Acyl Carrier Protein (ACP) Import into Chloroplasts Does Not Require the Phosphopantetheine: Evidence for a Chloroplast Holo-ACP Synthase

Fernandez, Michael D.; Lamppa, Gayle K.

doi:10.2307/3869134

Cited by 12 publications

(14 citation statements)

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“…However, together with the observation that in vitro translated prFD is in an apo form and imported FD is in the holo form, it is reasonable to propose that, in vivo, the iron-sulfur center is assembled inside chloroplasts after imported prFD has been processed to its mature size. A similar result has been reported for the holoenzyme assembly of aspartate aminotransferase imported into mitochondria in vivo (15) and the import of acyl carrier protein into isolated chloroplasts (16). Further support for our conclusion is that a partially purified enzyme from the stromal fraction of spinach chloroplasts, which is devoid of rhodanese activity, has been reported to reconstitute the iron-sulfur center into authentic apo-FD (17).…”

Section: Methodssupporting

confidence: 76%

Metal-ion-center assembly of ferredoxin and plastocyanin in isolated chloroplasts.

Theg

Bauerle

et al. 1990

Proc. Natl. Acad. Sci. U.S.A.

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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 plastocyanm 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 compete'nt to be assembled into holo forms.Most chloroplastic proteins are encoded in the nucleus and synthesized in the cytosol as higher molecular weight precursors with amino-terminal-extensions called transit peptides. They are imported into chloroplasts' posttranslationally. The import process is usually divided into the following steps: (i) binding of precursors to the outer envelope membrane; (ii) translocation of polypeptides across the outer and inner envelope membranes; (iii) removal of transit peptides by the stromal processing protease; and (iv) depending on the protein, either further sorting into other chloroplastic compartments and/or assembly into their respective complexes (1). Most import studies have been performed using an in vitro reconstituted system in which radiolabeled precursors are synthesized by in vitro transcription and translation from cloned genes and are subsequently imported into isolated chloroplasts.Precursors to ferredoxin (prFD) and plastocyanin (prPC) are two proteins that have been examined in transport studies (2). The mature forms of these proteins are metalloproteins that function in photosynthetic electron transport. One unsolved aspect of their biogenesis concerns assembly of their metal-ion prosthetic groups. Although these two proteins follow the general steps of import described above,' current evidence is inadequate to determine when during the import process prosthetic groups are added. An understanding of when the metal-ion centers are assembled will have important implications for possible import mechanisms. For example, if the metal-ion centers are added in the cytosol, the precursors would then have to carry the metal-ion centers across the envelope during import. If the metal-ion centers are assembled inside chloroplasts, this raises questions about whether assembly occurs before or after proteolytic processing. Imported precursors and mature-sized proteins may have different conformations and only one ofthem may be competent for prosthetic group assembly.Higher plant ferredoxin (FD) contains a 2Fe-2S-type ironsulfur center and functions exclusively in the stroma of chloroplasts. A pathway for introducing the iron-sulfur center into spinach FD in isolated chloroplasts has been described (3). Isolated chloroplasts were incubated with exogenous cysteine and the sulfur atoms ...

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Section: Methodssupporting

confidence: 76%

Metal-ion-center assembly of ferredoxin and plastocyanin in isolated chloroplasts.

Theg

Bauerle

et al. 1990

Proc. Natl. Acad. Sci. U.S.A.

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“…Import reactions were previously described (Fernandez and Lamppa, 1990). Chloroplasts were incubated with 3 /nL of radiolabeled recombinant precursor for 30 min at 26°C and stopped at 4°C.…”

Section: Chloroplast Import and Organelle-free Processing Assaysmentioning

confidence: 99%

Identification of a Chloroplast Coenzyme A-Binding Protein Related to the Peroxisomal Thiolases

Yang

Lamppa

1996

Plant Physiology

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A 30-kD coenzyme A (COA)-binding protein was isolated from spinach (Spinacea oleracea) chloroplast soluble extracts using affinity chromatography under conditions in which 95% of the total protein was excluded. The 30-kD protein contains an eight-aminoacid sequence, DVRLYYCA, that is identical t o a region in a 36-kD protein of unknown function that i s encoded by a kiwifruit (Actinidia deliciosa) cDNA. Southern blotting also detected a spinach gene that is related to the kiwifruit cDNA. The kiwifruit 36-kD protein that was synthesized in Escherícbia coli was imported into chloroplasts and cleaved to a 30-kD form; it was processed to the same size in an organelle-free assay. Furthermore, the kiwifruit protein specifically bound to COA. The kiwifruit protein contains a single cysteine within a domain that is related to the peroxisomal 6-ketoacyCCoA thiolases, which catalyze the COA-dependent degradative step of fatty acid P-oxidation. Within 50 amino acids surrounding the cysteine, considered to be part of the thiolase active site, the kiwifruit protein shows approximately 26% sequence identity with the mango, cucumber, and rat peroxisomal thiolases. N-terminal alignment with these enzymes, relative to the cysteine, indicates that the 36-kD protein is cleaved after serine-58 during import, agreeing with the estimated size (approximately 6 kD) of a transit peptide. The 30-kD protein is also related to the E. coli and mitochondrial thiolases, as well as t o the acetoacetylSoA thiolases of prokaryotes. Features distinguish it from members o f the thiolase family, suggesting that it carries out a related but nove1 function. The protein i s more distantly related to chloroplast P-ketoacyl-acyl carrier protein synthase III, the initial condensing enzyme of fatty acid synthetase that utilizes acetyl-COA.Plastids are multifunctional organelles that carry out numerous biochemical reactions in different organs in addition to photosynthesis in green tissues. The role of plastids as the primary source of fatty acids in the plant cell has been well documented. The major products of fatty acid synthesis are palmitic acid and oleic acid. Fatty acids are either transferred to glycerol-3-phosphate via an acyltransferase, directing them along a "prokaryotic" pathway within the plastid, or released by a thioesterase for organel-

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“…Procedures for the ACP import reactions were previously described (Fernandez and Lamppa, 1990). Chloroplasts were resuspended in buffer A (50 mM Hepes/KOH pH 8.0,0.33 M sorbitol, 10 mM methionine) at 900 pg chlorophyll/ml; 50 p1 was added to a 300-p1 reaction containing buffer A, 10 mM ATP, 10 mM MgCI,, and 25 pl translation products.…”

Section: In Vitro Import Assay Post-import Chase Organelle-free Modmentioning

confidence: 99%

“…Chloroplasts were again pelleted, resuspended in import buffer with and without 100 pM CoA, incubated for 30 min at 26°C reisolated, and the soluble proteins analyzed by SDSFAGE. Organelle-free assays were carried out as described (Fernandez and Lamppa, 1990). Radiolabeled pre-apoACP ( -L 3 X lo5 cpm) was added to a reaction mixture containing 20 mM Tris/HCI pH 8.0, 0.5 mM CoA and 60 pl of partially purified holo-ACP synthase (Yang and Lamppa, unpublished results) and incubated at 37°C for 90 min.…”

Section: In Vitro Import Assay Post-import Chase Organelle-free Modmentioning

confidence: 99%

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Acyl Carrier Protein (ACP) Import into Chloroplasts

Yang

Fernandez

Lamppa

1994

European Journal of Biochemistry

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During the import of the precursor for the acyl carrier protein (ACP) into chloroplasts, apoACP is converted to holoACP by the attachment of a phosphopantetheine group transferred from coenzyme A (CoA) by a chloroplast holoACP synthase [Fernandez, M. and Lamppa, G. (1990) Acyl carrier protein import into chloroplasts does not require the phosphopantetheine: evidence for a chloroplast holoACP synthase, Plant Cell 2, 195–‐2061. Here it is shown that exogenous addition of CoA to intact chloroplasts in the import assay stimulates the conversion of apoACP to holoACP. If adenosine 3′,5′‐bisphosphate [Ado(3′,5′)P2], the byproduct of the transfer reaction, was also included the extent of conversion was greatly reduced. CoA has its effect after ACP precursor (pre‐ACP) import and proteolytic removal of the transit peptide, thus indicating that the chloroplast holoACP synthase resides in the stroma where fatty acid synthase is found. When Ado(3′,5′)P2 was added alone to the import assay, it inhibited the synthesis of holoACP. Inhibition of the conversion of apo‐ to holoACP with Ado(3′,5′)P2 made it possible to examine whether the holoform of preACP could be imported into chloroplasts. Pre‐apoACP was synthesized in Escherichia coli and shown to be competent for import in an ATP‐ and temperature‐dependent manner. A partially purified chloroplast holoACP synthase converted 60–90% of the pre‐apoACP to pre‐holoACP. Pre‐holoACP incubated with chloroplasts in the presence of Ado(3′,5′)P2 yielded >60% holoACP, whereas the control reaction with pre‐apoACP gave primarily apoACP. Hence the phosphopantetheine prosthetic group of ACP does not block precursor movement through the translocation apparatus of the chloroplast envelope.

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Acyl Carrier Protein (ACP) Import into Chloroplasts Does Not Require the Phosphopantetheine: Evidence for a Chloroplast Holo-ACP Synthase

Cited by 12 publications

References 0 publications

Metal-ion-center assembly of ferredoxin and plastocyanin in isolated chloroplasts.

Metal-ion-center assembly of ferredoxin and plastocyanin in isolated chloroplasts.

Identification of a Chloroplast Coenzyme A-Binding Protein Related to the Peroxisomal Thiolases

Acyl Carrier Protein (ACP) Import into Chloroplasts

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