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• W2056574328 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.” 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 16Keegstra K. Cell. 1989; 56: 247-253Abstract Full Text PDF PubMed Scopus (112) Google Scholar and 18Keegstra K. Olsen L.J. Theg S.M. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1989; 40: 471-501Crossref Google Scholar). 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 (30Salomon M. Fischer K. Flugge U.-I. Soll J. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 5778-5782Crossref PubMed Scopus (95) Google Scholar; 27Li H.-M. Moore T. Keegstra K. Plant Cell. 1991; 3: 709-717Crossref PubMed Scopus (106) Google Scholar; 36Wu C. Ko K. J. Biol. Chem. 1993; 268: 19384-19391Abstract Full Text PDF PubMed Google Scholar; 10Fischer K. Weber A. Arbinger B. Brink S. Eckerskorn C. Flugge U.-I. Plant Mol. Biol. 1994; 25: 167-177Crossref PubMed Scopus (35) Google Scholar). 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 (5Cline K. Plant Physiol. 1988; 86: 1120-1126Crossref PubMed Google Scholar; 26Lamppa G.K. J. Biol. Chem. 1988; 263: 14996-14999Abstract Full Text PDF PubMed Google Scholar; 34Van den Broeck G. Van Houtven A. Van Montagu M. Herrera-Estrella L. Plant Sci. 1988; 58: 171-176Crossref Scopus (9) Google Scholar; 35Viitanen V. Doran E.R. Dunsmuir P. J. Biol. Chem. 1988; 263: 15000-15007Abstract Full Text PDF PubMed Google Scholar; 13Hand J.M. Szabo L.J. Vasconcelos A.C. Cashmore A.R. EMBO J. 1989; 8: 3195-3206Crossref PubMed Scopus (40) Google Scholar; 24Kohorn B.D. Tobin E.M. Cell. 1989; 1: 159-166Google Scholar; 28Li H.-M. Sullivan T.D. Keegstra K. J. Biol. Chem. 1992; 267: 18999-19004Abstract Full Text PDF PubMed Google Scholar). 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 (22Ko K. Ko Z.W. J. Biol. Chem. 1992; 267: 13910-13916Abstract Full Text PDF PubMed Google Scholar). 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 Rbcs1 1The abbreviations used are: Rbcsthe small subunit of ribulose-1,5-bisphosphate carboxylasePkapyruvate kinase A formPkgpyruvate kinase G formDhfrmouse cytosolic dihydrofolate reductaseDSPdithiobis(succinimidylpropionate)DTSSP3, 3′-dithiobis(sulfonylsuccinimidylpropionate)IgGimmunoglobulin Gbpbase pair(s)PAGEpolyacrylamide gel electrophoresis. and plastocyanin can be imported into both chloroplasts and leucoplasts (4Boyle S.A. Hemmingsen S.M. Dennis D.T. Plant Physiol. 1986; 81: 817-822Crossref PubMed Google Scholar; 12Halpin C. Musgrove J.E. Lord J.M. Robinson C. FEBS Lett. 1989; 258: 32-34Crossref Scopus (17) Google Scholar) as can an amyloplast protein precursor, the waxy polypeptide (20Klosgen R.B. Saedler H. Weil J.-H. Mol. & Gen. Genet. 1989; 217: 155-161Crossref PubMed Scopus (38) Google Scholar; 19Klosgen R.B. Weil J.-H. Mol. & Gen. Genet. 1991; 225: 297-304Crossref PubMed Scopus (25) Google Scholar). 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 8Dahlin C. Cline K. Plant Cell. 1991; 3: 1131-1140Crossref PubMed Google Scholar 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. the small subunit of ribulose-1,5-bisphosphate carboxylase pyruvate kinase A form pyruvate kinase G form mouse cytosolic dihydrofolate reductase dithiobis(succinimidylpropionate) 3, 3′-dithiobis(sulfonylsuccinimidylpropionate) immunoglobulin G base pair(s) polyacrylamide gel electrophoresis. 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) (2Blakeley S.D. Plaxton W.C. Dennis D.T. Plant Physiol. 1991; 96: 1283-1288Crossref PubMed Scopus (26) Google Scholar, 3Blakeley S.D. Gottlob-McHugh S. Wan J. Crews L. Miki B. Ko K. Dennis D.T. Plant Mol. Biol. 1995; 27: 79-89Crossref PubMed Scopus (14) Google Scholar) 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 32Smeekens S. Bauerle C. Hageman J. Keegstra K. Weisbeek P. Cell. 1986; 46: 365-375Abstract Full Text PDF PubMed Scopus (203) Google Scholar. Plastid envelopes were subfractionated using discontinuous sucrose gradients (17Keegstra K. Yousif A. Methods Enzymol. 1986; 118: 316-325Crossref Scopus (142) Google Scholar). Castor leucoplasts and pea chloroplasts, membrane fractions, and stromal extracts were subjected to denaturing SDS-polyacrylamide gel electrophoresis (25Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207538) Google Scholar) and electrophoretically transferred onto nitrocellulose membranes (33Towbin H. Staehelin Y. Gordon J. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 4350-4354Crossref PubMed Scopus (44939) Google Scholar). The protein blots were processed according to 14Hoffman N.E. Pichersky E. Malik V.S. Castresana C. Ko K. Darr S.C. Cashmore A.R. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 8844-8848Crossref PubMed Scopus (87) Google Scholar. 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. 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-. 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-. 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. 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. 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-. 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-. 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-. 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. 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. 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. 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. 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 11Greenwood J.S. Bewley J.D. Can. J. Bot. 1982; 60: 1751-1760Crossref Google Scholar. 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 × 107 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 21Ko K. Cashmore A.R. EMBO J. 1989; 8: 3187-3194Crossref PubMed Scopus (68) Google Scholar. Intact pea chloroplasts were isolated from 14-day-old seedlings as described by 1Bartlett S.G. Grossman A.R. Chua N.-H. Edelman M. Hallick R.B. Chua N.-H. Methods in Chloroplast Molecular Biology. Elsevier Biomedical Press, Amsterdam1982: 1081-1091Google Scholar. Approximately 8 × 107 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, 35S-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 37Wu C. Seibert F. Ko K. J. Biol. Chem. 1994; 269: 32264-32271Abstract Full Text PDF PubMed Google Scholar. For binding assays, the ionophore, nigericin (400 nM final concentration), was used in place of ATP (7Cline K. Werner-Washburne M. Lubben T. Keegstra K. J. Biol. Chem. 1985; 260: 3691-3696Abstract Full Text PDF PubMed Google Scholar). 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 EN3HANCE (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. 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) (9Dreses-Werringloer U. Fischer K. Wachter E. Link T.A. Flugge U.-I. Eur. J. Biochem. 1991; 195: 361-368Crossref PubMed Scopus (54) Google Scholar) 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). 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 (31Simcox P.D. Garland W. Deluca V. Canvin D.T. Dennis D.T. Can. J. Bot. 1979; 57: 1008-1014Crossref Google Scholar). The developmental pattern of mature Pkg was similar to that of Pka (Fig. 1C). 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 (15Joyard J. Billecocq A. Bartlett S.G. Block M.A. Chua N.-H. Douce R. J. Biol. Chem. 1983; 258: 10000-10006Abstract Full Text PDF PubMed Google Scholar; 6Cline K. Werner-Washburne M. Andrews J.T. Keegstra K. Plant Physiol. 1984; 75: 675-678Crossref PubMed Google Scholar). 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. 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. 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 for" @default.
• W2056574328 created "2016-06-24" @default.
• W2056574328 creator A5005688243 @default.
• W2056574328 creator A5016262550 @default.
• W2056574328 creator A5044302672 @default.
• W2056574328 creator A5055192012 @default.
• W2056574328 date "1995-07-01" @default.
• W2056574328 modified "2023-10-18" @default.
• W2056574328 title "Import Characteristics of a Leucoplast Pyruvate Kinase Are Influenced by a 19-Amino-acid Domain within the Protein" @default.
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