Acyl carrier protein (ACP) import into chloroplasts does not require the phosphopantetheine: evidence for a chloroplast holo-ACP synthase.
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.
Fernandez
M D
MD
Department of Biochemistry and Molecular Biology, University of Chicago, Illinois 60637.
Lamppa
G K
GK
D000213:Acyl Carrier Protein / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D001483:Base Sequence; D002736:Chloroplasts / Q000201:enzymology / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology; D008969:Molecular Sequence Data; D016296:Mutagenesis / Q000235:genetics; D010204:Pantetheine / Q000031:analogs & derivatives* / Q000378:metabolism; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism; D010770:Phosphotransferases / Q000378:metabolism*; D010944:Plants / Q000201:enzymology / Q000235:genetics / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D011498:Protein Precursors / Q000378:metabolism; D017855:Transferases (Other Substituted Phosphate Groups)
D000213:Acyl Carrier Protein; D011498:Protein Precursors; D010204:Pantetheine; D010770:Phosphotransferases; D017855:Transferases (Other Substituted Phosphate Groups); C021364:holo-(acyl-carrier-protein) synthase; D010727:Phosphoric Diester Hydrolases; C003129:4'-phosphopantetheine