From the
Two cDNA clones encoding distinct forms of plastid pyruvate
kinase (designated Pka and Pkg) have recently been characterized. Pkg
is found in both leucoplasts and chloroplasts, whereas Pka is present
only in leucoplasts. The precursors of these proteins have different in vitro import characteristics. The Pkg precursor behaves
like a typical stromal protein precursor with both types of plastid. In
contrast, Pka precursors accumulate on the outer envelope membrane of
leucoplasts under the same assay conditions and require a higher level
of ATP for import into the organelle. Interestingly, the binding of Pka
precursors to chloroplasts cannot be detected at any tested level of
ATP even though the precursors are imported into the organelle at
higher concentrations of ATP. Various N-terminal deletions and chimeric
fusions were used to examine the translocation signaling mechanism of
the Pka precursor. The N-terminal 83-amino- acid segment of Pka
contains a transit peptide that is capable of directing dihydrofolate
reductase and the mature body of Pkg into both types of plastid. Unlike
the complete Pka precursor, these fusion proteins behave like typical
stromal protein precursors. The behavior of the Pka transit peptide is
influenced by a 19-amino- acid domain
(-P-S-S-I-E-V-D-A-V-T-E-T-E-L-K-E-N-G-F-) located immediately
downstream of the N-terminal 83- residue segment. Deletion of this
domain from Pka alters its import properties such that it resembles a
typical stromal protein precursor. Re-introduction of the 19- residue
domain into the Dhfr fusion protein alters its import characteristics
to resemble that of the complete Pka precursor. This 19-amino-acid
domain can also influence the function of transit sequences from other
precursors when it is placed immediately behind the transit peptide.
These results suggest that this 19-amino-acid domain plays an important
role in governing the import characteristics of the Pka precursor. We
have named this 19-residue segment the ``import modifying
domain.''
Nuclear-encoded plastid proteins are usually synthesized as
precursors in the cytosol and then targeted to the organelle. The
translocation of protein precursors into the plastid is a complex
process involving steps such as unfolding, binding to receptors on the
outer envelope, and translocation across the two envelope membranes
(for reviews see Keegstra(1989) and Keegstra et al. (1989)). A
low level of ATP (0.1 mM) is needed for high affinity binding
of precursors to the plastid and a higher ATP level (1 mM) is
required for completing translocation. The primary signal for directing
precursor proteins through the translocation pathway usually resides in
the amino terminus and is termed the transit peptide. Generally, the
transit peptide contains sufficient information for the correct
targeting of proteins into the plastid and for intraorganellar sorting.
Although most plastid protein precursors are targeted to the
organelle as described above, some variations have been reported. Four
chloroplast outer envelope proteins (Om6.7, Om14, Sce70, and Omp24)
possess an uncleavable ``transit peptide'' at the amino
terminus, have ATP-independent uptake (with the exception of Omp24),
and do not require protease-sensitive receptors. This suggests the
presence of a pathway different from that used by other plastid
proteins (Salomon et al., 1990; Li et al., 1991; Wu
and Ko, 1993; Fischer et al., 1994). The transit peptide of
the maize inner envelope protein Bt1 and the Cab protein functions
primarily as a stromal targeting sequence. Additional information
targeting Bt1 to the inner envelope and Cab to the thylakoid is located
in the mature region of the protein (Cline, 1988; Lamppa, 1988; Van den
Broeck et al., 1988; Viitanen et al., 1988; Hand et al., 1989; Kohorn et al., 1989; Li et
al., 1992). Removal of carboxyl domains of chloroplast precursor
proteins has, in most cases, a dramatic effect on different stages of
translocation such as import, processing, and intraorganellar
targeting, suggesting that carboxyl-terminal sequences in the mature
part of the proteins can influence the function of the transit peptide
(Ko and Ko, 1992). Hence, the signal determining a protein's
import characteristics is highly complex and can be influenced by
information in various parts of the polypeptide. Our current
understanding of this phenomenon is mainly derived from proteins
targeted to chloroplasts. It has been suggested that similar mechanisms
operate in other types of plastid, e.g. leucoplasts, but there
may also be unique signals for each functionally and structurally
distinct plastid type. It is known that chloroplast protein precursors
such as Rbcs
Leucoplasts are found primarily
in nonphotosynthetic tissues such as the endosperm of developing castor
seeds. Leucoplasts are metabolically active and are the site of fatty
acid biosynthesis in developing oil seeds. In order to supply
substrates and cofactors for the production of fatty acids, these
organelles contain a glycolytic pathway, including the enzyme pyruvate
kinase, that catalyzes the irreversible reaction from
phosphoenolpyruvate and ADP to pyruvate and ATP. In this study, the
availability of cDNA clones encoding two distinct forms of leucoplast
pyruvate kinase (designated Pka and Pkg) (Blakeley et al.,
1991, 1995) allowed us to study the mechanism by which leucoplastic
proteins are imported into two different types of plastids,
chloroplasts and leucoplasts. The import results provide evidence that
Pka and Pkg behave differently toward the two types of plastid. We have
identified a 19-amino-acid domain located immediately downstream of the
Pka transit sequence which appears to alter the import characteristics
of Pka in response to energy status and plastid type.
Antibodies against castor Pka and tobacco Pkg were generated
using fusion proteins as described by Wu and Ko(1993). The resulting
fusion proteins contain carboxyl-terminal sequences of each pyruvate
kinase and the T7 gene 10 polypeptide. Preimmune IgGs were collected
prior to the injection of each rabbit.
Subfractionation of leucoplasts and chloroplasts was
according to Smeekens et al.(1986). Plastid envelopes were
subfractionated using discontinuous sucrose gradients (Keegstra and
Yousif, 1986). Castor leucoplasts and pea chloroplasts, membrane
fractions, and stromal extracts were subjected to denaturing
SDS-polyacrylamide gel electrophoresis (Laemmli, 1970) and
electrophoretically transferred onto nitrocellulose membranes (Towbin et al., 1979). The protein blots were processed according to
Hoffman et al.(1987). Primary antibody reactions were detected
using alkaline phosphatase-conjugated anti-rabbit IgGs (Promega).
All DNA manipulations were performed according to established
protocols. Castor Pka was subcloned into pGEM4 as a 2130-bp EcoRI cDNA fragment and was designated pCPka. SP6 RNA
polymerase-generated Pka transcripts from pCPka did not give rise to in vitro translation products. Replacement of the
5`-untranslated sequence of castor Pka with the corresponding region
from tobacco Pka did, however, result in successful in vitro translation. The 5` sequence replacement was accomplished by
inserting a 74-bp EcoRI-AluI DNA fragment encoding
the 5`-untranslated sequence and first two amino acids of the tobacco
Pka cDNA clone into the EcoRI and BamHI sites of
pGEM4 (Promega). The BamHI site was converted into a blunt
end. The resulting vector was called pT5Pka. A 2120-bp BamHI-SacI DNA fragment encoding castor Pka and its
3`-untranslated sequence was subsequently inserted into pT5Pka. This
final construct was designated pTCPka. This cloning scheme did not
affect the amino acid sequence of the castor Pka protein. Tobacco Pkg
was subcloned into pGEM5 as a 2000-bp PstI-NotI cDNA
fragment and was designated pTPkg.
The construction scheme for
amino-terminal deletions of Pka and Pkg is illustrated in Fig. 5.
A total of seven amino-terminal deletions were constructed for pTCPka.
Five of the deletions resulted in the removal of 30, 83, 102, 110, and
141 amino acids from the amino terminus of pTCPka and were designated
N1 to N5, respectively. The other two deletions resulted in the removal
of 19 and 58 amino acids from residue 83 to 102 and from 83 to 141 at
the amino terminus and were designated N6 and N7, respectively. The 19
amino acids are -P-S-S-I-E-V-D-A-V-T-E-T-E-L-K-E-N-G-F- (for the
complete DNA sequence of Pka, see Blakeley et al.(1991)). In vitro transcription of these deletions was initiated from
the SP6 promoter.
The chimeric constructs described in this section are
illustrated in Fig. 7. In vitro transcription of these
fusion constructs was initiated from the SP6 promoter.
All of the constructs presented here were analyzed with
restriction enzymes and sequencing to ensure that the fusion points
were in-frame using established protocols.
The growth conditions for pea plants were the same as
described by Ko and Cashmore(1989). Intact pea chloroplasts were
isolated from 14-day-old seedlings as described by Bartlett et
al.(1982). Approximately 8
Import assays were assembled in 0.3-ml volumes as
described in Bartlett et al.(1982). A typical import reaction
contained intact plastids,
The
amount of Pka precursor and mature forms of Pka and Pkg was determined
at various stages of castor endosperm development (Fig. 1C). The precursor form of Pka was detected at
stage 4, was present from stage 4 to 7 (Fig. 1C, lanes 3-6), and decreased by stage 8 (Fig. 1C, lane 7). Mature Pka could be detected
at stage 1-2, increased to a maximum at stage 4, and remained
relatively constant until stage 7, after which it declined (Fig. 1C, lanes 1-3 and 7). The
level of mature Pka correlates with maximal fatty acid biosynthesis
which occurs between stages 4 and 6 (Simcox et al., 1979). The
developmental pattern of mature Pkg was similar to that of Pka (Fig. 1C).
The import of Pka into leucoplasts is
different from both Pkg and Rbcs. Pka precursors associated with the
leucoplast envelope both in the presence of nigericin (Fig. 2A, lane 2) or 1 mM ATP (Fig. 2A, lane 4). These precursors were
sensitive to both thermolysin and trypsin (Fig. 2A, lanes 3, 5, and 6) and co-fractionated with
the membranes (Fig. 2A, lane 10). Hence, unlike
Pkg and Rbcs, Pka precursors accumulate on the outside surface of the
leucoplast in the presence of 1 mM ATP. At this ATP
concentration, Pka was not transported into leucoplasts even after 60
min (data not shown). It is probable that this form represents the
66-kDa immunoreactive band detected in the outer envelope. When the
concentration of ATP was increased to 3 mM, about 50% of the
Pka precursors were imported and processed to a protease-resistant
63-kDa protein in the stroma (Fig. 2A, lanes
4-7 and 9). The imported Pka co-migrated with the
major 63-kDa immunoreactive band found in the leucoplast stroma (Fig. 1A, lanes 4 and 10). Therefore,
a transit peptide of approximately 3 kDa is removed upon import
suggesting that Pka is a precursor protein with a transit signal. The
requirement of a higher level of ATP for Pka uptake suggests that Pka
possesses atypical import characteristics in the leucoplast when
compared with Pkg and Rbcs.
The import of Pkg and Pka into pea
chloroplasts was also studied. Pkg precursors were associated with the
chloroplast envelope in the presence of nigericin and were susceptible
to thermolysin (Fig. 2B, lanes 2 and 3). In the presence of 1 mM ATP, most Pkg precursors
were imported and processed to a thermolysin- and trypsin-resistant
55-kDa stromal form. Little of the bound precursor remained (Fig. 2B, lanes 4-7 and 9).
These import data are consistent with the immunoblots, where Pkg is
found in the chloroplast (Fig. 1A, lanes 7 and 11).
Unlike Pkg, Pka did not bind to the chloroplast
envelope even in the presence of nigericin (Fig. 2B, lanes 2 and 3), but was imported into the stroma and
processed to a protease-resistant 63-kDa form when the assays contained
1 mM ATP (Fig. 2B, lanes 4-10).
The lack of detectable binding even when import was inhibited by
nigericin is atypical for protein precursors destined for the
chloroplast.
The Rbcs precursor is imported
into leucoplasts at 1 mM ATP, but at a relatively lower level
which is only slightly enhanced at higher ATP concentrations (Fig. 3A). Both DSP and DTSSP gave rise to cross-linked
complexes between Lom70 and Rbcs precursors or its intermediate forms (Fig. 4C, lanes 2 and 3). The mature
form of Rbcs did not react even when the membrane-permeable
cross-linker DSP was used. Immunoprecipitated products were not
observed with anti-Cim37 IgGs (Fig. 4C, lanes 4 and 5). These results suggest that the Rbcs precursor and
the intermediate forms are also in close proximity to Lom70 during
translocation.
The import competence of the
truncated Pka proteins was tested in both leucoplasts and chloroplasts.
Truncated Pka proteins N1 to N5 were not able to bind to or import into
either type of plastid (N2 is used as an example in Fig. 6A), suggesting that the information for directing
the import of Pka resides within the amino-terminal 30 amino acids.
This was further corroborated by the N6 and N7 import results. Both N6
and N7 were imported, indicating that the import signal is located in
the amino-terminal 83-amino-acid segment. In contrast to full-length
Pka, both N6 and N7 bound to leucoplasts and chloroplasts in the
presence of nigericin (lane 2 of Fig. 6, B and C) and were imported into both types of plastid in the
presence of 1 mM ATP (lanes 4-7 and 9 of Fig. 6, B and C). The imported forms
were approximately 3 kDa smaller than the precursors, and approximately
50% of the precursors were imported. These results indicate that N6 and
N7 possess import properties that are different from the full-length
Pka but similar to typical plastid protein precursors with respect to
the ATP requirement and the behavior of the precursors toward the two
types of plastid.
Removal of an amino-terminal 87-amino-acid segment from Pkg
(NPkg) (Fig. 5A and lane 11 of Fig. 5B) abolished the import into either type of
plastid (Fig. 6A). The NPkg protein is smaller than
imported mature Pkg (Fig. 5B, lane 12),
suggesting that the amino-terminal 87-residue region possesses the
transit peptide. These results confirm that a transit peptide resides
within the amino-terminal 87 residues of Pkg and is essential for its
import.
Pka1-Pkg behaved like a typical protein precursor (data
not shown). The imported form was approximately 3 kDa smaller than the
precursor, confirming that the Pka transit signal is capable of
importing Pkg. Approximately 60% and 100% of the Pka1-Pkg precursors
presented were translocated into leucoplasts and chloroplasts,
respectively. The similar level of import found with Pka1-Pkg versus Pkg suggests that the Pka transit peptide is as
efficient as the Pkg transit peptide in directing the Pkg protein into
the plastids. It also confirms that the function of the Pka transit
peptide is modified by the 19-residue element.
The function of the
Pkg transit peptide was modified upon inclusion of the 19-amino-acid
domain in the protein, Pkg-Pka. Pkg-Pka bound to leucoplasts in the
presence of nigericin or ATP and was imported into the leucoplast and
processed in the presence of 1 mM ATP (Fig. 8A). The imported protein was approximately 6 kDa
smaller than the Pkg-Pka precursor, consistent with the removal of the
Pkg transit peptide. Hence, the Pkg transit peptide is capable of
directing the import of Pka into the leucoplast even with the
19-amino-acid domain. However, the efficiency of import was reduced
with less than 30% of the Pkg-Pka precursors imported (Fig. 8, A, lane 4, and C); the remainder accumulated
on the surface of the leucoplast (Fig. 8A, lanes 4 and 10). This compares with 60% or more import of the Pkg
precursor under the same conditions (Fig. 2A, lane
4) suggesting that the Pkg transit peptide is influenced at least
to some extent by the 19-residue domain of Pka. Therefore, in both
proteins (Pka and Pkg), the 19-residue domain reduced the amount of
import and enhanced the accumulation of the precursor on the surface of
the leucoplast. The import of Pkg-Pka was not increased at higher ATP
concentrations or at longer time intervals (longer than 15 min) as was
the case for Pkg or Rbcs (Fig. 8C and 9).
Inclusion of the 19-residue domain (Pka2-Dhfr) altered the import
characteristics of the precursor so that it resembles import of Pka (Fig. 8). Hence, Pka2-Dhfr bound to leucoplasts in the presence
of nigericin but was only imported when the ATP concentration was
elevated to 3 mM with about 50% of the Pka2-Dhfr being
imported (Fig. 8, A and C). Pka2-Dhfr did not
bind to chloroplasts in the presence of nigericin or at any ATP
concentration but was imported into chloroplasts at ATP concentrations
greater than 1 mM (Fig. 8, B and D).
These results confirm that the import of Pka is modified by a
19-residue domain immediately downstream to the transit sequence and
indicate that this domain is independent of other signals in the rest
of the Pka precursor.
The two plastid forms of pyruvate kinase, Pka and Pkg, have
distinct properties. Native Pka is present only in leucoplasts, whereas
Pkg is found in both chloroplasts and leucoplasts. Pka and Pkg are
immunologically distinct from cytosolic pyruvate kinase in developing
castor endosperm (Plaxton, 1989). Unlike most other plastid proteins
including Pkg, both the precursor and the mature form of Pka appear to
be present in leucoplasts at various stages of the developing castor
endosperm. The precursor form of Pka is associated with the surface of
the leucoplast, whereas the mature form is in the stroma. The mature
forms of Pka and Pkg accumulate in the stroma of the leucoplast in a
similar manner during seed development. The Pka precursor accumulates
when the level of mature Pka plateaus. Hence, the accumulation of the
precursor of Pka may result from a decline of the transport system for
Pka during later stages of endosperm development. It is not known if
the putative Pka precursor at the surface of the leucoplast has any
physiological function in vivo.
The in vitro import of Pka and Pkg precursors into leucoplasts and chloroplasts
is also different. Pka and Pkg are distinct plastidic forms since
cytosolic pyruvate kinase does not display any import activity.
The behavior of Pka toward chloroplasts is also atypical
since binding of Pka to chloroplasts could not be detected but the
precursor was imported at 1 mM ATP. Moreover, both precursor
and mature forms of Pka were not immunodetected in chloroplasts, which
is consistent with Northern analysis of castor leaves probed with Pka
from this plant (Blakeley et al., 1995). These results raise
the possibility that the distribution of proteins between different
plastid types may be controlled in part by the uptake process in
addition to the major control mechanism, tissue-specific gene
expression. Another possibility raised by these results is that these
two distinct forms of leucoplast pyruvate kinase (Pka and Pkg) may
proceed through slightly different translocation mechanisms and that
slightly different protein translocation mechanisms may exist in the
two types of plastid. Even though Pka is not immunologically found in
chloroplasts, the results indicate subtle differences between
chloroplasts and leucoplasts rather than no difference. This subtle
difference is also exhibited by Rbcs, indicating the preference for
chloroplasts.
A model for the uptake signaling
mechanism of Pka into leucoplasts and chloroplasts is presented in Fig. 10. In this model, the targeting of Pka to both types of
plastid and import into the stromal compartment is governed by a 3-kDa
transit peptide. A downstream 19-amino-acid domain, termed
``import modifying domain'' affects the function of the
transit peptide and in turn responds to ATP level and plastid type.
Whether the ``interaction'' between this domain and the
transit peptide is direct or indirect is not known. Examination of the
sequence of the 19 amino acids does not reveal any obvious features
that may suggest a mechanism. Preliminary results indicate that the
presence of the 19-amino-acid domain makes precursor proteins more
resistant to thermolysin. This resistance is overcome by high ATP
concentrations suggesting that there may be a conformational change in
response to ATP.
We thank Zdenka W. Ko for her excellent technical
assistance. We also would like to thank Yafan Huang, Chengbiao Wu, and
Lauralynn Kourtz for their helpful discussions.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(
)and plastocyanin can be imported
into both chloroplasts and leucoplasts (Boyle et al., 1986;
Halpin et al., 1989) as can an amyloplast protein precursor,
the waxy polypeptide (Klosgen et al., 1989; Klosgen and Weil,
1991). These data suggest that a basic import mechanism exists and that
different transit peptides can be recognized by all types of plastid.
However, only a very small set of protein precursors have been studied.
A study by Dahlin and Cline(1991) indicates that the import capability
of plastids changes during development. Import activity is high in
proplastids and declines gradually as the organelle matures, suggesting
that the plant cell regulates import activity in concert with the
demands of the developing plastids.
Preparation of Antibodies against Pka and Pkg
Subfractionation and Protein Analysis Procedures
Amino-terminal Deletions of Pka and Pkg
Figure 5:
Construction scheme for truncated Pka and
Pkg precursors. A, the construction details are described
under ``Materials and Methods.'' The given designations and
the number of residues deleted are summarized in the table. The import
competence of each construct is also presented in the table. The
restriction endonuclease sites used to construct the deletions are
depicted as follows: A, AvaII; B, BsaAI; E, EcoRI; H, HpaII; R, RsaI; S, SacI. Pka segments are
depicted as blank blocks (), and Pkg segments are bold-hatched (
). B, the in vitro translation products depicted in A were analyzed by
SDS-PAGE, and the resulting fluorogram is shown. The identity of the
radiolabeled products is indicated. Leucoplast import samples of Pka
and Pkg are also presented in lanes 1 and 12,
respectively, for comparison. Molecular mass markers are indicated in
kilodaltons.
N1 Deletion
A 1910-bp HpaII-SacI DNA
fragment from pTCPka was inserted into the BamHI and SacI sites of pT5Pka. The BamHI site was converted
into a blunt end by Klenow. The amino acid sequence at the fusion point
is M-S-R-Y-P-.
N2 Deletion
pCPka was subcloned into pGEM4 in
reverse such that the cDNA fragment is in the same direction as the T7
promoter. A 1806-bp BamHI-PstI DNA fragment was then
obtained from this resulting plasmid and inserted into BamHI
and PstI sites of pT5Pka. The amino acid sequence at the
fusion point is M-S-S-I-E-.
N3 and N4 Deletions
The 1727- and 1703-bp RsaI-HincII DNA fragments were obtained from pTCPka
and inserted into the HincII site of pT5Pka. The amino acid
sequences at the fusion points are M-S-T-R-R- and M-S-T-I-G- for N3 and
N4, respectively.
N5 Deletion
A BsaAI digestion and
religation were performed with the N3 construct removing a further 39
amino acids from the amino terminus. The amino acid sequence at the
fusion point is M-S-T-R-E.
N6 Deletion
A 240-bp BamHI-SacI
DNA fragment was obtained from the 5` end of pTCPka and inserted into
the BamHI and SacI sites of pT5Pka. This vector was
then used to subclone a 1485-bp XbaI fragment excised from N3.
This construct resulted in the removal of 19 amino acids from position
83 to 102. The amino acid sequence at the fusion point is P-S-R-T-R-.
N7 Deletion
A BsaAI digestion and
religation were performed with the N6 deletion construct to delete a
further 39 amino acids following the first 83 residues. N7 thus
resulted in the removal of 58 amino acids from residue 83 to 141. The
amino acid sequence at the fusion point is S-R-T-R-E-.
pNPkg Construct
A 59-bp SalI-BamHI DNA fragment encoding the 5`-untranslated
sequence and the first two residues of tobacco Pka was retrieved from
pT5Pka and inserted into pGEM4 behind the T7 promoter. This vector was
then used to subclone a 1664-bp SacI-NotI fragment
excised from pTPkg at the SacI and EcoRI sites. The
corresponding NotI and EcoRI sites were converted
into blunt ends by Klenow. As a result, 87 amino acids from the amino
terminus of tobacco Pkg were deleted. In vitro transcription
of pNPkg was initiated from the T7 promoter. The amino acid sequence at
the fusion point is M-S-N-S-P-.
Construction of Chimeric Fusion Proteins
Figure 7:
Construction scheme for the chimeric
fusion precursors Pka1-Pkg, Pkg-Pka, Pka1-Dhfr, and Pka2-Dhfr. A, the construction details are described under
``Materials and Methods.'' The number of amino acid residues
shown in parentheses represents the size of the amino-terminal
segment used to create the particular fusion protein. The same number
of amino-terminal amino acids of Pka was used for the construction of
Pka1-Pkg and Pka1-Dhfr. See text for details. The designation and
restriction sites used for the constructions are given in the figure.
The restriction endonuclease sites used to construct the fusions are
depicted as explained in Fig. 5. Pka segments are depicted as blank
blocks (), Dhfr as bold-hatched (
), and
Pkg segments as bold-hatched (
). B, the in vitro translation products depicted in A were
analyzed by SDS-PAGE, and the resulting fluorogram is shown. The lane
designations correspond to the number given to the molecule in A. Molecular mass markers are indicated in
kilodaltons.
Pka1-Pkg and Pka1-Dhfr Constructs
A 240-bp BamHI-SacI DNA fragment encoding the 5` end of castor
Pka was retrieved from pTCPka and inserted into the BamHI and SacI sites of pT5Pka. This new plasmid, in effect, encoded the
first 83 amino acids of castor Pka and was designated pT5Pka1. pT5Pka1
was then used to construct both Pka1-Pkg and Pka1-Dhfr. Pka1-Pkg was
made by inserting a 1664-bp SacI-NotI DNA fragment
encoding the mature body of tobacco Pkg into the SacI and XbaI sites of pT5Pka1. The NotI and XbaI
sites were converted into blunt ends. Pka1-Dhfr was constructed by
inserting an EcoRI DNA fragment encoding mouse cytosolic Dhfr
into the XbaI site of pT5Pka1. Both the EcoRI and XbaI sites were converted to blunt ends.
Pkg-Pka Construct
A 455-bp SalI-EcoRI DNA fragment encoding the first 87 amino
acids of tobacco Pkg was inserted into pGEM11Z(+). This new vector
was then used to subclone a 1760-bp SacI DNA fragment encoding
the mature body of castor Pka.
Pka2-Dhfr Construct
A 401-bp EcoRI-AvaII DNA fragment was retrieved from pTCPka
and inserted into pGEM4 at the EcoRI and SmaI sites.
The AvaII site was converted into a blunt end. The resulting
vector encoded the first 113 amino acids of castor Pka and was used to
subclone an EcoRI DNA fragment of Dhfr in the XbaI
site. Both the EcoRI and XbaI sites were converted to
blunt ends.
Protein Targeting to Leucoplasts and
Chloroplasts
The growth conditions for castor plants (Ricinus communis L. cv Baker 296) and isolation of intact
leucoplasts were carried out as described by Boyle et
al.(1986). Castor seeds (stages 4-6) were selected according
to the developmental profile described by Greenwood and Bewley(1982).
Protein content of the isolated leucoplasts was determined using the
BCA protein assay procedure (Sigma). The number of leucoplasts in the
suspension was determined using a Coulter counter (Coulter Electronics
Inc.). Approximately 8 10
leucoplasts were used for
each import reaction which is equivalent to about 200 µg of
protein.
10
chloroplasts
were used for each import reaction which is equivalent to 100 µg of
chlorophyll.
S-labeled translation products,
10 mM methionine, 10 mM cysteine, 50 mM HEPES-KOH, pH 8.0, 0.33 M sorbitol, and 1 mM ATP. Radiolabeled precursors were prepared as described by Wu et al.(1994). For binding assays, the ionophore, nigericin
(400 nM final concentration), was used in place of ATP (Cline et al., 1985). The assays were carried out under fluorescent
light or in the dark at room temperature for 30 min. Any modifications
to the typical import reaction conditions are noted, where appropriate,
in the text. Protease treatment, plastid reisolation, and
subfractionation of the organelles were performed according to Smeekens et al.(1986). Chemical cross-linking and immunoprecipitation
were performed as described by Wu and Ko (1993). The chemical
cross-linkers used were DSP (Sigma) and its water-soluble analog, DTSSP
(Pierce). The cross-linkers were reversed using dithiothreitol in the
loading dye. Samples were analyzed by SDS-PAGE and prepared for
fluorography using EN
HANCE (DuPont NEN) and exposed to
Kodak XAR x-ray films. The LKB ultrascan XL laser densitometer was used
to quantitate and analyze the resulting fluorograms.
Localization of Pka and Pkg Proteins in Castor
Leucoplasts and Pea Chloroplasts
The suborganellar location of
Pka and Pkg was determined by subfractionation and immunoblot analysis (Fig. 1A). Anti-Pka IgGs immunoreacted with a major
63-kDa stromal protein and a minor 66-kDa band in the membrane fraction
of leucoplasts (Fig. 1A, lanes 1, 3,
and 4). Immunoreactions were not observed in purified pea
chloroplasts or in castor leaf extracts (Fig. 1A, lanes 7 and 8), suggesting that Pka represents a
leucoplast-specific enzyme. In intact leucoplasts, the 66-kDa
polypeptide band is degraded by thermolysin suggesting that it is
located on the cytosolic side of the leucoplast envelope. In contrast,
the 63-kDa immunoreactive band is thermolysin-resistant indicating an
internal location (Fig. 1A, lane 2). Upon
subfractionation of the leucoplast envelope, the 66-kDa band
co-fractionated with the outer envelope (Fig. 1A, lanes 5 and 6). The identity of the inner and outer
leucoplast envelope was confirmed by IgGs against the 37-kDa
chloroplast inner membrane protein (Cim37) (Dreses-Werringloer et
al., 1991) and the 70-kDa outer envelope polypeptide (Com70) (Wu et al., 1994). In the case of leucoplasts, anti-Cim37 IgGs
immunoreacted with an inner membrane protein, and anti-Com70 antibodies
immunoreacted to a polypeptide in both outer and inner membrane (Fig. 1B, lanes 1-4). It is probable that
the 66-kDa immunoreactive band represents the precursor form of Pka and
that the 63-kDa protein band represents the imported, processed mature
form. This possibility is supported by the observation that the 66-kDa
band co-migrated with the in vitro Pka translation product (Fig. 1A, lanes 3 and 9) and evidence
from the in vitro import studies (Fig. 1A, lanes 10 and 11) described below. The in vitro Pka translation product was immunoprecipitated by the anti-Pka
IgGs but not by preimmune IgGs (Fig. 1A, lanes 12 and 13).
Figure 1:
Localization of Pka
and Pkg forms of plastid pyruvate kinase in leucoplasts and
chloroplasts. A, immunoblots probed with anti-Pka or anti-Pkg
IgGs. The order of lanes 1-8 is as follows: 1,
total castor leucoplast proteins; 2, total proteins from
thermolysin-treated leucoplasts; 3, total leucoplast membrane
fraction; 4, total leucoplast stromal fraction; 5,
outer leucoplast envelopes; 6, inner leucoplast envelopes; 7, total pea chloroplast proteins; and 8, castor leaf
extract. Lanes 9-13 represent a resulting fluorogram
from import reactions and immunoprecipitations of Pka and Pkg as
follows: 9, in vitro translation product; 10, total proteins from a leucoplast import assay; 11, total proteins from a chloroplast import reaction; 12, immunoprecipitated products from in vitro translated protein precursors using preimmune IgGs; and 13, immunoprecipitated products from in vitro translated protein precursors using specific IgGs. B,
immunoblot analysis of outer and inner leucoplast envelopes. Anti-Cim37
IgGs and anti-Com70 IgGs were used in lanes 1 and 2 and in lanes 3 and 4, respectively. Outer
envelope fractions are in lanes 1 and 3, and inner
envelopes are presented in lanes 2 and 4. C,
the protein profile of Pka and Pkg in developing castor endosperm. Lanes 1-7 in the immunoblots represent stages 1 and 2,
3, 4, 5, 6, 7, and 8-10. Samples presented in A and B were separated by 10% denaturing SDS-polyacrylamide gels. A
7.5% SDS-polyacrylamide gel was used in C. Molecular mass
markers are indicated in kilodaltons.
Anti-Pkg IgGs immunoreacted with a 55-kDa
band in purified castor leucoplasts, pea chloroplasts, and castor leaf
extracts (Fig. 1A, lanes 1, 7, and 8). This protein was found only in the stroma of leucoplasts (Fig. 1A, lanes 3-6). Hence, Pkg, unlike
Pka, is present in the stroma of both leucoplasts and chloroplasts. The in vitro Pkg translation product is approximately 61 kDa (Fig. 1A, lane 9), suggesting that a 6-kDa
transit peptide has been removed on uptake. In vitro, the Pkg
precursor is imported into both leucoplasts and chloroplasts and
processed to a 55-kDa form (Fig. 1A, lanes 10 and 11). The in vitro Pkg translation product
was immunoprecipitated by the anti-Pkg IgGs, but not by preimmune IgGs (Fig. 1A, lanes 12 and 13).
Targeting of Pka and Pkg to Leucoplasts and
Chloroplasts
The in vitro import ability of the castor
leucoplast was compared with the pea chloroplast by measuring the
uptake of the pea Rbcs precursor (Fig. 2A). A
post-treatment of the plastids with thermolysin or trypsin was employed
to distinguish externally located or intermembrane proteins from those
in the stroma (Joyard et al., 1983; Cline et al.,
1984). The Rbcs precursor associated with the leucoplast in the
presence of nigericin, an ionophore that dissipates gradients across
plastid membranes necessary for ATP production (Fig. 2A, lane 2). However, the assay did contain 25 µM ATP
from the translation mixture. The associated precursor was
thermolysin-sensitive (Fig. 2A, lane 3)
indicating a location on the outside face of the envelope. In the
presence of exogenously added ATP (1 mM), 30-40% of Rbcs
was imported into leucoplasts and processed to the mature form in the
stroma (Fig. 2A, lane 9). However, under these
conditions, most of the Rbcs was present as full-length precursors and
intermediate forms (Fig. 2A, lane 4) that
co-fractionated with the membranes (Fig. 2A, lane
10). The precursor form of Rbcs appeared to be resistant to
thermolysin (Fig. 2A, lane 5). Some of the
intermediate forms were cleaved by thermolysin resulting in distinct
smaller sized products (Fig. 2A, lane 5) that
co-fractionated with the membranes (Fig. 2A, lanes 7 and 8). However, the precursor and the intermediate forms
were trypsin-sensitive (Fig. 2A, lane 6)
whereas the mature form was resistant. Hence, although Rbcs is imported
by leucoplasts, the uptake appears to be different from that found in
chloroplasts suggesting some differences between the two types of
plastid. Nevertheless, isolated leucoplasts are capable of importing
proteins in a manner similar to that established for pea chloroplasts.
Figure 2:
Import of Rbcs, Pkg, and Pka precursors
into leucoplasts (A) and chloroplasts (B).
Information concerning the import reaction parameters are indicated and
is the same for each panel. The type of plastid tested is indicated by LEU (for leucoplasts) and CHL (for chloroplasts).
Total plastid proteins are shown in lanes marked LEU or CHL. The corresponding stromal and membrane fractions of the
same plastid type are indicated as STR and MEM,
respectively. The in vitro translation products are shown in lane 1 and indicated by TRA. The presence or absence
of nigericin (400 nM), post-uptake treatment with thermolysin
or trypsin are indicated by +/-. The concentration of
exogenous ATP (1 or 3 mM) added in each reaction in addition
to the 25 µM ATP contributed by the translation mixture is
indicated. Each lane contains either 2.5 µg of protein for the
leucoplast assays or 2 µg of chlorophyll for the chloroplast
samples. All the samples were analyzed by 10% denaturing SDS-PAGE. The
precursor and mature proteins are indicated in the figure as P and M, respectively.
The import of Pkg into leucoplasts is typical of stromal protein
precursors. Pkg precursors associated with leucoplasts in the presence
of nigericin and were sensitive to thermolysin (Fig. 2A, lanes 2 and 3). In the presence of 1 mM ATP,
approximately 60% of the Pkg precursors were imported into the stroma
and processed (Fig. 2A, lanes 4 and 9). The mature form was resistant to both thermolysin and
trypsin (Fig. 2A, lanes 5-7). The mature
form of Pkg is about 55 kDa, indicating that a 6-kDa transit peptide is
cleaved upon uptake. The imported Pkg co-migrated with the 55-kDa
immunoreactive stromal band (Fig. 1A, lanes 4 and 10).
Effect of ATP Concentration on the Import of Rbcs, Pka,
and Pkg
The effect of ATP concentration (0 to 3 mM) on
the import of Pka, Pkg, and Rbcs into leucoplasts and chloroplasts was
investigated (Fig. 3). The import reactions were carried out
under a dim green light to prevent light-dependent ATP production in
the chloroplast. The overall import profiles of Rbcs and Pkg were very
similar in both chloroplasts and leucoplasts. The amount of precursors
bound to chloroplasts increased between 0 to 0.1 mM ATP (Fig. 3B, lanes 1 and 2) and from 0 to
0.5 mM ATP for leucoplasts (Fig. 3A, lanes
1-4). Further increases in the ATP concentration resulted in
a decrease in bound precursor with a concomitant increase in the level
of import. The imported mature form of Pkg and Rbcs began to appear at
0.25 mM for chloroplasts (Fig. 3B, lane
3) and 1 mM for leucoplasts (Fig. 3A, lane 5). The level of imported products reached a steady state
beyond 1 mM ATP for chloroplasts (Fig. 3B, lanes 5-7) and 2 mM ATP for leucoplasts (Fig. 3A, lanes 6 and 7). Although the
import profiles for Rbcs and Pkg are similar, there are a number of
differences. Import of Rbcs appeared more preferential for chloroplasts
than for leucoplasts based on the ratio of precursor to mature form
observed in the assays. In addition, intermediates or aberrant
processing of Rbcs were clearly evident in leucoplasts but not in
chloroplasts. Intermediate or aberrantly processed forms of Pkg were
not detected in either plastid type.
Figure 3:
Import of Rbcs, Pkg, and Pka precursors
into leucoplasts (A) and chloroplasts (B) at
different ATP concentrations. The import reactions were conducted under
a dim green light. Lanes 1-7 represent 0-3 mM ATP used in the reactions.
The binding and import of Pka
displayed atypical characteristics in response to different ATP
concentrations. Binding of the Pka precursor to the chloroplast could
not be detected at any ATP concentration (Fig. 3B, lanes 1-7), and import occurred between 1 and 3 mM ATP (Fig. 3B, lanes 5-7). Hence, the
import of Pka into chloroplasts occurs without the typical binding step
and only when the ATP concentration is 1 mM. In contrast
to chloroplasts, Pka precursors bound to leucoplasts at all ATP
concentrations (Fig. 3A, lanes 1-7) but
were imported only at 2 to 3 mM ATP (Fig. 3A, lanes 6 and 7) suggesting that Pka requires higher
levels of ATP than other plastid proteins, such as Rbcs and Pkg, for
import into this organelle.
Chemical Cross-linking and Co-immunoprecipitation
Studies
To determine the location and the nature of the
association of Pka with the leucoplast envelope, the effects of
chemical cross-linkers and co-immunoprecipitation with anti-Com70 IgGs
were measured (Fig. 4). Com70 has recently been shown to be in
close proximity to a partially translocated precursor and may play a
role in an early step of the chloroplast protein import pathway (Wu et al., 1994).(
)Precursors halted at an
early stage of import can be chemically cross-linked to Com70, whereas
precursors imported cannot be cross-linked efficiently. The import
assays contained either 1 or 3 mM ATP, conditions which give
rise to externally and internally located Pka, respectively. The
resulting cross-linked complexes were immunoprecipitated with
anti-Com70 IgGs. Anti-Cim37 IgGs were used as a control. Immunoblots
demonstrate that the proteins corresponding to Com70 and Cim37
(designated as Lom70 and Lim37 in the leucoplast) are present in the
same membrane subfractions of castor leucoplasts (Fig. 1B) as observed in pea chloroplasts (Ko et
al., 1992) and probably serve similar roles. When the import
reactions contained 1 mM ATP, cross-linking occurred between
Pka and Lom70 with the two types of chemical cross-linkers (DSP and
DTSSP) (Fig. 4A, lanes 4 and 5).
Neither DSP nor DTSSP cross-linked the Pka precursor form to Lom70 when
3 mM ATP was used, indicating that Pka had been imported (Fig. 4A, lanes 8 and 9). These
results suggest that at 1 mM ATP, Pka is in close physical
proximity to Lom70 and can be cross-linked to it with the
membrane-impermeable cross-linker DTSSP, indicating that the Pka
precursor is external to the envelope under these conditions. At 3
mM ATP, the import of Pka is enhanced so that the precursor
proceeds rapidly through the early stages, reducing the amount
available for cross-linking to Lom70. These cross-linked products were
not precipitated with preimmune IgGs or anti-Cim37 IgGs (Fig. 4A, lanes 2 and 3 and 6 and 7).
Figure 4:
Chemical cross-linking and
co-immunoprecipitation of Pka, Pkg, and Rbcs to envelope proteins. The
resulting fluorograms are presented for Pka (A), Pkg (B), and Rbcs (C). The castor leucoplast import
system was used for this study (indicated as LEU in each of
the lanes). The in vitro translation products are shown in lane 1 of each panel and marked TRA. The
concentration of ATP added to the import reaction in addition to the
ATP contribution by the translation mixture is indicated. The type of
chemical cross-linker employed is indicated by DSP or DSSP (which
stands for DTSSP). The antiserum used in the particular experiment is
indicated by PRE- for preimmune IgGs, by 70KDa for
anti-Com70 IgGs, and by 37KDa for anti-Cim37
IgGs.
The effect of ATP concentration on the
cross-linking of Pkg with Lom70 was also determined. Pkg is imported
into leucoplasts at 1 mM ATP and appears to proceed rapidly
through the import pathway. Hence, Pkg was not cross-linked to Lom70 at
1 mM ATP, similar to Pka at 3 mM ATP (Fig. 4B). This provides further evidence that uptake of
Pka is different from that of Pkg.
Import of Amino-terminal Truncated Pka and Pkg
Proteins
Pka has an amino-terminal extension of 130 amino acids
when compared with the pyruvate kinase from Escherichia coli,
and it is assumed that this extension is involved in protein import. To
determine the signal(s) influencing Pka's import characteristics,
seven different amino-terminal deletions designated N1 to N7 were
constructed (Fig. 5). The size of N1 is approximately the same as
the mature Pka (Fig. 5B, lanes 1 and 3), suggesting that the processing site of the Pka precursor
is in the vicinity of the 30th amino acid. The predicted molecular mass
of the first 30 amino acids of the Pka precursor is about 3 kDa, which
is consistent with this cleavage site.
Figure 6:
Import of amino-terminal truncated Pka and
Pkg precursor proteins. The experiments were conducted and presented in
the same manner as described in Figs. 2 and 3. The resulting
fluorograms are presented for N2 and NPkg (A) and N6 and N7 (B-E). Lanes 7-10 of N7 are not shown in B and C because N6 and N7 behave in the same
way.
The difference between N6/N7 and Pka in ATP
requirement for import was evident when the assays were conducted at
increasing ATP concentrations (N6 is used as an example in Fig. 6, D and E). The amount of N6 bound to
chloroplasts increased with increasing ATP levels (up to 0.25
mM) (Fig. 6E, lanes 1-3). Above
0.5 mM ATP, binding decreased with a concomitant increase of
imported N6 (Fig. 6E, lane 4). Import reached a
maximal level at 2 mM ATP (Fig. 6E, lanes 6 and 7). In leucoplasts, the N6 precursor bound to the
organelle at all levels of ATP tested (Fig. 6D, lanes 1-7). Import occurred at 1 mM ATP (Fig. 6D, lane 5) and became maximal at 2
mM ATP (Fig. 6D, lanes 6 and 7). Hence, N6, which has 19 amino acids removed from the
carboxyl end of the amino-terminal extension, behaves similarly to Pkg,
suggesting that these 19 residues
(-P-S-S-I-E-V-D-A-V-T-E-T-E-L-K-E-N-G-F-) play a role in altering the
import characteristics of Pka. The removal of 39 additional amino acids
immediately downstream of the 19-residue region(N7) gave the same
results.
Import of Pka1-Pkg and Pkg-Pka Fusion Proteins
The
19-amino-acid region between the transit peptide and the pyruvate
kinase sequence appear to affect the function of the Pka transit signal
in response to ATP concentration and plastid type. To determine whether
this region specifically affects Pka or if it can influence the
behavior of other protein precursors, two chimeric fusion proteins
designated Pka1-Pkg and Pkg-Pka were made by fusing the mature Pkg
protein (NPkg) to the amino-terminal 83 amino acids of Pka (designated
Pka1-Pkg) and by joining the amino-terminal 87 residues of Pkg to the
mature Pka protein(N2) (designated Pkg-Pka) (molecules 1 and 2 in Fig. 7A; Fig. 7B, lanes 1 and 2).
Figure 8:
Import of fusion proteins Pkg-Pka and
Pka2-Dhfr into leucoplasts and chloroplasts. The import experiments
were conducted and presented in the same manner as described in Figs. 2
and 3. The resulting fluorograms are presented for Pkg-Pka (A-D) and Pka2-Dhfr (A-D).
Import of
Pkg-Pka into chloroplasts gave results similar to Pka alone. Pkg-Pka
did not bind to chloroplasts in the presence of nigericin or at any ATP
concentration (Fig. 8, B, lanes 2-4, and D). It was imported into the stroma and processed at 1 mM ATP (Fig. 8B), and unprocessed Pkg-Pka was not
detected in any chloroplast subfraction. Import at increasing ATP
concentrations was also similar to Pka (Fig. 8D). These
results indicate that the Pkg transit peptide is capable of directing
the import of Pka, but its function is modified by the presence of the
19-amino-acid domain.
Import of Pka-Dihydrofolate Reductase Fusion
Proteins
Two foreign chimeric fusion proteins, Pka1-Dhfr and
Pka2-Dhfr, were constructed to test whether the amino-terminal 83 amino
acids containing the transit peptide of Pka and the downstream
19-residue domain exerted the same influence on the import
characteristics of a foreign protein as it did in Pka or Pkg-Pka. These
two constructs were made by fusing mouse dihydrofolate reductase (Dhfr)
to either the amino-terminal 83 or 114 amino acids of Pka (molecules 3 and 4 in Fig. 7A and Fig. 7B, lanes 3 and 4). Pka1-Dhfr
behaved like a typical plastid protein precursor (data not shown),
further demonstrating that the first 83-amino-acid segment of Pka
contains a transit peptide capable of directing the import of foreign
proteins into the stromal compartment of both types of plastid.
The uptake of Pkg is similar to other proteins, such as Rbcs, in
that it binds with nigericin or ATP and imports into the stroma of both
types of plastid and is processed to the mature form at 1 mM ATP. Pka is imported differently from either Pkg or Rbcs in
response to ATP concentration and plastid type. Pka binds to
leucoplasts in the presence of nigericin but import occurs only at ATP
concentrations greater than 2 mM. The external location of Pka
on the leucoplast envelope at lower ATP levels was confirmed by its
sensitivity to thermolysin and chemical
cross-linking/co-immunoprecipitation experiments. The in vitro import results also correlate with the developmental profile of
Pka. The observed alterations in Pka's import competence may be a
response to cellular ATP levels. The presence of the externally located
Pka precursors may result from lower ATP levels in the vicinity of the
leucoplast at later stages of endosperm development. Thus, a potential in vivo consequence of this in vitro effect of ATP
levels on Pka uptake may be a mechanism for redirecting Pka from
external to internal sites and vice versa. The redistribution
of Pka may be related to the developmental changes in the castor
endosperm.
The Transit Peptide of Pka Is Capable of Directing Proteins
into Two Different Types of Plastid
Pka precursors from which 30
to 141 amino acids had been removed from the amino terminus did not
bind to either leucoplasts or chloroplasts and were not imported.
Hence, the targeting of Pka is dependent on a transit signal located at
the amino terminus. Excision of segments of Pka immediately after the
amino-terminal 83 amino acids (N6 and N7 constructs) did not abolish
the import of Pka, further suggesting that the first 83 amino acids
contain a transit peptide. The amino-terminal 83-amino-acid segment
could also target mouse Dhfr to both types of plastid whereas Dhfr
itself is not imported (Wu and Ko, 1993; data not shown). This region
could also facilitate the import of the mature body of Pkg into both
types of plastid. Hence, the amino-terminal 83-amino-acid domain of Pka
contains a transit peptide capable of directing proteins across the two
envelope membranes to the stromal compartment in both leucoplasts and
chloroplasts. The subsequent 19-amino-acid segment, immediately
following the carboxyl end of the amino-terminal 83-amino-acid region
of the Pka precursor, appears to modify the transport properties of the
transit peptide.
Modification of Pka Import by a Domain Downstream of the
Transit Peptide
The atypical nature of Pka transport appears to
result from a 19-amino-acid domain downstream from the transit peptide.
The presence of this domain increases the concentration of ATP required
for import and prevents binding of the precursor to chloroplasts. The
import profile of Pka becomes typical of most plastid proteins upon
removal of this domain, suggesting that it affects the function of the
transit peptide. The influence of the 19-amino-acid domain can be
overcome by higher levels of ATP. In vivo, this may be a
mechanism for regulating the import of Pka in response to cellular ATP
status associated with the biosynthetic activity of the tissue.
Insertion of the 19-amino-acid domain into other precursor proteins,
including ones from non-plant sources, makes their transport properties
resemble that of Pka, suggesting that this region is not dependent on
other areas of the Pka protein for its action. Hence, the introduction
of the 19-amino-acid segment into fusion constructs such as Pkg-Pka or
Pka2-Dhfr converts the import behavior of the fusion constructs from
the typical pattern to that of Pka. Similar energy requirements and the
same response to plastid type as seen with Pka can therefore be
transposed into other proteins.
(
)Removal of this domain
abolishes any resistance to thermolysin at all tested levels of ATP
(0-3 mM). However, the change in import characteristics
cannot be attributed simply to ATP levels alone since ATP
resupplementation of leucoplasts derived from various developmental
stages does not alter the immunologically detectable pattern of
Pka.
Furthermore, the possibility that ATP simply drives
the generic unfolding of Pka necessary for import is also not the
complete answer since urea-induced unfolding of Pka does not relieve
the higher ATP requirement for import.
Moreover, the import
of Pka into leucoplasts and chloroplasts is different, suggesting that
factors in addition to ATP are involved in the uptake of Pka.
Figure 10:
Model for the import signaling
information of Pka. The Pka precursor is represented in various
domains. The number of amino acid residues in the amino-terminal
domains is indicated. Features essential to the import characteristics
of the Pka precursor are labeled.
The
results presented in this paper indicate that leucoplasts and
chloroplasts respond to targeting signals differently, suggesting
differences in the import machinery of these two organelles, and these
may contribute to the regulation of protein import. These differences
and the mechanism by which the 19-amino-acid domain affects protein
uptake into both plastids is being investigated further.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.