(Received for publication, April 4, 1997, and in revised form, June 16, 1997)
From the Plant Science Center, Cornell University, Ithaca, New York 14853
Nucleus-encoded chloroplast proteins that reside
in the thylakoid lumen are synthesized as precursors with bipartite
transit peptides that contain information for uptake and
intra-chloroplast localization. We have begun to apply the superb
molecular and genetic attributes of Chlamydomonas to study
chloroplast protein import by creating a series of deletions in the
transit peptide of plastocyanin and determining their effects on
translocation into isolated Chlamydomonas chloroplasts.
Most N-terminal mutations dramatically inhibited in vitro
import, whereas replacement with a transit peptide from the -subunit
of chloroplast ATPase restored uptake. Thus, the N-terminal region has
stroma-targeting function. Deletions within the C-terminal portion of
the transit peptide resulted in the appearance of import intermediates,
suggesting that this region is required for lumen translocation and
processing. Thus, despite its short length and predicted structural
differences, the Chlamydomonas plastocyanin transit peptide
has functional domains similar to those of vascular plants. Similar
mutations have been analyzed in vivo by transforming
altered genes into a mutant defective at the plastocyanin locus
(K. L. Kindle, manuscript in preparation). Most mutations affected
in vitro import more severely than plastocyanin
accumulation in vivo. One exception was a deletion that
removed residues 2-8, which nearly eliminated in vivo
accumulation but had a modest effect in vitro. We suggest that this mutant precursor may not compete successfully with other proteins for the translocation pathway in vivo. Apparently,
in vivo and in vitro analyses reveal different
aspects of chloroplast protein biogenesis.
The majority of chloroplast proteins are encoded in the nucleus, synthesized in the cytosol as precursors, and directed to the organelle by a stroma-targeting domain within an N-terminal extension known as the transit peptide (TP).1 Studies using vascular plant chloroplasts have shown that transport across the plastid envelope requires binding to specific receptors and translocation through a general import channel. A subset of imported proteins is integrated into or transported across the chloroplast thylakoid membrane, by one of at least four distinct mechanisms. For recent reviews on chloroplast protein import see Refs. 1-4.
The mechanism of routing proteins to the chloroplast envelope membranes
and translocating them into the stroma appears to be unique, since
aside from Hsp70s, none of the currently identified components is
related to proteins associated with other membrane translocation
pathways (1). Chloroplast protein import is initiated by specific
contact between the stroma-targeting domain of the precursor and
components of the import receptor, an interaction that requires GTP and
ATP (5-9). This is followed by translocation across the envelope into
the chloroplast stroma in a process that requires higher levels of ATP
and may be assisted by chaperones (10-13). That the stroma-targeting
domain is necessary and sufficient for sorting to the envelope and
translocation into the stroma has been demonstrated both in
vivo and in vitro (for numerous examples, see Ref. 14).
Competition studies in which synthetic peptides or precursors were
added to in vitro chloroplast protein import assays have
demonstrated that a single pathway is used to translocate most
precursors across the envelope (15-18). Although there is no primary
amino acid sequence consensus in the stroma-targeting domains of
transit peptides, they share certain features that are apparently
important for efficient plastid targeting. Generally they are enriched
in hydroxylated amino acid residues and deficient in acidic residues.
They have an N-terminal region devoid of Gly, Pro, and charged
residues; a middle region rich in Ser, Thr, Lys, and Arg; and a
C-terminal region predicted to form a -strand, which is terminated
by the stromal processing consensus site (I/V)X(A/C)
A, where
indicates the cleavage site (19). Removal of the
stroma-targeting domain by the stromal processing protease generates
mature stromal proteins or intermediates that are subsequently targeted
to other chloroplast locations (20, 21).
Lumen resident proteins contain bipartite transit peptides, which consist of an N-terminal stroma-targeting domain fused to a lumen-targeting domain. The lumen-targeting domain is required for translocation across the thylakoid membrane (for reviews see Refs. 1, 4, and 14) and contains a hydrophobic region similar to the signal sequences required for bacterial protein secretion (22). Transport into the lumen can be mediated by at least two mechanisms that are utilized by specific subsets of lumen resident proteins (18, 23). Two different pathways have also been described for integration of proteins into the thylakoid membrane (24-27). The function and evolution of these parallel targeting pathways into and across the thylakoid membrane remain to be elucidated.
Most chloroplast protein import studies have relied on in
vitro assays in which isolated chloroplasts are incubated with
proteins that have been synthesized in vitro. However, it is
unlikely that the complexity of the in vivo milieu is fully
reproduced in vitro. In fact, very different results were
obtained when two constructs in which the small subunit of
ribulose-bisphosphate carboxylase (SSU) TP coding region was fused to
that of neomycin phosphotransferase (NptII) were tested
in vivo and in vitro (28, 29). Disparate results
were also obtained when the plastocyanin TP was fused to -lactamase
(30). The interpretation of these results is complicated because they
involved chimeric proteins, and different species were used for the
in vitro and in vivo analyses.
Spinach or pea chloroplasts have generally been used for in vitro experiments because of their ease of handling and physiological longevity after isolation (31). Unfortunately, neither pea nor spinach is especially tractable for in vivo studies. To examine the role of the transit peptide in chloroplast protein import both in vitro and in vivo, using a native chloroplast protein in a homologous system, we have chosen to study plastocyanin (PC) import in the unicellular eukaryotic alga, Chlamydomonas reinhardtii.
The isolation of biochemically active chloroplasts from
Chlamydomonas is not as well-documented as in pea or
spinach. However, energy-requiring uptake of the SSU into these
chloroplasts has been reported (32, 33). Chlamydomonas
transit peptides are shorter than those in vascular plants, and the
N-terminal region of the stroma-targeting domain, defined for vascular
plant precursors as deficient in Gly, Pro, and charged residues, is
either extremely short or missing. Furthermore,
Chlamydomonas stroma-targeting domains have a central region
with predicted -helical secondary structure, a characteristic of
mitochondrial presequences (34). Indeed, the Chlamydomonas
SSU transit peptide had mitochondrial targeting function when expressed
in yeast (35). These observations raise the question of whether uptake
and processing of proteins by Chlamydomonas chloroplasts are
analogous to these processes in vascular plants. Current data on this
point are contradictory. For example, the specificity of the
Chlamydomonas stromal processing peptidase has been reported
to be the same as (36) and different from (37) the pea enzyme.
Furthermore, although Chlamydomonas pre-SSU and
pre-CF1-
were imported into spinach and pea
chloroplasts, both incorrectly and correctly processed products were
observed (37-39). On the other hand, a synthetic TP from
CF1-
inhibited the uptake of pea pre-SSU into pea
chloroplasts (40), suggesting that there must be functional
similarities between the transit peptides.
To determine whether similar domains exist in Chlamydomonas transit peptides and those of vascular plants, we have examined in vitro import of plastocyanin (PC) precursors with a variety of deletions in the transit peptide. PC resides in the thylakoid lumen. The Chlamydomonas plastocyanin TP sequence has regions similar to those identified in the bipartite TPs of lumen resident proteins in vascular plants, although positively charged residues are not excluded from the N terminus of the stroma-targeting domain (41). The results presented below demonstrate that Chlamydomonas chloroplasts import PC in a transit peptide- and energy-requiring process. Furthermore, the Chlamydomonas PC transit peptide appears to contain functional domains similar to those of vascular plants.
We have compared the results of this in vitro analysis to those obtained in vivo by transforming genes with identical deletions in the coding region of the plastocyanin TP into a mutant defective at the PC structural gene locus.2 Although there are similarities in the results obtained with the two approaches, there are also several intriguing differences, which suggest that these complementary analyses may reveal different aspects of the chloroplast protein import pathway.
A full-length PC cDNA clone
(PC6-2) was obtained from Sabeeha Merchant (UCLA; see Ref. 41). The
downstream EcoRI site was removed by partially digesting the
DNA with EcoRI, creating blunt ends with the Klenow fragment
of DNA polymerase I, and religating the plasmid. EcoRI and
XbaI digestion released the cDNA insert, which was
subcloned into pGEM7Zf(+) (Promega, Madison, WI) digested with the same
enzymes. A series of transit peptide deletions were constructed in the
genomic petE gene as described in detail
elsewhere.2 Briefly, an EcoRI site was
introduced 60 base pairs upstream of the initiation codon, and
NheI sites were individually introduced at several locations
encoding Ala-Ser in the transit peptide. A series of deletions within
the transit peptide was constructed by combining
EcoRI-NheI fragments from appropriate pairs of
plasmids. In addition, a deletion mutation removing residues 2-8 was
made by site-directed mutagenesis. These deletions were subcloned into the cDNA context by the following procedure. A BbsI site
in the first exon of the genomic clone is located 314 nucleotides
downstream of the introduced EcoRI site. The
EcoRI-BbsI fragments encompassing the transit
peptide deletions were subcloned into the PC cDNA, replacing the
EcoRI-BbsI fragment in the cDNA; this
resulted in clones that are 31 base pairs longer than PC6-2 at the 5
end, as shown in Fig. 1. Several chimeric
genomic petE genes have been constructed
previously3 by making
translational fusions between the promoter, 5
-untranslated region, and
26- or 29 amino acid residues of the transit peptide-coding region of
atpC to two different locations in the PC transit peptide. (atpC encodes CF1-
, the
-subunit of
chloroplast ATPase.) The chimeric transit peptide-coding regions were
cloned into the petE cDNA context as
EcoRI-BbsI fragments, as described above.
Plasmid DNAs were isolated (42), linearized with XbaI, and transcribed with SP6 polymerase as described (43) except that the RNAs were resuspended in half the reported volume. These RNAs were translated with a wheat germ system as described previously (43) in the presence of [35S]methionine (Amersham Corp. SJ235).
Chlamydomonas Culture Conditions and Preparation of ChloroplastsC. reinhardtii CC-406
(cw15mt) was obtained from the
Chlamydomonas Genetics Center (Duke University).
Chloroplasts were isolated using a modification of methods previously
reported to yield a high fraction (90%) of intact chloroplasts (33,
44). Cells grown to different concentrations (1-5 × 106 cells/ml) under photoautotrophic or mixotrophic
conditions yielded chloroplasts with similar import efficiencies.
Therefore, cells were grown in liquid HSA medium (45) under 16 h
room light:8-h dark cycles to a concentration of 3-4 × 106 cells/ml, collected by centrifugation at 2800 × g for 5 min, and washed in 50 ml of 20 mM
Hepes/Na salt, pH 7.5 (Sigma). After centrifugation for 4 min at
2000 × g, cells were resuspended at a concentration of
2 × 108 cells/ml in breaking buffer (0.3 M sorbitol, 50 mM Hepes/Na salt, pH 7.5, 2 mM EDTA, 1 mM MgCl2) plus 1%
bovine serum albumin (fraction V from Sigma) and incubated on ice for
15 min. The algae were loaded into an ice-cold Kontes press,
equilibrated for 3 min to 35 p.s.i. N2, and lysed by
rapid depressurization. Lysed cells (3 ml) were loaded onto Percoll
step gradients made of 4 ml each of 45 and 70% Percoll in a 15-ml
COREX tube (44). After 15 min centrifugation at 2000 × g in a swinging bucket rotor, chloroplasts were collected
from the 45/70% Percoll interface, washed in 3 volumes of breaking
buffer, and harvested by centrifugation for 3 min at 1400 × g. Generally, less dense cultures required higher breaking
pressure to achieve maximal yields of chloroplasts. These preparations
are enriched in chloroplasts, but not pure, since immunoblot analysis
indicated substantial levels of the mitochondrial ATPase subunit
F1-
and low molecular weight G proteins, which are
localized to Golgi and flagellar membranes (Ref. 46; data not shown).
Final yields averaged about 24%, so that a 2-liter culture produced
plastids equaling about 3 mg of chlorophyll. Chloroplasts equivalent to
37.5 µg of chlorophyll were used in each assay, as described below;
this allowed an average of 40 assays/liter of cultured cells.
Chloroplasts were resuspended in
import buffer (0.33 M sorbitol, 50 mM
Hepes/KOH, pH 8.0) at 375 µg of chlorophyll/ml. Translation products
were diluted 1:3 in import buffer containing 30 mM
methionine. Equal amounts of [35S]methionine-labeled
precursors were used for each import assay, as determined by
quantifying bands from SDS-PAGE by PhosphorImager analysis (Molecular
Dynamics, Inc., Sunnyvale, CA). Import assays were conducted at
24 °C in a volume of 150 µl, which included 25 µl of diluted
translation products and chloroplasts equivalent to 37.5 µg of
chlorophyll, with or without 10 mM Mg-ATP for 15 min,
illuminated by white light unless indicated otherwise (43). Assays were
also performed in the dark in foil-wrapped tubes without added ATP.
After incubation, intact chloroplasts were reisolated by centrifugation
through a 35% Percoll cushion following treatment with 95 µg/ml
thermolysin for 40 min at 4 °C (43), as indicated. Plastids were
washed in import buffer, pelleted by centrifugation, and stored at
20 °C. Samples were resuspended in 18 µl of 10 mM
EDTA, an equal volume of 2 × SDS-PAGE buffer was added, and they
were then heated for 10 min at 67 °C. Aliquots of 7.5 µl were
loaded into polyacrylamide gels (43), which were electrophoresed, dried, and exposed to PhosphorImager screens. In calculating import efficiency, the methionine content of the precursor (2 residues) and
the fully processed mature protein (1 residue) was taken into account.
Since relatively few in vitro chloroplast protein import studies have been reported with Chlamydomonas chloroplasts, we first sought to establish that the system had characteristics expected of a bona fide in vitro import assay, i.e. that uptake of proteins by isolated chloroplasts is energydependent, requiring either white light or ATP, and that after uptake into the chloroplast the precursors are protected from added protease by the plastid envelope. We first characterized the uptake of the wild-type PC precursor into isolated Chlamydomonas chloroplasts in these respects.
Fig. 2A shows that white light
or additional ATP was required for uptake and processing of
plastocyanin (compare lanes 4-6). In the presence of 10 mM ATP and white light, Chlamydomonas
chloroplasts converted about 14% of the input wild-type PC precursor
to the mature size in a 15-min assay, and this polypeptide was
resistant to thermolysin. Import was linear with time for 15-20 min
(Fig. 2B). Approximately twice as much mature protein was
produced when 10 mM ATP was added to an import reaction
performed in white light, compared with white light alone (compare Fig.
2A, lanes 6 and 7). When import assays
were conducted in foil-wrapped tubes without added ATP and plastids
were treated with thermolysin following the import period, neither
mature PC nor PC precursor was detected (Fig. 2A, lane 4).
This demonstrates that PC precursor was susceptible to thermolysin
degradation and that there was insufficient ATP in the extract to
support detectable import. However, protease-resistant precursor was
detected in the light in the absence of added ATP, as shown in Fig.
2A, lane 6, and Fig. 2C, where it accounted for one-third of the total protease-protected plastocyanin species. When
ATP was added to the assay, the amount of mature PC increased, and the
amount of protease-protected PC precursor decreased (Fig. 2C). In white light alone, the total amount of
protease-protected PC species was about 75% as high as the maximal
level, when additional ATP was included. Although adding ATP above 1 mM did not further increase the total amount of
protease-protected PC, the fraction that was mature increased slightly
above 2.5 mM. This suggests that a first step in
plastocyanin import, conversion to a protease-protected form, requires
less ATP than subsequent steps for translocation into the thylakoid
lumen and processing to the mature size.
Effect of Transit Peptide Deletions on in Vitro Import
A
series of deletions have been introduced into the petE gene,
which resulted in in-frame deletions of 3-28 amino acid residues from
the plastocyanin transit peptide.2 These deletions were
subcloned into a cDNA context, so that they could be used to
synthesize radioactively labeled precursors for in vitro
chloroplast protein import assays. In vitro translation products from these clones yielded a small amount of an additional protein that migrated more slowly during SDS-PAGE. (Compare the translation products from the clone with the genomic 5-untranslated region (gPC in Figs. 2 and 3A) to those from the
cDNA clone (cPC in Fig. 4A).) Nonetheless,
the precursor produced from wild-type gPC imported into isolated
Chlamydomonas chloroplasts with an efficiency equivalent to
the protein produced from the original cDNA (compare Figs.
2B and 4A).
Fig. 3 shows that most transit peptide
deletions resulted in a dramatic reduction in the amount of PC imported
into isolated Chlamydomonas chloroplasts. Only deletions
near the N terminus (2-8 and
10-12) allowed the uptake and
processing of significant amounts of plastocyanin. The
13-19
precursor retained residual import activity, as demonstrated by the
appearance of a small amount of mature PC. When the
30-37 precursor
was incubated with Chlamydomonas chloroplasts, two to three
smaller bands resulted, as shown in Fig. 3A, as well as
protease-resistant precursor. The appearance of protease-resistant
precursor suggests that import into the stroma may be impaired, and the
accumulation of intermediates suggests that stromal processing may be
aberrant and/or that lumen translocation may be inhibited. The lowest
band may represent a small amount of mature PC, although it migrates
very close to a band of unknown origin that sticks to the outside of
chloroplasts in vitro and is present among translation
products from this particular cDNA.
An import time course of the wild-type, 2-8, and
10-12
precursors is shown in Fig. 4. Although
the overall uptake and processing of the
10-12 and wild-type
precursors appear similar, the final amount of mature-sized protein was
reduced by about half for the
10-12 precursor. The inhibition
caused by this 3 amino acid deletion varied from one experiment to the
next; mature PC produced from the
10-12 precursor ranged from 33 to
70% of the wild-type level. From the
2-8 cDNA construct, a low
molecular weight translation product close to the size of mature PC was
synthesized in vitro. However, this translation product did
not seem to bind to the outside of the plastid or to be taken into the
chloroplasts (Fig. 4). The time course of import appeared somewhat
different for the
2-8 precursor. After 2 min, the amount of mature
PC was similar to wild type, but at later time points, the amount that
accumulated was significantly reduced. In different experiments the
2-8 precursor was imported and processed to approximately 5-25%
the extent of the wild-type precursor.
We next asked whether a portion of the transit peptide
from a protein normally residing in the Chlamydomonas stroma
could replace the N-terminal portion of the plastocyanin TP. If
Chlamydomonas TPs have domains similar to those of vascular
plants, it was expected that the N terminus of CF1-
could replace the N terminus of the PC transit peptide and effect its
import into isolated chloroplasts. The stromal processing protease site
in Silene plastocyanin has been localized between Lys-41 and
Ala-42 (20). A putative site for processing of Chlamydomonas
PC by the stromal peptidase may occur either after Lys-22, if the
processing site is similar to Silene PC, or perhaps after
Ala-23, since the site LKA
A is related to the stromal processing
consensus site (V/I)X(A/C)
A (19). As shown in Fig.
5C, two portions of the CF1-
transit peptide, containing either 26 or 29 amino acid residues, were fused to the
plastocyanin TP at two points, either N-terminal or C-terminal to the
presumed stromal processing site, at amino acid residues 20 or 30, respectively. The sites of the fusions are indicated by the name of the
construct, so that in
26-20PC, 26 amino acid residues from
CF1-
were fused to the plastocyanin TP at amino acid
residue 20. As shown in Fig.
5A, the
26-20PC and
29-20PC precursors were imported and processed to the mature size,
although in the latter case, an intermediate species also accumulated. The
10-19 PC precursor, which showed no import activity in the in vitro assay, has a TP deletion that removes fewer amino
acid residues than the 20 or 30 amino acid residues eliminated from the
PC TP in the chimeric constructs. Together, these results indicate that
the N-terminal 20 amino acid residues of the TP are necessary for
import of PC into Chlamydomonas chloroplasts and that a
portion of the CF1-
TP is sufficient to restore this function. This suggests that the N terminus of the PC TP is a stroma-targeting domain whose function can be replaced by the N
terminus of the CF1-
TP.
Neither the 26-30PC nor
29-30PC chimeric precursors were
imported and processed to the mature size upon incubation with Chlamydomonas chloroplasts. While import of the shortest
precursor (
26-30PC) did not result in any processed plastocyanin,
the addition of three amino acids from CF1-
(in the
29-30PC precursor) allowed the production of a protein intermediate
in size between the precursor and mature PC. Thus, the site of the
fusion to the PC transit peptide is an important determinant of
chimeric TP function.
To compare the sizes of the products created during in vitro
import of precursors containing alterations in the transit peptide, the
processed proteins were electrophoretically separated in adjacent wells, as shown in Fig. 5B. Proteins intermediate in size
between the precursor and mature plastocyanin were produced with
30-37,
29-20PC, and
29-30PC. The protein produced
from the
29-30PC precursor migrated between the mature-sized and
intermediate-sized proteins produced from the
29-20PC precursor.
The most prominent protease-resistant protein arising from the
30-37 precursor appears to migrate with the
intermediate-sized protein found in
29-20PC.
We have demonstrated energy- and transit
peptide-dependent import of a lumen resident protein into
Chlamydomonas chloroplasts. The import efficiency of PC into
Chlamydomonas chloroplasts averaged 14% of the input
precursor. This is inefficient in comparison to import into pea
chloroplasts, where, for example, Bauerle et al. (47) found
that 34-50% of different PC precursors was imported. We have also
tested the import efficiency of the CF1- precursor and
found that about 10% was taken up and processed.3
Goldschmidt-Clermont et al. (33) reported that 5-15% of
the SSU precursor was imported in vitro by
Chlamydomonas chloroplasts. Improvements in the
Chlamydomonas in vitro chloroplast protein import assay
might increase our ability to detect low level import of mutant
precursors.
Import of proteins into isolated pea chloroplasts has been divided into several steps that have different energy requirements. Initial binding of pre-SSU to the chloroplast requires low concentrations of GTP and ATP (50-100 µM), whereas translocation across the envelope is maximal with >1 mM ATP (10, 48, 49). Recently, Scott and Theg (50) have described a precursor-sized intermediate that accumulates at low ATP levels. This intermediate has apparently crossed the outer envelope membrane because it is protected from added protease and yet has not entered the stroma because its transit peptide has not been removed. The protease-protected precursor that we have observed may represent a similar translocation intermediate, since it was more abundant in the light without extra ATP. The ratio of mature to unprocessed, protease-protected precursor increased as more ATP was added, suggesting that low levels of ATP drive precursor uptake, but complete translocation into the lumen and processing into a mature-sized protein require more energy.
The lack of a discernible PC intermediate during in vitro
import of the wild-type precursor is not too surprising. Using pea chloroplasts, the amount of PC intermediate that was detected depended
on the import conditions and the precursor (47). It is possible that
the Chlamydomonas stroma contains proteases that digest
intermediates during post-import manipulations. However, intermediate-sized PC species that were presumably defective in lumen
translocation were observed following import of 30-37 pre-PC and
the
29-20PC and
29-30PC chimeric precursors. Therefore, it
seems more likely that lumen translocation is so rapid in
Chlamydomonas that wild-type intermediates are translocated
and processed during post-import protease treatment and chloroplast
isolation. It is clear from pulse-labeling experiments that PC
precursor import proceeds through an intermediate in vivo
(51).2 Perhaps treatments that rapidly stop the in
vitro import reaction (for example HgCl2) would be
useful in Chlamydomonas as they have been in pea to identify
pathway intermediates of proteins destined for the thylakoid (52,
53).
Our results show that the N-terminal half of the plastocyanin TP is
required for import into Chlamydomonas chloroplasts, since no import was detected with the 10-19 precursor. There is no single
part of the first 19 amino acid residues that is critical for
stroma-targeting function, since individual deletions between residues
2-8, 10-12, and 13-19 all allowed at least residual import. This
suggests that either there are redundant stroma-targeting elements or,
perhaps more likely, that each part of this region contributes to the
overall efficiency of chloroplast uptake. It should be noted, in
contrast, that even short deletions at the extreme N terminus of the
Silene pratensis ferredoxin TP eliminated in
vitro import into pea chloroplasts (54). The reason that a similar
deletion in the
2-8 precursor had a relatively small effect in the
Chlamydomonas in vitro import assay is unknown but may
relate to the lack of an uncharged N-terminal domain in the Chlamydomonas PC transit peptide. When the N-terminal 20 amino acid residues were replaced by regions of the CF1-
transit peptide, a mature-sized PC was produced upon import,
demonstrating that PC stroma-targeting function can be replaced by the
CF1-
transit peptide.
Deletion of part of the hydrophobic portion at the C-terminal end of
the transit peptide in 30-37 led to accumulation of at least two
intermediate-sized proteins, which were localized to the stroma in
preliminary experiments (data not shown). These observations suggest
that the
30-37 precursor is translocated across the chloroplast
envelopes and processed in the stroma but that lumen translocation and
processing by the thylakoid protease do not occur. Overall, the
functional domains in the Chlamydomonas PC transit peptide
therefore appear to be similar to those defined in vascular plant TPs
(55).
The mutations described in this paper have also been tested in
vivo, by transforming a series of mutant petE genes
into a strain defective at the plastocyanin structural gene
locus.2 Fig. 3 shows a summary of the in vitro
and in vivo results for the deletion mutants. Although these
data were consistent in many cases, deletions generally had a more
severe impact on in vitro chloroplast protein import than on
accumulation in vivo. For example, although no import was
detected with the 10-19 precursor in vitro, PC
accumulated to about 15% of the wild-type level in transformants expressing this mutant gene. In vitro import may be
sensitive to even subtle defects in envelope translocation, whereas
envelope translocation may not normally be the rate-limiting step for
PC accumulation. Indeed, envelope translocation is thought not to limit
the targeting of lumen resident proteins during in vitro import into intact chloroplasts, since saturation of thylakoid transport can occur when large amounts of precursor are present (18).
Envelope translocation may limit PC accumulation in vivo only when it is very severely impaired.
In contrast, the 2-8 precursor imported relatively well in
vitro, but very little mature PC accumulated in vivo in
transformants expressing the corresponding mutant gene at a high level.
Since pulse-labeling experiments indicated that the
2-8 precursor
was synthesized and relatively stable in these transformants, we
suggest that it may be less efficient in binding to or translocation
across the plastid envelope and therefore unable to compete with other wild-type precursors in vivo. Apparently there is a unique
requirement for the extreme N terminus of the TP in vivo
that is not reproduced by in vitro uptake into isolated
chloroplasts.
C-terminal deletions in the PC transit peptide had a very severe effect
on accumulation of PC species in vivo, whereas
protease-protected PC intermediates were readily observed when the
30-37 precursor was imported in vitro. Since
pulse-labeling experiments indicated that an intermediate species was
produced in vivo,2 the protein is probably
translocated across the envelope, but lumen translocation is blocked.
In vivo, the intermediate is presumably too unstable to
accumulate to a detectable level. We speculate that the effects of
C-terminal transit peptide deletions are probably similar in
vivo and in vitro, but the short duration of the
in vitro assays allows easier detection of the intermediate
species.
A comparison of the in vivo and in vitro analyses
of the chimeric constructs is shown in Fig. 5C.
Interestingly, mature PC accumulated to nearly the wild-type level when
the 26-20PC construct was expressed in vivo and to about
half the wild-type level for the
26-30PC and
29-20PC
constructs.3 No intermediate species were observed for
these constructs, suggesting that if stromal intermediates are
generated in vivo, they are too unstable to accumulate. For
the
29-30PC construct, trace amounts of an intermediate species
considerably smaller than the one observed in vitro
accumulated. In vitro, the
29-30PC precursor appeared to
be imported and processed to an intermediate that probably lacks a
functional lumen-targeting domain. It presumably fails to
accumulate to a significant level in vivo because it remains
in the stroma and is degraded. The
26-30PC precursor imported
poorly in vitro and remained unprocessed, yet substantial mature plastocyanin accumulated in transformants that expressed the
mutant gene. We suggest that even if envelope translocation is
inefficient in vivo, it is sufficient to support substantial accumulation of plastocyanin. The precursor may also remain unprocessed in vivo, but in any case, it is apparently competent for
lumen translocation and thylakoid processing.
Clearly, the nature of in vitro and in vivo
analysis of chloroplast import and targeting is quite different. An
in vitro assay occurs over a period of minutes in isolated
chloroplasts. The precursor has no competition from other precursors
and is present in nonphysiological quantities. Chloroplast protein
accumulation in transgenic organisms occurs over a period of hours to
days and depends on many processes in addition to protein import,
including transcription, translation, and, in the case of many
chloroplast proteins, assembly with cofactors and other polypeptides,
as well as turnover of the protein and/or complex. A small change in
in vivo chloroplast protein import might not limit
accumulation of the mature protein and therefore be undetectable.
Moreover, mislocalized precursors and intermediates may be unstable
in vivo and therefore not detected except in pulse-labeled
cells (56, 57).2 However, experiments examining cytosolic
processes that regulate chloroplast protein import (58-60) and
temporal or spatial coupling between synthesis and import should be
more accessible in vivo. Moreover, differences between
in vivo and in vitro results, such as those noted
here for the 2-8 deletion, may provide a window into the
integration of the import pathway with cellular metabolism. The ability
to compare in vivo and in vitro results in a
homologous system provides a unique opportunity to study chloroplast
protein import in a genetically tractable organism.
We are indebted to K. Cline for expert advice on developing the in vitro assay and critical review of the manuscript. We are also grateful to members of the Stern and Kindle groups for helpful discussions and to D. Higgs and R. Drager for comments on the manuscript.