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Plant Physiol. (1998) 118: 691-699 Domains of a Transit Sequence Required for in Vivo Import in Arabidopsis Chloroplasts1
Department of Molecular Cell Biology, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands
Nuclear-encoded precursors of chloroplast proteins are synthesized with an amino-terminal cleavable transit sequence, which contains the information for chloroplastic targeting. To determine which regions of the transit sequence are most important for its function, the chloroplast uptake and processing of a full-length ferredoxin precursor and four mutants with deletions in adjacent regions of the transit sequence were analyzed. Arabidopsis was used as an experimental system for both in vitro and in vivo import. The full-length wild-type precursor translocated efficiently into isolated Arabidopsis chloroplasts, and upon expression in transgenic Arabidopsis plants only mature-sized protein was detected, which was localized inside the chloroplast. None of the deletion mutants was imported in vitro. By analyzing transgenic plants, more subtle effects on import were observed. The most N-terminal deletion resulted in a fully defective transit sequence. Two deletions in the middle region of the transit sequence allowed translocation into the chloroplast, although with reduced efficiencies. One deletion in this region strongly reduced mature protein accumulation in older plants. The most C-terminal deletion was translocated but resulted in defective processing. These results allow the dissection of the transit sequence into separate functional regions and give an in vivo basis for a domain-like structure of the ferredoxin transit sequence.
Among eukaryotes perhaps the most complex subcellular organization
is found in higher plants. In addition to routing cytosolically synthesized proteins to peroxisomes, mitochondria, and the secretory machinery, plants also route proteins to their plastids. The targeting of proteins in the plant cell is therefore of particular interest. Because most chloroplast proteins are nuclear encoded and synthesized in the cytoplasm, chloroplast biogenesis depends upon a protein-import apparatus that recognizes these proteins and translocates them across
the chloroplast's double-membrane envelope (for reviews, see de Boer
and Weisbeek, 1991 In unraveling the molecular mechanism of translocation it will be
essential to understand the interactions between the import machinery
and the transit sequence of the precursor. Such an understanding requires that the content and architecture of the topogenic information in the precursor are known. When analyzing this information only in
vitro, possible shortcomings of the experimental system are difficult
to discriminate from differences in topogenic information. In the
intact plant cell mistargeting needs to be prevented or minimized,
which may require the presence of additional information. In addition,
developmental aspects of targeting and routing in different tissues
need to be understood. Thus, the in vitro analysis of topogenic
information may result in a simplified and incomplete picture. This
prompted us to analyze the information content of a transit sequence
both in vitro and in vivo, in a homologous system.
We chose as a marker for transport, pre-Fd from Silene
pratensis, which has been extensively characterized. In vitro this precursor is imported without the need for cytosolic factors, indicating that the precursor directly recognizes the chloroplast envelope (Pilon et al., 1992a The use of transgenic plants in this study allowed only a limited set
of deletion mutants to be analyzed. As the amino-terminal sequence of
the protein might be very important for the efficiency of translation,
deletions in the first five amino acids of the transit sequence were
not used. Deletions that affect the sequence of the mature part of the
protein were also excluded. The four deletion mutants that were chosen
span the transit sequence from residues 6 to 42 and are comparable in
length. The analysis of import with pea chloroplasts had indicated that
each deleted region contains a specific function of the transit
sequence. Deletion mutant Arabidopsis was used as a model plant. The endogenous Arabidopsis Fd
has a higher electrophoretic mobility than the S. pratensis protein and therefore can be distinguished on western blots. Also, for
this plant species efficient transformation protocols are available
(Valvekens et al., 1988 The in vivo import results of the deletion mutants are consistent with
the presence of separate, functional domains in the transit sequence of
Fd, whereas in vitro the deletion mutants could not be imported in
Arabidopsis chloroplasts.
Reagents
Plant Growth Conditions Arabidopsis was grown in soil in growth chambers with 16 h of light and 8 h of darkness at 22°C and 70% RH.Chloroplast Isolation from Arabidopsis To obtain a preparation with maximal import efficiency the procedure of Somerville et al. (1981) was modified. Chloroplasts were
isolated from 3- to 4-week-old Arabidopsis ecotype Colombia-0. Typically, 40 g fresh weight of plant material from rosette leaves was used, divided in four portions, and ground in a polytron blender in
position 3 for 5 s in 100 mL of ice-cold grinding buffer (330 mM sorbitol, 20 mM tricine/KOH, pH 8.4, 5 mM EGTA, 5 mM EDTA, 10 mM
NaCO3, 0, 1% BSA, and 330 mg/L isoascorbate).
The suspension was filtered through two layers of Miracloth
(Calbiochem) between two layers of cheesecloth, resulting in the crude
homogenate. From this point all procedures were done at 0°C to 4°C.
The suspension was centrifuged for 5 min at 3000 rpm in an HB-4 rotor
(Sorvall) to pellet the crude chloroplast fraction.
In Vitro Import Experiments Isolated chloroplasts were centrifuged for 2 min at 2,000 rpm at 4°C in a swing-out centrifuge, and the pellet was resuspended in ice-cold import buffer (330 mM sorbitol, 5 mM Hepes/KOH, pH 8.0, 10 mM NaHCO3, 8 mM MgCl2, and 0.1% BSA). To each sample containing 20 µg of chlorophyll, 1.5 mM DTT (final concentration), 1.2 mg of antipain, and 5 mM ATP (final concentration) were added, making the total volume 100 µL. The samples were kept on ice in the dark for 20 min. Fifty microliters of translation mixture in import buffer containing approximately 200,000 TCA-precipitable cpm was added. The samples were then incubated at 25°C in the light to allow import. After 30 min the samples were placed on ice. From this point all procedures were performed at 4°C under dim-green light. The samples were split into two equal aliquots. One aliquot was treated with 7.5 µL of thermolysin (4 mg/mL thermolysin in import buffer and 10 mM CaCl2) at 4°C for 30 min, after which time EDTA in import buffer was added to a final concentration of 5 mM. To the other aliquot EDTA was added directly. The samples were centrifuged for 2 min at 2,000 rpm in a swing-out centrifuge. The pellet was washed once with grinding buffer. Finally, the chloroplasts were lysed in sample buffer (125 mM Tris/HCl, pH 6.8, 5% SDS [v/w], 10% glycerol [v/v], and 5% -mercaptoethanol [v/v]), followed by heating to 95°C for 5 min.
Analysis by SDS-PAGE and fluorography and quantification were performed
essentially as described (Pilon et al., 1990; Knight and Gray, 1995).
Plasmid Constructions Standard procedures were used for restriction enzyme digestions, analysis of restriction sites, ligations, and transformation of Escherichia coli. The NcoI-BamHI fragments from plasmids pETFD-wt, -323, -342, -361, and -372 containing pre-Fd were cloned in pMOG18 (Sijmons et al., 1990) under the control
of the constitutively active cauliflower mosaic virus 35S promoter and
enhancer, in front of a nopaline synthase termination signal to allow
expression in plants, resulting in plasmids pWA1, -2, -3, -4, and -5. The EcoRI-HindIII fragments were subcloned in the
binary vector pMOG23 (Sijmons et al., 1990), resulting in plasmids pC1,
-2, -3, -4, and -5 to enable plant transformation. The sequence of the
inserts in pC1-5 was confirmed by dideoxy sequencing.
Plant Transformation Constructs pC1-5 were transformed to Agrobacterium tumefaciens strain LBA-4404 containing helper plasmid pTiAch5 (Hoekema et al., 1983), using a freeze-thaw method as
described (An et al., 1988). The intactness of the transformed binary
vectors in A. tumefaciens was determined by plasmid
isolation and restriction mapping as described (An et al., 1988).
Arabidopsis seeds ecotype C24 were used in a root-explant
transformation procedure according to the method of Valvekens (1988).
Plants were regenerated from kanamycin-resistant callus. Calli
transformed with the different constructs regenerated with comparable
efficiencies, although the number of transgenic lines varied per
construct; for each construct 14 to 39 independent transgenic lines
were regenerated and self-crossed to generate the seeds for the
F1 line. The F1 plants were
tested for the presence of S. pratensis pre-Fd by immunoblotting and were self-crossed. These plants were used in Figure
3. F2 plants were germinated on Murashige and
Skoog medium containing 125 mg/L kanamycin to select homozygous lines.
Homozygous F2 lines were used in Figures 4 and 5.
Integration of the T-DNA was determined by Southern blotting.
Preparation of Plant Extracts Plant leaf material, 0.1 to 0.5 g fresh weight, was ground on ice in a microcentrifuge tube in extraction buffer (100 mM NaCl, 50 mM Tris/HCl, pH 7.5, 0, 5% [v/v] Triton X-100, 10 mM-mercaptoethanol, and 1 mM PMSF). The
extract was centrifuged for 5 min in an Eppendorf centrifuge to pellet
the insoluble fraction. The soluble fraction was mixed with sample
buffer, and 10 µg of protein was applied to SDS-PAGE and
immunoblotting was performed essentially as described (Pilon et al.,
1990), except that blots were developed with enhanced chemiluminescence
reagent. Blots were quantified with a personal densiometer (model SI,
Molecular Dynamics, Sunnyvale, CA). All values were averaged over two
experiments and corrected for the amount of endogenous Arabidopsis Fd
that was detected and correlated to a range of known amounts of
purified pre-Fd present on the same blot.
Northern and Southern Blotting Total RNA was isolated and applied to northern blotting for the quantitative data in Figure 5, as described (Quaedvlieg et al., 1995).
DNA was precipitated out of the remaining solution, digested with
HindIII, and applied to Southern blotting. Blots were probed
with a random-primed BamHI-BstEII fragment from
pETFD-wt corresponding to the mature sequence of S. pratensis pre-Fd or an 18S rRNA (Pruitt and Meyerowitz, 1986)
probe, which corrected for loading differences. Bands were quantified
with a phosphor imager (model SI, Molecular Dynamics).
General Methods Published methods were used for SDS-PAGE (Laemmli, 1970), protein
assays (Bradford, 1976), and in vitro transcription and translation
(Pilon et al., 1995).
Fd Precursors In this study the information content of the Fd transit sequence was analyzed both in vivo and in vitro. The transit sequence of the S. pratensis Fd wild-type precursor and the four deletion mutant precursors 6-14, 15-25, 26-34, and 35-42 are
given in Figure 1.
In Vitro Protein Uptake Assay with Arabidopsis Chloroplasts It has been reported that, in general, plastids from younger tissues are most active for import (Dahlin and Cline, 1991). Pea
chloroplasts are usually isolated from 10- to 12-d-old seedlings. The
small size of Arabidopsis, however, made it necessary to use 3- to
4-week-old seedlings to obtain reproducible yields and intactness. Relative to pea, Arabidopsis chloroplasts appeared to have a higher buoyant density. Arabidopsis chloroplasts were pelleted through a
Percoll solution of 60% (v/v), whereas pea chloroplasts could only
be pelleted through a concentration of 40% (v/v) (not shown). Typically, our preparations contained more than 90% intact
chloroplasts. Arabidopsis chloroplasts were more fragile compared with
pea chloroplasts, which forced us to reduce the number of manipulations
in subsequent import assays (see ``Materials and Methods'').
The Expression of S. pratensis Fd Forms
in Transgenic Plants
Localization of S. pratensis Fd Processing is one indication of import by the chloroplast. However, to determine the intracellular localization of the newly introduced Fd, chloroplasts were isolated from the transgenic plants. For these experiments two independent lines with good expression of the S. pratensis Fd (see Fig. 3) were selected for each construct. Because very young Arabidopsis plants are too small to allow reproducible isolation of intact chloroplasts, 3-week-old seedlings were used for these experiments, the same age as the seedlings used in the in vitro experiments. Equal amounts of chloroplasts, as determined by chlorophyll content, were applied to immunoblotting both as crude homogenate and as purified intact chloroplasts (see "Methods and Materials"). The results are shown in Figure 4. The untransformed control plants only showed the endogenous Arabidopsis Fd that resides in the chloroplast, indicating that the method of determining the localization is suitable. Two unspecific bands marked with an asterisk were detected in the untransformed controls. These bands could also be detected in some samples from the transgenic plant lines. In transgenic plants expressing the wild-type pre-Fd from S. pratensis, mature-sized Fd was detected in all samples with approximately the same intensity in the crude homogenate and the isolated chloroplasts. With deletion mutant35-42, a precursor and a mature-sized band were
detected both in the crude homogenate and in the isolated chloroplasts. With deletion mutant 26-34, no S. pratensis Fd could be
detected in the crude homogenate or in the isolated chloroplasts. With deletion mutant 15-25 mature-sized S.
pratensis Fd was detected with the same intensity in both
fractions. In transgenic plants expressing the deletion mutant,
6-14 precursor-sized S. pratensis Fd could only be
detected in the crude homogenate but not in the chloroplast fraction.
Expression of the S. pratensis Fd Transgene To determine whether the amounts of S. pratensis Fd in plant homogenates from 3-week-old (Fig. 4) and 1-week-old seedlings (Fig. 3) were correlated with transcript levels of the introduced S. pratensis Fd gene, total RNA was isolated and applied to a northern blot. The filter was incubated with a probe that hybridizes to the mRNA of the mature part of S. pratensis Fd and quantified (see Fig. 5A). All lines showed a significant detectable expression of the S. pratensis Fd gene, whereas in untransformed plants, no expression could be detected (not shown). The expression of the S. pratensis Fd gene was comparable after 1 or 3 weeks at the mRNA level. Only in line 28 from plants expressing deletion mutant6-14 was the expression lower in
3-week-old seedlings. This indicates that the difference in import
pattern of the precursors was not caused by a difference in transgene
expression. The absence of the mutant 26-34 S. pratensis
Fd protein in 3-week-old seedlings was not caused by the lack of
expression of the S. pratensis Fd gene and therefore had to
originate from posttranscriptional processes.
In this study the topogenic information of a chloroplast transit
sequence was analyzed both in vitro and in vivo in the same plant
species, Arabidopsis. In vitro the full-length S. pratensis Fd precursor was imported in an ATP-dependent fashion by isolated Arabidopsis chloroplasts. In vivo the full-length precursor was imported, and the protein was processed to the mature size and localized in the chloroplast in transgenic Arabidopsis. Four mutants with deletions in adjacent regions of the transit sequence were analyzed for their in vitro and in vivo import competence. In vitro
none of these mutant precursors was imported by Arabidopsis chloroplasts. In contrast, in transgenic plants expressing these mutant
precursor constructs, more subtle effects were observed. Full loss of
import was observed for deletion mutant S. pratensis Fd Forms in Transgenic Plants The amount of precursor-sized protein that could be detected in 3-week-old plants expressing deletion mutants6-14, 15-25, and
26-34 decreased compared with 1-week-old seedlings. Since the
expression on mRNA level was comparable, this can be caused by a
difference in translation efficiency, a decreased precursor stability,
or an altered import properties of the chloroplast. This does not
affect the accumulation of mature-sized wild-type S. pratensis Fd, but it affects the accumulation of mature-sized Fd
of deletion mutant 26-34.
Difference between in Vitro and in Vivo Import There is a striking discrepancy between the in vitro and in vivo import properties of the deletion mutants in Arabidopsis chloroplasts. Deletion mutants15-25 and 35-42 were imported by the
chloroplast in 3-week-old-seedlings in vivo, but not in vitro by
chloroplasts isolated from seedlings of the same age. Mature-sized
protein from deletion mutant 26-34 could only be detected in
1-week-old seedlings in vivo, no import was observed in 3-week-old
seedlings either in vitro or in vivo. The lack of import of the mutants
in vitro results most likely from both intrinsic properties of these
precursor proteins, which reduces their ability to be imported, and the
relatively low translocation efficiency of an in vitro import system.
In vivo protein accumulation in chloroplasts occurs constantly and the
organelle is maintained in its native cellular environment in
transgenic plants. For stable precursors in vivo import can be less
efficient and still not limit mature-sized protein accumulation.
Difference between Pea and Arabidopsis Chloroplasts Pea and Arabidopsis chloroplasts differed in their capacity to import two of the deletion mutants in vitro. Deletion mutants26-34
and 35-42 could be imported by isolated pea chloroplasts, although
with a reduced efficiency (Pilon et al., 1995). No detectable import
occurred into Arabidopsis chloroplasts. In all of the experiments a Fd
precursor from S. pratensis was used for import in pea and Arabidopsis chloroplasts, which might also affect the import
efficiency. Differences in import capacities might partly be explained
by the difference in age of the plants used to isolate the chloroplasts for the in vitro import assays. For practical reasons the Arabidopsis plants were older than the pea plants at the time of chloroplast isolations. Previously, Dahlin and Cline (1991) showed that older plastids have a reduced import capacity in vitro. However, our isolated
Arabidopsis chloroplasts, despite their age, were still capable of
importing the wild-type precursor. Thus, it appears that Arabidopsis
chloroplasts are more stringent than pea chloroplasts in their
requirement for the transit sequence structure
Domain Structure of the Transit Sequence of Fd The N-terminal uncharged region of the Fd transit sequence (residues 6-14) contains essential information for chloroplast targeting. This region was also found to interact with lipids of the chloroplast envelope (Pilon et al., 1995). Surprisingly, this region is
the least conserved part of the Fd transit sequence. The only features
shared by other Fd transit sequences in this region are the hydrophobic
nature of these amino acids and the lack of charged residues.
2 Present address: Colorado State University, Department of Biology, Anatomy/Zoology Building, Fort Collins, CO 80523. * Corresponding author; e-mail w.a.rensink{at}bio.uu.nl; fax 31-30-251-3655. Received May 11, 1998;
accepted July 16, 1998.
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J. Huang, J. P. Taylor, J.-G. Chen, J. F. Uhrig, D. J. Schnell, T. Nakagawa, K. L. Korth, and A. M. Jones The Plastid Protein THYLAKOID FORMATION1 and the Plasma Membrane G-Protein GPA1 Interact in a Novel Sugar-Signaling Mechanism in Arabidopsis PLANT CELL, May 1, 2006; 18(5): 1226 - 1238. [Abstract] [Full Text] [PDF]
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P.-A. Vidi, M. Kanwischer, S. Baginsky, J. R. Austin, G. Csucs, P. Dormann, F. Kessler, and C. Brehelin Tocopherol Cyclase (VTE1) Localization and Vitamin E Accumulation in Chloroplast Plastoglobule Lipoprotein Particles J. Biol. Chem., April 21, 2006; 281(16): 11225 - 11234. [Abstract] [Full Text] [PDF]
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D. W. Lee, S. Lee, G.-j. Lee, K. H. Lee, S. Kim, G.-W. Cheong, and I. Hwang Functional Characterization of Sequence Motifs in the Transit Peptide of Arabidopsis Small Subunit of Rubisco Plant Physiology, February 1, 2006; 140(2): 466 - 483. [Abstract] [Full Text] [PDF]
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A. Fu, S. Park, and S. Rodermel Sequences Required for the Activity of PTOX (IMMUTANS), a Plastid Terminal Oxidase: IN VITRO AND IN PLANTA MUTAGENESIS OF IRON-BINDING SITES AND A CONSERVED SEQUENCE THAT CORRESPONDS TO EXON 8 J. Biol. Chem., December 30, 2005; 280(52): 42489 - 42496. [Abstract] [Full Text] [PDF]
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T. Inaba, M. Alvarez-Huerta, M. Li, J. Bauer, C. Ewers, F. Kessler, and D. J. Schnell Arabidopsis Tic110 Is Essential for the Assembly and Function of the Protein Import Machinery of Plastids PLANT CELL, May 1, 2005; 17(5): 1482 - 1496. [Abstract] [Full Text] [PDF]
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S. E. Abdel-Ghany, P. Muller-Moule, K. K. Niyogi, M. Pilon, and T. Shikanai Two P-Type ATPases Are Required for Copper Delivery in Arabidopsis thaliana Chloroplasts PLANT CELL, April 1, 2005; 17(4): 1233 - 1251. [Abstract] [Full Text] [PDF]
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 O. S. Harb, B. Chatterjee, M. J. Fraunholz, M. J. Crawford, M. Nishi, and D. S. Roos Multiple Functionally Redundant Signals Mediate Targeting to the Apicoplast in the Apicomplexan Parasite Toxoplasma gondii Eukaryot. Cell, June 1, 2004; 3(3): 663 - 674. [Abstract] [Full Text] [PDF]
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 A. Schulz, J. Knoetzel, H. V. Scheller, and A. Mant Uptake of a Fluorescent Dye as a Swift and Simple Indicator of Organelle Intactness: Import-competent Chloroplasts from Soil-grown Arabidopsis J. Histochem. Cytochem., May 1, 2004; 52(5): 701 - 704. [Abstract] [Full Text] [PDF]
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K. H. Lee, S. J. Kim, Y. J. Lee, J. B. Jin, and I. Hwang The M Domain of atToc159 Plays an Essential Role in the Import of Proteins into Chloroplasts and Chloroplast Biogenesis J. Biol. Chem., September 19, 2003; 278(38): 36794 - 36805. [Abstract] [Full Text] [PDF]
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T. Shikanai, P. Muller-Moule, Y. Munekage, K. K. Niyogi, and M. Pilon PAA1, a P-Type ATPase of Arabidopsis, Functions in Copper Transport in Chloroplasts PLANT CELL, June 1, 2003; 15(6): 1333 - 1346. [Abstract] [Full Text]
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M. Pilon, J. D. Owen, G. F. Garifullina, T. Kurihara, H. Mihara, N. Esaki, and E. A.H. Pilon-Smits Enhanced Selenium Tolerance and Accumulation in Transgenic Arabidopsis Expressing a Mouse Selenocysteine Lyase Plant Physiology, March 1, 2003; 131(3): 1250 - 1257. [Abstract] [Full Text] [PDF]
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 J. Bauer, A. Hiltbrunner, P. Weibel, P.-A. Vidi, M. Alvarez-Huerta, M. D. Smith, D. J. Schnell, and F. Kessler Essential role of the G-domain in targeting of the protein import receptor atToc159 to the chloroplast outer membrane J. Cell Biol., December 9, 2002; 159(5): 845 - 854. [Abstract] [Full Text] [PDF]
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E. A.H. Pilon-Smits, G. F. Garifullina, S. Abdel-Ghany, S.-I. Kato, H. Mihara, K. L. Hale, J. L. Burkhead, N. Esaki, T. Kurihara, and M. Pilon Characterization of a NifS-Like Chloroplast Protein from Arabidopsis. Implications for Its Role in Sulfur and Selenium Metabolism Plant Physiology, November 1, 2002; 130(3): 1309 - 1318. [Abstract] [Full Text] [PDF]
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C.-W. Sun, L.-J. Chen, L.-C. Lin, and H.-m. Li Leaf-Specific Upregulation of Chloroplast Translocon Genes by a CCT Motif-Containing Protein, CIA 2 PLANT CELL, September 1, 2001; 13(9): 2053 - 2061. [Abstract] [Full Text] [PDF]
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L. M.A. Dirk, M. A. Williams, and R. L. Houtz Eukaryotic Peptide Deformylases. Nuclear-Encoded and Chloroplast-Targeted Enzymes in Arabidopsis Plant Physiology, September 1, 2001; 127(1): 97 - 107. [Abstract] [Full Text] [PDF]
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R. A. Ivey III, C. Subramanian, and B. D. Bruce Identification of a Hsp70 Recognition Domain within the Rubisco Small Subunit Transit Peptide Plant Physiology, April 1, 2000; 122(4): 1289 - 1300. [Abstract] [Full Text]
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W. A. Rensink, D. J. Schnell, and P. J. Weisbeek The Transit Sequence of Ferredoxin Contains Different Domains for Translocation across the Outer and Inner Membrane of the Chloroplast Envelope J. Biol. Chem., March 31, 2000; 275(14): 10265 - 10271. [Abstract] [Full Text] [PDF]
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P. C. Sehnke, R. Henry, K. Cline, and R. J. Ferl Interaction of a Plant 14-3-3 Protein with the Signal Peptide of a Thylakoid-Targeted Chloroplast Precursor Protein and the Presence of 14-3-3 Isoforms in the Chloroplast Stroma Plant Physiology, January 1, 2000; 122(1): 235 - 242. [Abstract] [Full Text] [PDF]
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C. Dabney-Smith, P. W. J. van den Wijngaard, Y. Treece, W. J. Vredenberg, and B. D. Bruce The C Terminus of a Chloroplast Precursor Modulates Its Interaction with the Translocation Apparatus and PIRAC J. Biol. Chem., November 5, 1999; 274(45): 32351 - 32359. [Abstract] [Full Text] [PDF]
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A. V. Vener, A. Harms, M. R. Sussman, and R. D. Vierstra Mass Spectrometric Resolution of Reversible Protein Phosphorylation in Photosynthetic Membranes of Arabidopsis thaliana J. Biol. Chem., March 2, 2001; 276(10): 6959 - 6966. [Abstract] [Full Text] [PDF]
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Y. Usui, M. Nakase, H. Hotta, A. Urisu, N. Aoki, K. Kitajima, and T. Matsuda A 33-kDa Allergen from Rice (Oryza sativa L. Japonica). cDNA CLONING, EXPRESSION, AND IDENTIFICATION AS A NOVEL GLYOXALASE I J. Biol. Chem., March 30, 2001; 276(14): 11376 - 11381. [Abstract] [Full Text] [PDF]
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Abstract
Introduction
)-ppp-(5)-G, and
Percoll were from Pharmacia. T7 polymerase transcription kits were from Epicentre Technologies (Madison, WI). Plasmid pET11-d was purchased from Novagen (Madison, WI). Wheat-germ lysate translation kits were
from Promega. Goat-anti-rabbit antibody with horseradish peroxidase
conjugate were from Bio-Rad. Enhanced chemiluminescence western
blotting reagents and all radiochemicals were from Amersham. All other
chemicals were of the highest grade available.
6 chloroplasts. Typically, 150 to 250 µg of
chlorophyll was obtained per isolation. Chloroplasts were stored on ice
in the dark and used for import assays within 2 h after isolation.
