell. Nuclear-encoded chloroplast proteins are
predominantly synthesized as larger molecular weight precursors containing an N-terminal transit peptide. The transit peptide (tp)1 is a cleavable
N-terminal signal sequence that mediates precursor binding and import
into the chloroplast via an ATP-dependent mechanism. Following transport of the precursor through the chloroplast envelope, the transit peptide is cleaved by the stroma-processing peptidase to
generate the mature protein. Transport of precursors across the
chloroplast envelope has been shown to involve distinct protein complexes within the outer membrane (Toc, translocation
apparatus of the outer membrane of
chloroplasts) and inner membrane (Tic, translocation apparatus of the inner membrane
of chloroplasts) (1-4). These two complexes are believed
to interact at inter-membrane contact sites, which enables
precursor translocation in a single coordinated step (5, 6).
Recently, an inner membrane localized anion channel,
PIRAC (Protein Import
Related Anion Channel), has been
shown to be involved in chloroplast protein import. PIRAC is a
50-picoSiemen anion channel that becomes inactivated during chloroplast
protein transport via a mechanism that requires both a functional
transit peptide and a stromal ATP source (7). The exact role of PIRAC
in protein import into the chloroplast is not defined as yet, however,
at least three different mechanisms may explain channel inactivation: (i) PIRAC may function as the protein import channel in the inner membrane of chloroplasts, and inactivation represents a shift from ion
conductance to protein conductance; (ii) PIRAC may be intimately
associated with translocation components of the inner membrane such
that a precursor-induced conformational change results in inactivation
of the ion channel; or (iii) the formation of contact sites between Toc
and Tic during translocation causes transient inactivation of the ion
channel. Further investigation is needed to elucidate the precise role
of PIRAC in protein import into the chloroplast.
Current models suggest that transit peptides are both necessary and
sufficient to direct the targeting and import of precursor proteins
into the chloroplast. However, direct experimental evidence is not
entirely consistent with these models. For example, the attachment of
transit peptides to unrelated precursors confers proper targeting to
some but not all proteins (8-11). In addition, no common motif has
been identified among the several hundred transit peptides that have
been sequenced (12). To explain this apparent enigma, it has been
postulated that a common secondary or tertiary structure involving the
mature domain might provide both targeting specificity and
translocation efficiency (6). Consistent with this notion, chimeras
constructed between the transit peptide and various portions of the
mature domain have been shown to improve import efficiency (8, 10).
Also, deletion of C-terminal sequences from various precursors has been
shown to interfere with one or more steps of the translocation pathway, depending on the precursor studied and the extent of the C-terminal deletion (11). These studies collectively suggest that contributions from the mature domain play an important role in facilitating the
interaction of transit peptides with the translocation apparatus. However, a more systematic and quantitative study is needed to identify
mature domain sequences that directly influence chloroplast import.
In this work, we have evaluated the import activity of the precursor to
the small subunit of Rubisco (prSSU), the mature domain (mSSU), the
full-length transit peptide, (SS-tp), and three prSSU mutants that lack
52, 67, or 74 C-terminal amino acids, respectively. Using these
proteins as competitive inhibitors of the radiolabeled precursor during
import, we demonstrated that the transit peptide is absolutely required
for interaction with the translocation apparatus. However, a specific
region within the mature domain strongly influences this interaction.
In addition, we demonstrated that sequences within the C terminus of
prSSU also influence the interaction between the transit peptide and
PIRAC. The findings indicate that the C terminus of prSSU plays an
important role in protein translocation.
| |
MATERIALS AND METHODS |
Plant Growth and Chloroplast Isolation--
Dwarf pea seedlings
(Pisum sativum, Laxton Progress number 9) were grown, and
chloroplasts isolated as described by Bruce et al. (13).
Chloroplasts for electrophysiology measurements were isolated from pea
leaves by cutting with a razor blade in 2.5 mM Tes/KOH, pH
7.2, 225 mM sorbitol, 25 mM KCl, and 2 mM CaSO4. This preparation was transferred
directly to a 2-ml chamber, which was mounted on a light microscope to
allow visual selection of single intact chloroplasts.
Generation of C-terminal Deletions--
The full-length
precursor was isolated from pET11-prSSU (14) using BamHI and
NcoI, and subcloned into the same sites of pET23d (Novagen).
Exonuclease III digestion was performed using the Erase-a-Base kit
(Promega). To determine the extent of the deletions, direct colony
polymerase chain reaction was performed using T7 promoter
primers. Plasmid DNA was isolated and sequenced in both directions
using an ABI automated sequencer. Three deletion mutants, prSS52
(52), prSS67 (67), and prSS74 (74) were generated that
lacked 52, 67, and 74 amino acids, respectively, at the C terminus. The
74 deletion contained an additional 6 amino acids (ATVVRM) at the C
terminus due to read through into vector sequences. All other regions
of the precursor were unchanged.
Preparation of Precursor Proteins and the Transit
Peptide--
Recombinant proteins were expressed and isolated from the
BL21(DE3) strain of Escherichia coli as described by
Pinnaduwage and Bruce (15). The final inclusion body pellet was then
solubilized in 8 M urea + 50 mM dithiothreitol.
SS-tp was expressed and purified from the BL21 strain of E. coli as described by Pinnaduwage and Bruce (14).
In Vivo Radiolabeling of prSSU and Deletions--
prSSU and
prSSU deletion proteins were in vivo radiolabeled in
E. coli. The cells were grown for 8 h in Dulbecco's
modified Eagle's medium (BioWhittaker) without cysteine and
methionine, followed by incubation with Tran35S-Label
metabolic labeling reagent (ICN). The inclusion bodies were isolated
using BugBusterTM protein extraction reagent (Novagen) and
solubilized in 8 M urea + 50 mM dithiothreitol.
Specific activity was 1-5 × 106 dpm/µg protein.
In Vitro Protein Import Assays--
Kinetic import assays were
performed by incubating increasing concentrations of
35S-labeled prSSU with freshly prepared chloroplasts (50 µg of chlorophyll) in the presence of 1× Import Buffer (330 mM sorbitol, 50 mM HEPES-KOH, pH 8.0)
containing 5 mM Mg-ATP, 10 mM dithiothreitol.
Urea concentration was kept constant at 300 mM and was
shown not to affect import (data not shown). Addition of
35S-prSSU started the assay. After 10 min at room
temperature, the assay was terminated by rapid dilution in ice-cold 1×
Import Buffer (5-fold) and immediately placing the samples on ice in
the dark. Intact plastids were re-isolated according to Bruce et
al. (13) and were prepared for SDS-PAGE.
In Vitro Protein Import Competition Assays--
Competition
import assays were performed by incubating 100 nM
35S-prSSU and increasing concentrations of cold competitor
proteins with freshly prepared chloroplasts as described above.
Competitions proceeded for 15 min at room temperature. To start the
assay, 35S-prSSU and the competitor protein were added in
rapid succession (<3 s between additions) (15). Import assays were
performed as described above.
Time Course of Import and Degradation--
Time course import
assays of 35S-precursors were performed using 300 nM of each precursor as described above. Assays were
performed at room temperature in the dark. Addition of the
35S-precursor started the assay, and 100-µl samples were
removed at timed intervals, diluted 10-fold with ice-cold 1× Import
Buffer, and placed on ice in the dark. Intact chloroplasts were
re-isolated from each sample as described by Bruce et al.
(13). These samples represent the re-purified chloroplast sample. Also
at each time interval, 50 µl of the import reaction was removed and
immediately mixed with 50 µl of 2× SB and boiled for 3 min. These
samples represent the total import reaction.
Data Analysis--
Data analysis was performed by electronic,
filmless autoradiography (Instant-Imager, Packard
Instruments), and quantification of signal by InstantImager
analysis software (Packard Instruments). Signals were reported as
counts per minute (cpm) and converted to molecules of
mSSU/chloroplast/min using the counting efficiency of the instrument,
the specific activity of the precursor, the cysteines and methionines
per molecule, the number of chloroplasts per ml of import reaction, and
the length import time. The data were analyzed using GraphPad PrismTM
(GraphPad Software, Inc.) computer software for enzyme kinetics and the
Km and Vmax determined using
nonlinear regression. These calculations were performed unless
otherwise noted.
Dixon Analysis--
For Dixon analysis, competition import
reactions were performed as described above with either 60 nM or 180 nM 35S-precursor plus
increasing concentrations of cold competitor. Rates were calculated in
molecules of mSSU/chloroplast/min as described above, and the inverse
plotted as a function of inhibitor concentration. Determination of
Ki values was performed by linear regression
analysis of the plot of 1/V versus [I] at the two fixed
prSSU concentrations where the intersection of the lines is given as
Ki.
Electrophysiological Measurements--
Standard patch clamp
technique (16) was used to measure current across the chloroplast
envelope. Electrodes were made from borosilicate glass by a two-step
pull and extensively fire-polished. Electrodes were filled with buffer
containing 2.5 mM Tes/KOH, pH 7.2, 250 mM KCl,
and 2 mM CaSO4, leading to a 10-fold KCl
gradient. Electrode resistances were typically around 30 M
. To
clarify the inactivation of PIRAC during protein import, proteins were added to the pipette filling solution (i.e. cytoplasmic
equivalent) to a concentration of 50 nM. ATP was added to
the bath solution (stroma equivalent) to 0.5 mM for >5 min
before electrophysiological experiments were started.
Currents were measured with an Axopatch 200B patch clamp amplifier
(Axon Instruments). The data were filtered at a cut-off frequency of 1 kHz, using an 8-pole Bessel filter (internal filter of the Axopatch
200B). The filtered data were digitized at 10 kHz using a CED 1401+ and
analyzed with the Patch and Voltage Clamp software (Cambridge
Electronic Design) (16). Potentials are given with regard to the
pipette interior, the bath was kept at ground, using a 250 mM KCl agar bridge. To determine the distributions of open
and closed time durations of PIRAC, a module was developed in the
matrix-calculating software Matlab (Mathworks, Inc.). This module uses
the 50% threshold method to identify transitions of the channel
between the open and the closed state. The distributions were fitted
with multiexponential probability density functions using the maximum
likelihood method (17).
Protein Measurements--
Purified proteins were quantified by
commercial Bradford reagent (Bio-Rad). Quantification of total
chloroplast protein was performed using the BCA protein quantification
assay (Pierce).
| |
RESULTS |
Generation and Purification of C-terminal Deletions--
To
determine the effect of prSSU C-terminal sequences in chloroplast
protein import, serial C-terminal deletions were generated and
characterized. Out of the several hundred clones originally generated,
only a small subset was characterized further, based on deletion length
and ease of purification from inclusion bodies when expressed in
E. coli (data not shown). The location and size of these
deletions are shown in Fig. 1. Variants
of prSSU that lack 52, 67, and 74 amino acids at the C terminus were
denoted 52, 67, and 74. We were unable to detect a deletion
that yielded an insoluble protein shorter in length than the 74
deletion, suggesting that sequences within this region are directly
involved in inclusion body formation. The purification of prSSU, mSSU, GST-tp, and SS-tp was reported previously (15). The 52, 67, and
74 deletions were isolated by a similar method and analyzed for
purity by SDS-PAGE (Fig. 2). The apparent
molecular masses of 52, 67, and 74 are 18.9, 16.7, and 9.8 kDa, respectively, which differ from the calculated molecular masses of
15.3, 12.5, and 11.6 kDa, respectively, for the deletions. We have no
explanation for these abnormal electrophoretic mobilities.
View larger version (13K):
[in this window]
[in a new window]
|
Fig. 1.
Comparison of mSSU, prSSU, GST-tp, SS-tp, and
C-terminal deletions. The position and length of the transit
peptide and mature domain has been drawn to scale. The region
corresponding to the full-length transit peptide is shown in
gray, and the region corresponding to the mature domain is
shown in black. The small white block between GST
and SS-tp represents two amino acids (GS) introduced during
subcloning. Glutathione S-transferase protein is shown with
the diagonal pattern and has been condensed for space
constraints. The amino acid sequences for prSSU, mSSU, and the
deletions (prSS52, prSS67, and prSS74) were obtained from DNA
sequence analysis of the cloned genes. The actual molecular weight is
shown in Daltons on the right.
|
|
View larger version (65K):
[in this window]
[in a new window]
|
Fig. 2.
Purification of E. coli
expressed prSSU, mSSU, 52,
67, 74, GST-tp, and
SS-tp. Overexpressed prSSU, mSSU, 52, 67, and 74 were
purified to near homogeneity. SDS-PAGE and Coomassie Brilliant Blue
stain of purified precursors (5 µg of total protein loaded). Purified
protein loaded in each lane is shown at the top of the
figure.
|
|
In Vitro Import Kinetics--
To define the kinetic parameters of
prSSU import in vitro, the rate of import (processing to
mSSU) as a function of prSSU concentration was determined. The amount
of 35S-prSSU substrate was varied from 10 nM to
1.6 µM in a 10-min in vitro import assay (Fig.
3A). This time point was
within the linear range of the time course of import (data not shown).
Three independent import assays were performed and the amount of mSSU product was directly quantified (Fig. 3B). The average
product formed at each substrate concentration was analyzed by
nonlinear regression (Fig. 3B), which permitted the
determination of Km (282 ± 55 nM)
and Vmax (~9,887 ± 735 molecules of
mSSU/chloroplasts/min) values. Although the Km did
not deviate significantly between separate chloroplast preparations,
the Vmax was found to vary between chloroplast
preparations by ~2-fold.
View larger version (30K):
[in this window]
[in a new window]
|
Fig. 3.
In vitro import kinetics.
A, electronic autoradiogram of a representative import
reaction as a function of increasing concentrations of
35S-prSSU. The concentration of prSSU used in each import
reaction is shown at the top of the gel. Import reactions
were conducted as described under "Materials and Methods."
Chloroplast loading was normalized by BCA protein measurements.
B, quantitative analyses of import data using increasing
concentrations of 35S-prSSU. The inset shows the
data represented as an Eadie-Scatchard analysis. Data are presented as
mean ± S.D.
|
|
Import Competitions and IC50 Determination for prSSU,
mSSU, GST-tp, and SS-tp--
To determine which sequences of prSSU are
both necessary and sufficient for chloroplast import, purified prSSU,
mSSU, GST-tp, and SS-tp were used as competitive inhibitors of
35S-prSSU import in vitro. The concentration of
35S-prSSU used in the competition assays was 100 nM, which is well below the measured Km.
Fig. 4A illustrates
representative import assays for each competitor, and direct
quantification of three independent assays for each competitor is shown
in Fig. 4B. Both mSSU and GST-tp failed to inhibit import of
35S-prSSU, even at the highest concentrations tested. The
inability of GST-tp to compete for the import of prSSU suggests that
the transit peptide requires a free N terminus to productively engage the translocation apparatus. Because both prSSU and its full-length transit peptide effectively compete for import (Fig. 4A),
these data clearly indicate that transit peptide is both necessary and sufficient to direct import into chloroplasts in vitro.
However, the IC50 for SS-tp (570 ± 111 nM) was higher than the IC50 for unlabeled
prSSU (80 ± 15 nM), indicating that the transit
peptide alone engages the import machinery less effectively than the
full-length precursor. Taken together, these data suggest that
sequences in the mature domain of prSSU act in cis to
modulate or enhance the import activity of the transit peptide.
View larger version (34K):
[in this window]
[in a new window]
|
Fig. 4.
Import competitions and IC50
determination for prSSU, mSSU, GST-tp, and SS-tp. A,
SDS-PAGE analysis of import competitions between 35S-prSSU
and cold competitors mSSU, GST-tp, SS-tp, and prSSU. The concentration
of competitor in each reaction is shown at top of the
figure. The 35S-precursor was 100 nM for all
reactions. Reactions proceeded for 10 min and were halted by rapid
dilution. B, graphical analysis of import competitions
between radiolabeled prSSU and competitors mSSU ( ), GST-tp ( ),
SS-tp ( ), and prSSU ( ). Samples were normalized to a no
competitor control. All except GST-tp represent the average of three
independent experiments. The data were analyzed as described under
"Materials and Methods."
|
|
Import Competitions and IC50 Determination of
C-terminal Deletions--
To identify the regions within the mature
domain of prSSU that facilitate import, prSSU C-terminal deletions
(52, 67, and 74) were used as competitive inhibitors in
35S-prSSU import assays. All three deletions were tested
with the same preparation of chloroplasts to minimize experimental
variability. Decreasing levels of mSSU accumulation with increasing
competitor concentration indicated that all three precursor mutants
were able to compete with 35S-prSSU for import into the
chloroplast (Fig. 5A).
However, the longer precursor mutants competed more effectively than
the shorter mutants. Quantification of three independent experiments
(Fig. 5B) resulted in IC50 calculations of
33 ± 15.3, 145 ± 26.9, and 305 ± 40.8 nM
for the 52, 67, and 74 mutants, respectively. Plotting these
values as a function of mature domain length illustrates the
relationship between precursor length and activity as a competitive inhibitor (Fig. 6). Although removal of
the last 52 amino acids from the C terminus of prSSU only slightly
changes the affinity of the precursor for the translocation apparatus,
removal of an additional 15 amino acids increases the IC50
from 33 ± 15.3 nM to 145 ± 26.9 nM
and removal of an additional seven amino acids increases the
IC50 further to 305 ± 40.8 nM. These
findings suggest that sequences between the C termini of the 52 and
74 mutants contribute significantly to the process of chloroplast
protein targeting/translocation.
View larger version (35K):
[in this window]
[in a new window]
|
Fig. 5.
Import competitions and IC50
determination of C-terminal deletion. A, SDS-PAGE
analysis of import competitions between 35S-prSSU and
truncated precursors. The concentration of competitor in each reaction
is shown at top of the figure. The 35S-precursor
was 100 nM for all reactions. Reactions proceeded for 10 min and were halted by rapid dilution. B, graphical analysis
of import competitions between 35S-prSSU and truncated
precursors 52 ( ), 67 ( ), and 74 (). Samples were
normalized to a no competitor control. The data were analyzed as
described under "Materials and Methods."
|
|
View larger version (16K):
[in this window]
[in a new window]
|
Fig. 6.
Relationship of mature domain length and
IC50. Plot of IC50 of competitor as a
function of length of the mature domain of SS-tp (2 amino acids in
mature domain), 74 (46 amino acids in mature domain), 67 (53 amino acids in mature domain), 52 (68 amino acids in mature domain),
and prSSU (120 amino acids in mature domain), and their respective
IC50 values from Table I. Graph was generated using
Kaleidagraph (Abelbeck Software). Data are presented as mean ± S.D.
|
|
Dixon Analysis of Ki Values for prSSU and C-terminal
Deletions--
To determine the inhibition constant,
Ki, for prSSU and the three C-terminal deletion
mutants, Dixon analysis was performed using 35S-prSSU as
substrate and unlabeled prSSU, 52, 67, or 74 as competitive
inhibitors of import. 35S-prSSU was fixed at 60 and 180 nM, and the inhibitor concentration was varied from 50 nM to 1.2 µM (Fig.
7A). Data were fit by linear regression (Fig. 7B) and the intersection point of the lines
is defined as Ki. The Ki values
for prSSU (105 nM), 52 (66 nM), 67 (110 nM), and 74 (437 nM) are consistent with the
IC50 values for each competitor (Table
I). Successive deletion of sequences
within the prSSU mature domain results in less effective import
inhibition, suggesting that an element within the mature domain
modulates targeting and/or translocation efficiency.
View larger version (43K):
[in this window]
[in a new window]
|
Fig. 7.
Dixon analysis of Ki
values for prSSU and C-terminal deletions. A,
representative SDS-PAGE analysis of import competitions between
35S-prSSU and prSSU. The concentration of
35S-precursor was fixed at either 180 or 60 nM
while varying the concentrations of the competitor as shown at the
top of the figure. Truncated precursors were also used as
competitors (data not shown; see Table I for Ki
values). Reactions proceeded for 10 min and were halted by rapid
dilution. B, graphical analysis of import competitions
between radiolabeled prSSU (60 nM, ; 180 nM,
 ) and prSSU. The data were analyzed using GraphPad PrismTM (GraphPad
Software, Inc.) computer software for enzyme kinetics by linear
regression.
|
|
View this table:
[in this window]
[in a new window]
|
Table I
Inhibition of import is affected by regions in the mature domain
Comparison of precursor length and import competence. IC50 and
Ki were calculated by GraphPad PrismTM of previously
described data. IC50 is defined as the concentration of
inhibitor required to inhibit 50% of activity. Ki
is defined as the concentration of competitor required to bind half the
total binding sites on an enzyme at equilibrium in the absence of
substrate.
|
|
Attenuated import competition by the prSSU deletion mutants could
result from disrupted binding at the chloroplast surface, impaired
translocation through Toc and Tic, or some combination of these
processes. To explore a binding-specific effect, binding studies were
performed with prSSU and the precursor mutants at low levels of ATP
(data not shown). The binding results demonstrated that removal of 52, 67, and 74 amino acids from the C terminus did not significantly affect
binding of these precursors to the chloroplast presumably via Toc components.
Import Time Course of prSSU and C-terminal Deletions--
Except
for mSSU and GST-tp, all of the proteins tested have been shown to
compete for the import of 35S-prSSU. To directly determine
whether the C-terminal deletion mutants of prSSU are competent for
chloroplast import in vitro, the truncated precursors were
metabolically labeled with 35S and used as substrates in
import time course experiments. Monitoring the level of
35S-labeled protein remaining in both the reaction mixture
and within re-isolated chloroplasts permits direct quantification of
the substrate remaining at each time point, as well as the amount of
imported/processed mature form. In this way, both the import rate and
the extent of mSSU accumulation for each precursor may be determined
and directly compared.
The four autoradiogram panels on the left of Fig.
8A indicate the precursor pool
size remaining in each import reaction as a function of import time.
Quantification of these data are shown in Fig. 8B, where the
amount of precursor was normalized to the 1 min time point allowing
comparison between the different precursors. All four proteins, prSSU,
52, 67, and 74, were imported at a similar rate, as seen in
Fig. 8A and quantified in Fig. 8B. Re-isolation
of the chloroplasts, however, demonstrated that only full-length prSSU
and the 52 precursor imported and accumulated as mature processed
proteins. Although the level of 67 and 74 precursors clearly
decreased as a function of import time, these mutants failed to
accumulate to detectable levels as a processed form in the stroma.
Taken together, these results indicate that all four precursor proteins
import successfully into the chloroplast but that the 67 and 74
truncated precursors are subject to rapid degradation following import.
Neither the identity nor the sub-organellar location of the protease
responsible for this degradation is known.
View larger version (46K):
[in this window]
[in a new window]
|
Fig. 8.
Import time course of prSSU and C-terminal
deletions. A, SDS-PAGE analysis of time course import
reactions of 35S-prSSU and 35S-truncated
precursors (52, 67, and 74). The concentration of
35S-precursor was 300 nM for all reactions.
Left panels: 5 µl of total import reaction from time
points indicated at the top of the figure. Import reactions
proceeded for the indicated time and were halted by dilution (1:2) with
1× SSB and boiling immediately. Right panels: 35 µg of
protein from re-isolated chloroplasts from time points indicated at the
top of the figure. Reactions proceeded for the indicated
time and were halted by rapid dilution. B (left),
graphical analysis of import time course showing decrease of
precursors: prSSU (), prSS52 ( ), prSS67 (), and
prSS74 (*); (right), graphical analysis of import time
course showing increase of accumulation of mature protein: mSSU (),
mSS52 (), mSS67 (*), and mSS74 (). All data were
analyzed using GraphPad PrismTM (GraphPad Software, Inc.) computer
software for enzyme kinetics by linear regression.
|
|
Interaction of prSSU and C-terminal Deletions with
PIRAC--
Thus far, we have demonstrated that sequences
within the mature domain of prSSU enhance the efficiency of transit
peptide-mediated import into chloroplasts. These findings implicate an
interaction of the precursor with transport complexes, Toc and Tic.
However, the anion channel, PIRAC, is also involved in chloroplast
protein import since a functional transit peptide and full-length
precursor have been shown to inactivate the channel (18). To evaluate whether precursor interaction with PIRAC is also modulated by sequences
in the mature domain, single channel recordings of PIRAC conductance
were measured in the presence of prSSU, mSSU, and the prSSU C-terminal
deletion mutants. Fig. 9A
shows the single channel recordings of PIRAC conductance for a control
excised patch and for an excised patch after protein addition. Fig.
9B represents the all points amplitude histogram of these
recordings. Addition of prSSU to the envelope patch decreases the open
probability (PO) of the channel from 0.81 ± 0.04 in the control situation to 0.17 ± 0.08, indicating that
prSSU induces channel inactivation. In contrast, addition of mSSU
induced no significant change in PIRAC activity, as indicated directly
in Fig. 9A, and in the failure of this protein to decrease
the PO measured in the all points amplitude
histogram in Fig. 9B. The deletion mutants of prSSU, however, induced PIRAC inactivation in a manner that was proportional to the length of the mature domain of the precursor. The
PO of PIRAC in the presence of 52, 67, and
74 was 0.45 ± 0.07, 0.53 ± 0.07, and 0.71 ± 0.07, respectively (Table II). These data
indicate that the transit peptide is necessary for PIRAC inactivation, as was shown previously (7, 18), and that the removal of the C terminus
of the precursor attenuates this interaction.
View larger version (29K):
[in this window]
[in a new window]
|
Fig. 9.
Interaction of prSSU and C-terminal deletions
with PIRAC. A, single channel recordings of the
absence (control) or presence of 50 nM protein
(mSSU, prSSU, prSS52, prSS67, or prSS74) in the pipette
filling solution. Recordings were made at a holding potential of 20
mV. B, all point amplitude histograms of single channel
recordings of PIRAC in the absence and presence of the different
proteins.
|
|
View this table:
[in this window]
[in a new window]
|
Table II
Effect of precursor addition on the Po and duration times for
PIRAC
Comparison of the probability of channel opening in the absence
(control) or presence of 50 nM protein (mSSU, prSSU,
prSS52, prSS67, and prSS74). Durations of the closed
( C) and open (O) states are also given as the
mean ± S.D.
|
|
Under control conditions (i.e. in the absence of any
precursor) and in the presence of mSSU, three closed channel states
with characteristic mean durations of 0.35 ± 0.03, 2.41 ± 0.98, and 29.1 ± 6.8 ms and two open channel states with mean
durations of 0.88 ± 0.34 and 47.2 ± 9.6 ms have been
detected for PIRAC (Table II) (18). However, in the presence of prSSU,
an additional closed state (4C) with a much longer
duration was detected (18). The duration of this new long-lived closed
state of PIRAC is 989 ± 214 ms. All of the truncated precursors
induced a similar long-lived closed state with a mean duration of
~1300 ± 400 ms (Table II). The observation that full-length
prSSU and the deletion mutants decreased the PO
of PIRAC, yet did not significantly change the duration of
4C, suggests that these precursors interact with PIRAC
via a common mechanism but that their affinity for the channel is
proportional to the length of the mature domain. Whether the difference
in affinities results from an attenuation of direct precursor
interaction with PIRAC or, alternatively, is a secondary result of a
diminished interaction with components of either Toc and/or Tic will
require additional study.
| |
DISCUSSION |
Kinetic Analysis of prSSU--
The precursor for the small subunit
of Rubisco is one of the most well studied precursors, yet kinetic
analysis of its in vitro import has not been reported
previously. Compared with the few other precursors whose import kinetic
parameters have been determined experimentally (prLHCP, prFd,
prOEE23, and prOEE33), the Km of prSSU
import is intermediate between the value reported for prFd (120 nM) and prOEE23 (400 nM). The
Vmax of prSSU import is also comparable to that
reported for prLHCP. In contrast, the Vmax of
prSSU import is significantly less than the Vmax
reported for either prFd (~22,000 molecules/chloroplast/min) (19) or prOEE23 (>40,000 molecules/chloroplast/min) (20). These
differences may reflect genuine disparities in the rate or efficiency
of import for the various precursors, or alternatively, they may
represent differential responses to chemical denaturation agents used
for solubilization of the precursor. As discussed previously by Pilon et al. (19), however, the rates of translocation measured
in vitro are quite close to the rates required in
vivo for normal maintenance of chloroplast biogenesis, which lends
credibility to the use of in vitro import studies as a means
of exploring the mechanism of chloroplast protein transport.
Effect of GST Fusion on SS-tp Activity--
An interesting and
significant finding from our in vitro import studies is that
the transit peptide of prSSU failed to function as a competitive
inhibitor of import when GST was fused to the N terminus of the transit
peptide. This finding suggests that addition of a 26.6-kDa folded
fusion protein inhibits interaction of the N terminus of the transit
peptide with one or more components of the targeting/translocation
apparatus. Although the precise mechanism of this apparent interaction
is unknown, one possibility is that the committed step in
binding/translocation occurs when the N terminus of the precursor
traverses both envelope membranes and gains accessibility to the
stroma. Once the N terminus of the transit peptide stretches into the
stroma, binding by one or more molecular chaperones may prevent escape
and provide a mechanism for unidirectional ATP-dependent
translocation. Evidence from our laboratory indicates that chloroplast
transit peptides function in vitro as substrates for hsp70
(21), and that >80% of stromal-targeted precursors in the CHLPEP data
base contain high affinity hsp70 binding sites at their N termini (21,
22). If this interaction occurs in vivo, then binding by
hsp70 in either the stroma (23) or the intermembrane space (3) would
require a free transit peptide N terminus, and fusion to GST would
predictably block import.
An alternative explanation for the lack of import activity by GST-tp is
that the GST domain may interfere with interactions between the transit
peptide and lipid components of the chloroplast envelope. Two
independent laboratories have shown that the N terminus of transit
peptides are capable of interacting directly with chloroplast-specific lipids (15, 24). The GST-tp fusion protein used in this study was shown
previously to inhibit the interaction of SS-tp with liposomes whose
composition mimics the outer envelope (15). Regardless of the molecular
mechanism, however, our findings clearly suggest that accessibility of
the N terminus of SS-tp is required for interaction with one or more
components of the chloroplast translocation apparatus. Several
investigations into the role of different domains of chloroplast
transit peptides have also shown that the N terminus is required for
precursor import in vitro (24, 25), in vivo (26,
27), and for directing the import of certain fusion proteins in
vitro (8). An intriguing aspect of these reports is that N
terminus of some transit peptides is often the least conserved region
of the entire presequence (26).
Diminished Activity of SS-tp versus prSSU--
Although popular
models contend that the transit peptide is both necessary and
sufficient to support the import of chloroplast-destined precursors
(6), several reports indicate that the addition of mature domain
sequences increases the targeting/translocation activity of certain
transit peptides (8-10). We also found that sequences within the
mature domain of prSSU enhance import efficiency, only when fused
in cis with the transit peptide. The minimal requisite structural elements for engaging the translocation apparatus are contained within the transit peptide, however, not the mature domain.
In direct support of this conclusion, short synthetic peptides derived
from the SS-tp sequence competitively inhibit prSSU import (28), and
similarly, the full-length ferredoxin transit peptide has been shown to
import into chloroplasts with no associated passenger protein (29). The
full-length precursor was a more effective inhibitor of in
vitro chloroplast import, however, and deletion of specific mature
domain sequences within the context of prSSU significantly impaired
this activity, strongly implicating this mSSU sequence in a beneficial
cis interaction with the transit peptide during chloroplast
targeting/translocation. The physical/chemical basis for this
interaction remains unknown, however.
Effect of C-terminal Deletions--
Both the IC50 and
the Ki values calculated from our experiments
indicate that removal of sequences at the C terminus of prSSU decrease
the affinity of the protein for one or more components of the
translocation apparatus. This effect is not strictly a linear function
of precursor length, however, as shown in Fig. 6. Removal of the last
52 amino acids of prSSU may have enhanced import potential, yet removal
of a few additional sequences progressively and significantly weakened
the ability of the precursors to compete for import. In addition, the
52, 67, and 74 mutations did not affect precursor binding to
the chloroplast. Unlike full-length prSSU and the 52 mutant,
however, the 67 and 74 precursors did not accumulate in the
stroma, suggesting that the decrease in the pool amount may be due to
rapid degradation. These observations contrast with the findings of a
previous study of prSSU import, which indicate that removal of 26 and
47 amino acids from the C terminus completely abolish import (11). Even
at the earliest point in import time course experiments, the authors
did not detect binding or import by the mutant precursors and
attributed no import to abolished binding as opposed to rapid
degradation. One major difference between that report (11) and our
study is that we used purified proteins rather than in vitro
translated protein. A potential explanation for the discrepancies in
the findings from these two studies, therefore, is that components in
the wheat germ extract may interfere with the import of the mutant precursors.
Stromal Assembly versus Degradation of the Truncated Small
Subunit--
Direct import of our radiolabeled prSSU mutants into
isolated chloroplasts indicated that wild-type prSSU and the 52
mutant clearly engaged the translocation apparatus, traversed the
chloroplast envelope, and were properly processed into stable
appropriately-sized mature forms. In contrast, neither the precursor,
mature form, nor discreet degradation products of the radiolabeled
67 and 74 mutants were detected. The rate at which all four
precursors decreased in the total import reaction was comparable,
however, suggesting that each precursor was successfully imported into the chloroplast. Degradation of the 67 and 74 precursors outside the chloroplast is highly unlikely since the organelle preparations were Percoll-purified and extensively washed. A probable explanation is
that the 67 and 74 mutants were degraded in the stroma following import. Mistargeted proteins, incorrectly translated proteins, nonfunctional proteins, or overproduced subunits are proteolytically degraded in the chloroplast to prevent accumulation of nonfunctional or
potentially toxic subunits. Analysis of the Rubisco holoenzyme three-dimensional structure (30) indicates that the deletions in 67
and 74 remove significant structural elements that intimately contact adjacent large subunits. The 52 mutation, however, removes only one unstructured domain that may not be required for folding and/or holoenzyme assembly. The failure of m67 and m74 to
assemble properly into the holoenzyme may result, therefore, in rapid
degradation via a post-translocational mechanism.
The protease responsible for m67 and m74 degradation is not
known, however, a likely candidate is the plastid homologue (ClpC/P) to
the bacterial Clp family of serine proteases. ClpC is the regulatory
subunit and ClpP the proteolytic subunit. Although both subunits are
stromally localized (31), they have also been shown to co-purify with
the chloroplast translocation apparatus (32, 33) and were implicated in
degradation of mistargeted aberrant prOE33. (34). Further
experiments are needed to determine what protease is responsible for
proteolytic degradation of m67 and m74.
Effect on PIRAC--
Previous studies have demonstrated that PIRAC
is inactivated during ATP-dependent protein translocation
into the chloroplast (7, 18). This inactivation is reflected by the
presence of a precursor-induced long-lived closed state that is absent
in the control patches. The fact that PIRAC is localized in the inner envelope and is inactivated by cytosolic-localized precursors or
transit peptides in the presence of stromal-localized ATP suggests that
PIRAC is either a component of Tic or is in some way intimately associated with the Tic complex. All of the precursors and transit peptides tested to date (7, 18) induced a similar long-lived closed
state, suggesting that the duration of closure is independent of
transit peptide/precursor length. Some other dynamic aspect of the
translocon, as opposed to the time needed for completion of polypeptide
translocation, may determine the duration of PIRAC closure. The effect
of the C-terminal deletions of prSSU on the PO
of PIRAC may result from removing a critical structural element that is
required both for precursor interaction with Toc/Tic and PIRAC, or
alternatively, it may be a secondary effect of reduced affinity for a
component in the outer envelope, which precedes interaction with PIRAC.
Concluding Remarks--
Conflicting evidence in the literature has
debated the role of the C terminus of chloroplast precursor proteins in
targeting/translocation across the chloroplast envelope. In this first
study to directly compare the targeting/translocation activity of a
purified precursor, its mature domain, transit peptide, and multiple
C-terminal deletion mutants, we have verified a basic tenet of
chloroplast import models that the transit peptide is absolutely
necessary for import. Our data also demonstrate quantitatively that the
interaction of prSSU's transit peptide with the translocation
apparatus is positively modulated by sequences within the mature
domain, which function in cis. Moreover, we have partially
mapped the regions within the mature domain that are particularly
important for this interaction. In addition to facilitating a transit
peptide-mediated interaction with components of the translocation
apparatus, Toc and Tic, sequences within the mature domain of prSSU
also contribute to the precursor's affinity for PIRAC. The structural
basis for interactions between disparate regions of the precursor and
these distinct chloroplast membrane complexes remains to be determined.
| |
ACKNOWLEDGEMENTS |
B. D. B. and C. D. S. thank Dr. R. Miltenberger for critical reading of the manuscript. We
thank Dr. M. Salvucci for the gift of the cloned precursor of the small
subunit of Rubisco from tobacco.
| |
FOOTNOTES |
*
This work was supported in part by the National Science
Foundation Cell Biology Program (to B. D. B.) and the
University of Tennessee/Oak Ridge National Laboratory Science Alliance.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.
¶
Part of the joined program "Chloroplast Protein Import" in
collaboration with Prof B. de Kruijff and Prof. P. Weisbeek, supported by the foundation for Earth and Life Sciences with financial aid from
The Netherlands Organization for Scientific Research.
Undergraduate student supported by a National Science
Foundation Research Experience for Undergraduates Award (to B. D. B.) and by the John and Clara Hobby Scholarship.
**
To whom correspondence should be addressed. Tel.: 423-974-4082, E-mail: bbruce@utk.edu.
| |
ABBREVIATIONS |
The abbreviations used are:
tp, transit peptide;
Rubisco, ribulose-bisphosphate carboxylase/oxygenase;
Tes, N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid;
PAGE, polyacrylamide gel electrophoresis;
GST, glutathione
S-transferase.
| |
REFERENCES |
| 1.
|
Perry, S. E.,
and Keegstra, K.
(1994)
Plant Cell
6,
93-105[Abstract]
|
| 2.
|
Hirsch, S.,
Muckel, E.,
Heemeyer, F.,
Heijne, G. v.,
and Soll, J.
(1994)
Science
266,
1989-1992[Abstract/Free Full Text]
|
| 3.
|
Schnell, D. J.,
Kessler, F.,
and Blobel, G.
(1994)
Science
266,
1007-1012[Abstract/Free Full Text]
|
| 4.
|
Schnell, D. J.,
Blobel, G.,
Keegstra, K.,
Kessler, F.,
Ko, K.,
and Soll, J.
(1997)
Trend. Cell Biol.
7,
303-304[Medline]
[Order article via Infotrieve]
|
| 5.
|
Chen, X.,
and Schnell, D. J.
(1999)
Trends Cell Biol.
9,
222-227[CrossRef][Medline]
[Order article via Infotrieve]
|
| 6.
|
Bruce, B. D.,
and Keegstra, K.
(1994)
in
Advances in Molecular and Cell Biology: Molecular Processes of Photosynthesis.
(Barber, J., ed), Vol. 10
, pp. 389-430, Jai Press Inc., Greenwich, CT
|
| 7.
|
van den Wijngaard, P. W.,
and Vredenberg, W. J.
(1997)
J. Biol. Chem.
272,
29430-29433[Abstract/Free Full Text]
|
| 8.
|
Wasmann, C.,
Reiss, B.,
Bartlett, S.,
and Bohnert, H.
(1986)
Mol. Gen. Genet.
205,
446-453[CrossRef]
|
| 9.
|
Lubben, T. H.,
Gatenby, A. A.,
Ahlquist, P.,
and Keegstra, K.
(1989)
Plant Mol. Biol.
12,
13-18[CrossRef]
|
| 10.
|
Kavanagh, T. A.,
Jefferson, R. A.,
and Bevan, M. W.
(1988)
Mol. Gen. Genet.
215,
38-45[CrossRef][Medline]
[Order article via Infotrieve]
|
| 11.
|
Ko, K.,
and Ko, Z. W.
(1992)
J. Biol. Chem.
267,
13910-13916[Abstract/Free Full Text]
|
| 12.
|
von Heijne, G.,
Hirai, T.,
Kl sgen, R. B.,
Steppuhn, J.,
Bruce, B. D.,
Keegstra, K.,
and Herrmann, R.
(1991)
Plant Mol. Biol. Rep.
9,
104-126
|
| 13.
|
Bruce, B. D.,
Perry, S.,
Froehlich, J.,
and Keegstra, K.
(1994)
Plant Molecular Biology Manual
, pp. 1-15, Kluwer Academic Publishers, Belgium
|
| 14.
|
Klein, R. R.,
and Salvucci, M. E.
(1992)
Plant Physiol.
98,
546-553[Abstract/Free Full Text]
|
| 15.
|
Pinnaduwage, P.,
and Bruce, B. D.
(1996)
J. Biol. Chem.
271,
32907-32915[Abstract/Free Full Text]
|
| 16.
|
Hamill, O.,
Marty, A.,
Neher, E.,
Sakmann, B.,
and Sigworth, F.
(1981)
Pflügers Arch.
391,
85-100[CrossRef][Medline]
[Order article via Infotrieve]
|
| 17.
|
Colquhoun, D.,
and Sigworth, F.
(1995)
in
Single Channel Recording
(Sakmann, B.
, and Neher, E., eds), 2nd Ed.
, pp. 483-587, Plenum Press, New York
|
| 18.
| van den Wijngaard, P., Dabney-Smith, C., Bruce, B., and Vredenberg, W. (1999) Biophys. J., in press
|
| 19.
|
Pilon, M.,
Weisbeek, P. J.,
and de Kruijff, B.
(1992)
FEBS Lett.
302,
65-68[CrossRef][Medline]
[Order article via Infotrieve]
|
| 20.
|
Cline, K.,
Henry, R.,
Li, C.,
and Yuan, J.
(1993)
EMBO J.
12,
4105-4114[Medline]
[Order article via Infotrieve]
|
| 21.
| Ivey, R., and Bruce, B. (2000) Cell Stress Chaperones, in
press
|
| 22.
|
von Heijne, G.
(1992)
J Mol Biol
225,
487-494[CrossRef][Medline]
[Order article via Infotrieve]
|
| 23.
|
Marshall, J. S.,
DeRocher, A. E.,
Keegstra, K.,
and Vierling, E.
(1990)
Proc. Natl. Acad. Sci. U. S. A.
87,
374-378[Abstract/Free Full Text]
|
| 24.
|
Pilon, M.,
Wienk, H.,
Sips, W.,
de Swaaf, M.,
Talboom, I.,
van `t Hof, R.,
de Korte-Kool, G.,
Demel, R.,
Weisbeek, P.,
and de Kruijff, B.
(1995)
J. Biol. Chem.
270,
3882-3893[Abstract/Free Full Text]
|
| 25.
|
Lawrence, S. D.,
and Kindle, K. L.
(1997)
J. Biol. Chem.
272,
20357-20363[Abstract/Free Full Text]
|
| 26.
|
Rensink, W. A.,
Pilon, M.,
and Weisbeek, P.
(1998)
Plant Physiol.
118,
691-699[Abstract/Free Full Text]
|
| 27.
|
Kindle, K. L.,
and Lawrence, S. D.
(1998)
Plant Physiol.
116,
1179-1190[Abstract/Free Full Text]
|
| 28.
|
Perry, S. E.,
Li, H.-M.,
and Keegstra, K.
(1991)
Methods Cell Biol.
34,
327-344[Medline]
[Order article via Infotrieve]
|
| 29.
|
van `t Hof, R.,
and de Kruijff, B.
(1995)
J. Biol. Chem.
270,
22368-22373[Abstract/Free Full Text]
|
| 30.
|
Taylor, T.,
and Andersson, I.
(1997)
Biochemistry
36,
4041-4046[CrossRef][Medline]
[Order article via Infotrieve]
|
| 31.
|
Shanklin, J.,
DeWitt, N. D.,
and Flanagan, J. M.
(1995)
Plant Cell
7,
1713-1722[Abstract]
|
| 32.
|
Akita, M.,
Nielsen, E.,
and Keegstra, K.
(1997)
J. Cell Biol.
136,
983-994[Abstract/Free Full Text]
|
| 33.
|
Nielsen, E.,
Akita, M.,
Davila-Aponte, J.,
and Keegstra, K.
(1997)
EMBO J.
16,
935-946[CrossRef][Medline]
[Order article via Infotrieve]
|
| 34.
|
Halperin, T.,
and Adam, Z.
(1996)
Plant Mol. Biol.
30,
925-933[CrossRef][Medline]
[Order article via Infotrieve]
|
Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.
TOP
ABSTRACT
INTRODUCTION
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
