In vitro reconstitution of protein transport into chloroplasts.
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Biological Transport↗
Biomedical subjects
K Keegstra
Publications and source records attributed to K Keegstra.
Targeting of proteins to the outer envelope membrane uses a different pathway than transport into chloroplasts.
The chloroplastic envelope is composed of two membranes, inner and outer, each with a distinct set of polypeptides. Like proteins in other chloroplastic compartments, most envelope proteins are synthesized in the cytosol and post-translationally imported into chloroplasts. Considerable knowledge has been obtained concerning protein import proteins. We isolated a cDNA clone from pea that encodes a 14-kilodalton outer envelope membrane protein. The precursor form of this protein does not possess a cleavable transit peptide and its import into isolated chloroplasts does not require either ATP or a thermolysin-sensitive component on the chloroplastic surface. These findings, together with similar observations made with a spinach chloroplastic outer membrane protein, led us to propose that proteins destined for the outer membrane of the chloroplastic envelope follow an import pathway distinct from that followed by proteins destined for other chloroplastic compartments.
Adenosine Triphosphate↗
Current views on chloroplast protein import and hypotheses on the origin of the transport mechanism.
Most chloroplastic proteins are synthesized as precursors in the cytosol prior to their transport into chloroplasts. These precursors are generally synthesized in a form that is larger than the mature form found inside chloroplasts. The extra amino acids, called transit peptides, are present at the amino terminus. The transit peptide is necessary and sufficient to recognize the chloroplast and induce movement of the attached protein across the envelope membranes. In this review, we discuss the primary and secondary structure of transit peptides, describe what is known about the import process, and present some hypotheses on the evolutionary origin of the import mechanism.
Amino Acid Sequence↗
Identification of heat shock protein hsp70 homologues in chloroplasts.
Cytoplasmic members of the heat shock protein hsp70 family have recently been implicated in the transport of proteins to the endoplasmic reticulum and mitochondria. In addition, other hsp70 homologues have been found in the endoplasmic reticulum and mitochondria and, at least for the endoplasmic reticulum hsp70 homologue, may be involved in the proper folding and assembly of newly transported proteins. Since chloroplasts are an important site of protein transport in plant cells, we were interested in determining whether hsp70 proteins might be located in this organelle. By using immunoblotting techniques and two antibody preparations against hsp70 proteins, we have identified three chloroplastic proteins of approximately 70 kDa that are related to hsp70 proteins. One of these proteins was tightly associated with the outer envelope membrane and was not exposed at the outer surface of the chloroplasts. The other two were soluble proteins located in the stroma. Steady-state levels of the chloroplastic hsp70 homologues did not change after heat stress nor were any additional hsp70 homologues detected in chloroplasts isolated from heat-stressed plants. We discuss the possible functions of these hsp70 homologues in the transport of proteins into and within chloroplasts.
Antibodies↗
Metal-ion-center assembly of ferredoxin and plastocyanin in isolated chloroplasts.
Most chloroplastic proteins are cytosolically synthesized and posttranslationally transported to their proper locations. Two examples of this group of proteins are ferredoxin and plastocyanin, both of which are metal-containing components of the photosynthetic electron-transport chain. The import process for these two proteins includes the insertion of the metal ions to produce the holo forms of the proteins. We show here that in vitro translated precursor proteins of ferredoxin and plastocyanin are synthesized as apo forms and are assembled into their respective holo forms after being imported into isolated chloroplasts. We also provide evidence that only mature-sized proteins are competent to be assembled into holo forms.
Journal Article↗
ATP is required for the binding of precursor proteins to chloroplasts.
One of the first steps in the transport of nuclear-encoded, cytoplasmically synthesized precursor proteins into chloroplasts is a specific binding interaction between precursor proteins and the surface of the organelle. Although protein translocation into chloroplasts requires ATP hydrolysis, binding is generally thought to be energy independent. A more detailed investigation of precursor binding to the surface of chloroplasts showed that ATP was required for efficient binding. Protein translocation is known to require relatively high levels (1 mM or more) of ATP. As little as 50-100 microM ATP caused significant stimulation of precursor binding over controls with no ATP. Several different precursors were tested and all showed increased binding upon addition of low levels of ATP. Nonhydrolyzable analogs of ATP did not substitute for ATP, indicating that ATP hydrolysis was required for binding. A protonmotive force was not involved in the energy requirement for binding. Other (hydrolyzable) nucleotides could substitute for ATP but were less effective at stimulating binding. Binding was stimulated by ATP generated inside chloroplasts even when an ATP trap was present to destroy external ATP. We conclude that internal ATP is required for stimulation of precursor binding to chloroplasts.
Adenosine Triphosphate↗
Internal ATP is the only energy requirement for the translocation of precursor proteins across chloroplastic membranes.
The energy requirements for the import of nuclear-encoded proteins into isolated chloroplasts have been reinvestigated. We have shown that, in contrast to protein import into mitochondria, the translocation of the precursors to ferredoxin, plastocyanin (prPC) and the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (prSS) across all chloroplastic membranes is independent of a protonmotive force and requires only ATP. This extends previous works in which investigations were limited to prSS and demonstrates that our results are probably general to all chloroplastic protein precursors. Our results are particularly interesting for the import of prPC, since in addition to the two envelope membranes, this protein must traverse the energy-transducing thylakoid membranes en route to its proper location in the thylakoid lumen. This lack of involvement of a protonmotive force, specifically of a transmembrane electric potential, demonstrates that separate mechanisms operate during the import of proteins into chloroplasts and mitochondria. We also examined the question of whether ATP is utilized inside or outside of chloroplasts during protein import. Previous attempts to resolve this question have resulted in conflicting answers. We found, by two independent approaches, that ATP for protein import is utilized inside chloroplasts. The implications of these results on the possible mechanisms of protein import into chloroplasts are discussed.
Adenosine Triphosphate↗
Transport and routing of proteins into chloroplasts.
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Chloroplasts↗
Chloroplast protein import : quantitative analysis of precursor binding.
The first step of chloroplast protein import is binding of a precursor protein to the surface of the organelle. Precursor binding for the small subunit of ribulose-1,5-bisphosphate carboxylase to isolated pea chloroplasts was investigated using a receptor-ligand binding assay. Translocation of precursors was blocked by conducting the binding assays at 0 degrees C. Binding of precursor was judged to be receptor mediated by the following criteria: (a) precursor binding was saturable at between 1500 and 3500 molecules per chloroplast; (b) binding is a high affinity interaction with a dissociation constant of 6 to 10 nanomoles; (c) binding is physiologically productive since most of the bound precursors could be imported from the bound state; and (d) precursor binding was sensitive to both protease and the sulfhydryl modifying reagent N-ethylmaleimide. The effects of these two reagents differed in that protease reduced the total number of binding sites from the surface of chloroplasts but had little effect on binding affinity, whereas N-ethylmaleimide reduced the binding affinity but had little or no effect on receptor density.
Journal Article↗
Targeting of proteins into chloroplasts.
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Amino Acid Sequence↗
Imported large subunits of ribulose bisphosphate carboxylase/oxygenase, but not imported beta-ATP synthase subunits, are assembled into holoenzyme in isolated chloroplasts.
The large subunit of ribulose bisphosphate carboxylase from Anacystis nidulans 6301, and the beta subunit of chloroplast ATP synthase from maize, were fused to the transit peptide of the small subunit of ribulose bisphosphate carboxylase from soybean. These proteins were assayed for post-translational import into isolated pea chloroplasts. Both proteins were imported into chloroplasts. Imported large subunits were associated with two distinct macromolecular structures. The smaller of these structures was a hybrid ribulose bisphosphate carboxylase holoenzyme, and the larger was the binding protein oligomer. Time-course experiments following import of the large subunit revealed that the amount of large subunit associated with the binding protein oligomer decreased over time, and that the amount of large subunit present in the assembled holoenzyme increased. We also observed that imported small subunits of ribulose bisphosphate carboxylase, although predominantly present in the holoenzyme, were also found associated with the binding protein oligomer. In contrast, the imported beta subunit of chloroplast ATP synthase did not assemble into a thylakoid-bound coupling factor complex.
Journal Article↗
Specific Labeling of the Phosphate Translocator in C(3) and C(4) Mesophyll Chloroplasts by Tritiated Dihydro-DIDS (1,2-Ditritio-1,2-[2,2' -Disulfo-4,4' -Diisothiocyano] Diphenylethane).
The phosphate translocator protein of C(3) and C(4) mesophyll chloroplast envelopes was specifically labeled using the anion exchange inhibitor, 1,2-ditritio-1,2-(2,2' -disulfo-4,4' -diisothiocyano) diphenylethane ([(3)H](2)-DIDS). Intact mesophyll chloroplasts were isolated from the C(3) plants, Spinacia oleracea L. (spinach) and Pisum sativum L. (pea), and the C(4) plant, Zea mays L. (corn). Chloroplasts were incubated with 5 to 50 mum [(3)H](2)-DIDS and, in addition, pea chloroplasts were also incubated with pyridoxal phosphate/tritiated sodium borohydride. The chloroplasts were washed, the envelopes isolated and solubilized. Following sodium dodecyl sulfate polyacrylamide gel electrophoresis, label from bound [(3)H](2)-DIDS was detected only in the 28- to 30-kilodalton protein (proposed C(3) phosphate translocator) for both C(3) and C(4) chloroplasts, as demonstrated by fluorography. In contrast, when pyridoxal phosphate/tritiated sodium borohydride was used to label pea chloroplasts, radioactivity was detected in several other bands in addition to the 29-kilodalton polypeptide. These findings suggest that DIDS is a much more specific inhibitor than reagents previously employed to study the phosphate translocator and could be used to isolate and characterize the differences in the C(3) and C(4) phosphate translocator protein(s).
Journal Article↗
Evidence that a Chloroplast Surface Protein Is Associated with a Specific Binding Site for the Precursor to the Small Subunit of Ribulose-1,5-Bisphosphate Carboxylase.
Most chloroplast proteins are encoded by nuclear genes and synthesized in the cytoplasm as higher molecular weight precursors. These precursors are imported posttranslationally into the chloroplasts, where they are proteolytically processed, and sorted to their proper locations. The first step of this import process is thought to be the binding of precursors to putative receptors on the outer envelope membrane of chloroplasts. We have investigated the interaction of the precursor to the small subunit of ribulose-1,5-bisphosphate carboxylase with its putative receptor by using a heterobifunctional, photoactivatable cross-linker. The resulting cross-linked conjugate has a molecular weight of 86,000, and is present on the surface of chloroplasts as determined by its sensitivity to digestion with protease. Control experiments demonstrated that the label in the conjugate is derived from small subunit precursor and that the conjugate is formed only when modified precursor is reacted in the presence of chloroplasts. Based on these results, we postulate that a protein on the surface of chloroplasts is part of the receptor which interacts with the small subunit precursor.
Journal Article↗
The role of the transit peptide in the routing of precursors toward different chloroplast compartments.
The role of the transit peptide in the routing of imported proteins inside the chloroplast was investigated with chimeric proteins in which the transit peptides for the nuclear-encoded ferredoxin and plastocyanin precursors were exchanged. Import and localization experiments with a reconstituted chloroplast system show that the ferredoxin transit peptide directs mature plastocyanin away from its correct location, the thylakoid lumen, to the stroma. With the plastocyanin transit peptide-mature ferredoxin chimera, a processing intermediate is arrested on its way to the lumen. We propose a two domain hypothesis for the plastocyanin transit peptide: the first domain functions in the chloroplast import process, whereas the second is responsible for transport across the thylakoid membrane. Thus, the transit peptide not only targets proteins to the chloroplast, but also is a major determinant in their subsequent localization within the organelle.
Amino Acid Sequence↗
Efficient in vitro import of a cytosolic heat shock protein into pea chloroplasts.
In order to further our understanding of the targeting of nuclear-encoded proteins into intracellular organelles, we have investigated the import of chimeric precursor proteins into pea chloroplasts. Two different chimeric precursor proteins were produced by in vitro expression of chimeric genes. One chimeric precursor contained the transit peptide of the small subunit of soybean ribulose 1,5-bisphosphate carboxylase and the mature peptide of the same protein from pea. The second contained the same transit peptide plus 13 amino acids of the pea mature peptide fused to a cytosolic heat shock protein. The extent of import and binding of the two chimeric proteins was examined by using quantitative assays and was compared to the import of pea small subunit precursor. Both precursor proteins imported well into pea chloroplasts, although the extent of import observed with the chimeric small-subunit-heat shock precursor was less than that observed with the soybean-pea small subunit precursor. The heat shock protein alone did not import into nor bind to chloroplasts. The binding of both the chimeric small-subunit-heat shock protein and the soybean-pea small subunit precursor to chloroplasts was physiologically significant, as shown by the fact that when chloroplasts with bound precursors were isolated, these bound precursors could subsequently be imported.
Journal Article↗
Adenine nucleotide translocase-dependent anion transport in pea chloroplasts.
Pea chloroplasts were found to take up actively ATP and ADP and exchange the external nucleotides for internal ones. Using carrier-free [14C]ATP, the rate of nucleotide transport in chloroplasts prepared from 12-14-day-old plants was calculated to be 330 mumol ATP/g chlorophyll/min, and the transport was not affected by light or temperature between 4 and 22 degrees C. Adenine nucleotide uptake was inhibited only slightly by carboxyatractylate, whereas bongkrekic acid was nearly as effective an inhibitor of the translocator in pea chloroplasts as it was in mammalian mitochondria. There was no counter-transport of adenine nucleotides with substrates carried on the phosphate translocator including inorganic phosphate, 3-phosphoglycerate and dihydroxyacetone phosphate. However, internal or external phosphoenolpyruvate, normally considered to be transported on the phosphate carrier in chloroplasts, was able to exchange readily with adenine nucleotides. Furthermore, inorganic pyrophosphate which is not transported by the phosphate carrier initiated efflux of phosphoenolpyruvate as well as ATP from the chloroplast. These findings illustrate some interesting similarities as well as differences between the various plant phosphate and nucleotide transport systems which may relate to their role in photosynthesis.
Adenine Nucleotides↗
Precursors to two nuclear-encoded chloroplast proteins bind to the outer envelope membrane before being imported into chloroplasts.
Precursor forms of chloroplast proteins synthesized in cell-free translation systems can be imported posttranslationally into isolated, intact chloroplasts. Radiochemically pure precursors to the small subunit of ribulose-1,5-bisphosphate carboxylase and to the light-harvesting chlorophyll a/b protein have been prepared by in vitro translation of hybrid-selected mRNA and used to study this import process. If chloroplasts are pretreated with the uncoupler nigericin, import does not occur, but the precursors bind to the chloroplast surface. Reincubation of the precursor-chloroplast complex in the presence of ATP results in import of bound precursors. The binding appears to be mediated by proteins of the outer chloroplast envelope membrane because pretreatment of chloroplasts with protease inhibits their ability to bind as well as to import precursors. These results indicate that at least a portion of the observed binding is to functional receptor proteins involved in the import process.
Adenosine Triphosphate↗
l-Aspartate Transport into Pea Chloroplasts : Kinetic and Inhibitor Evidence for Multiple Transport Systems.
The kinetics of l-aspartate transport into pea chloroplasts was studied in the presence and absence of transport inhibitors to determine whether multiple aspartate carriers exist. Transport was measured by the silicone oil centrifugation technique. Reciprocal plots of concentration-dependent transport rates were biphasic, indicating the presence of two transport components, distinguishable on the basis of their affinity for aspartate. These transport components, called high affinity and low affinity transport could also be distinguished on the basis of their apparent substrate saturability and their sensitivity to media pH. The apparent K(m) for high affinity transport was 30 micromolar. The K(m) for low affinity transport was not determined. To test whether these transport components could also be distinguished on the basis of inhibitor sensitivity and to assess the value of inhibitors for distinguishing multiple aspartate translocators, a survey of several classes of potential inhibitors was conducted. High affinity aspartate transport was inhibited by p-chloromercuribenzenesulfonate and mersalyl, both sulfhydryl-reactive reagents; diethyl pyrocarbonate, a histidine-reactive reagent; and nigericin and carbonyl cyanide m-chlorophenylhydrazone, both ionophores. Low affinity aspartate transport was not inhibited by p-chloromercuribenzenesulfonate or nigericin, but preliminary results suggest it was sensitive to diethyl pyrocarbonate. Because the high and low affinity transport components could be distinguished not only by their sensitivity to media pH and substrate saturability, but also by their sensitivity to various inhibitors, we concluded that they may represent different transport systems or carriers.
Journal Article↗