Import of the acyl carrier protein (ACP) precursor into the chloroplast resulted in two products of about 14 kilodalton (kD) and 18 kD when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Time course experiments indicate that the latter is a modification derivative of the 14-kD peptide after the removal of the transit peptide. Substitution of serine 38 by alanine, eliminating the phosphopantetheine prosthetic group attachment site of ACP, produced a precursor mutant that gave rise to only the 14-kD peptide during import, showing that the modified form depends on the presence of serine 38. Furthermore, these results demonstrate that the prosthetic group is not essential for ACP translocation across the envelope or proteolytic processing. Analysis of the products of import by nondenaturing, conformationally sensitive gels showed reversal of the relative mobility of the 14-kD peptide and the modified form, raising the possibility that the modification is the addition of the phosphopantetheine. Proteolytic processing and the modification reaction were reconstituted in an organelle-free assay. The addition of coenzyme A to the organelle-free assay completely converted the 14-kD peptide to the modified form at 10 micromolar, and this only occurred with the wild-type substrate. Reciprocally, treatment of the products of a modification reaction with Escherichia coli phosphodiesterase converted the modified ACP from back to the 14-kD peptide. These results strongly support the conclusion that there is a holo-ACP synthase in the soluble compartment of the chloroplast capable of transferring the phosphopantetheine of coenzyme A to ACP.

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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lmport of the acyl carrier protein (ACP) precursor into the chloroplast resulted in two products of about 14 kilodalton (kD) and 18 kD when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Time, course experiments indicate that the latter is a modification derivative of the 14-kD peptide after the removal of the transit peptide. Substitution of serine 38 by alanine, eliminating the phosphopantetheine prosthetic group attachment site of ACP, produced a precursor mutant that gave rise to only the 14-kD peptide during import, showing that the modified form depends on the presence of serine 38. Furthermore, these results demonstrate that the prosthetic group is not essential for ACP translocation across the envelope or proteolytic processing. Analysis of the products of import by nondenaturing, conformationally sensitive gels showed reversal of the relative mobility of the 14-kD peptide and the modified form, raising the possibility that the modification is the addition of the phosphopantetheine. Proteolytic processing and the modification reaction were reconstituted in an organelle-free assay. The addition of coenzyme A to the organelle-free assay completely converted the 14-kD peptide to the modified form at 10 micromolar, and this only occurred with the wild-type substrate. Reciprocally, treatment of the products of a modification reaction with Escherichia coli phosphodiesterase converted the modified ACP form back to the 14-kD peptide. These results strongly support the conclusion that there is a holo-ACP synthase in the soluble compartment of the chloroplast capable of transferring the phosphopantetheine of coenzyme A to ACP.