©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Import Characteristics of a Leucoplast Pyruvate Kinase Are Influenced by a 19-Amino-acid Domain within the Protein (*)

Jiangxin Wan , Stephen D. Blakeley , David T. Dennis , Kenton Ko (§)

From the (1)Department of Biology, Queen's University, Kingston, Ontario, Canada K7L 3N6

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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.''


INTRODUCTION

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()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.

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.


MATERIALS AND METHODS

Preparation of Antibodies against Pka and Pkg

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 and Protein Analysis Procedures

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).

Amino-terminal Deletions of Pka and Pkg

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.


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

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.


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.

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.

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.

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 10 chloroplasts were used for each import reaction which is equivalent to 100 µg of chlorophyll.

Import assays were assembled in 0.3-ml volumes as described in Bartlett et al.(1982). A typical import reaction contained intact plastids, 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 ENHANCE (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.


RESULTS

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).

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).

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).

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.

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.

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.

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.

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.


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.

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.

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).

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).


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.

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.


DISCUSSION

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 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 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.

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.

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.()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.


FOOTNOTES

*
This work was supported by grants from the Natural Sciences and Engineering Research Council of Canada. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence and reprint requests should be addressed. Tel.: 613-545-6155; Fax: 613-545-6617.

The abbreviations used are: Rbcs, the small subunit of ribulose-1,5-bisphosphate carboxylase; Pka, pyruvate kinase A form; Pkg, pyruvate kinase G form; Dhfr, mouse cytosolic dihydrofolate reductase; DSP, dithiobis(succinimidylpropionate); DTSSP, 3, 3`-dithiobis(sulfonylsuccinimidylpropionate); IgG, immunoglobulin G; bp, base pair(s); PAGE, polyacrylamide gel electrophoresis.

K. Ko, unpublished data.

J. Wan, S. D. Blakeley, D. T. Dennis, and K. Ko, unpublished data.


ACKNOWLEDGEMENTS

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.


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