THE PLANT CELL, Vol 2, Issue 3 195-206, Copyright © 1990 by American Society of Plant Biologists
Acyl Carrier Protein (ACP) Import into Chloroplasts Does not Require the Phosphopantetheine: Evidence for a Chloroplast Holo-ACP Synthase
M. D. Fernandez and G. K. Lamppa
Department of Biochemistry and Molecular Biology, University of Chicago, 920 East 58th Street, Chicago, Illinois 60637
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
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.
