ACYL CARRIER PROTEIN (ACP) IMPORT INTO CHLOROPLASTS - COVALENT MODIFICATION BY A STROMAL HOLOACP SYNTHASE IS STIMULATED BY EXOGENOUSLY ADDED COA AND INHIBITED BY ADENOSINE 3',5'-BISPHOSPHATE

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 chl oroplast holoACP synthase [Fernandez, M. and Lamppa, G. (1990) Acyl ca rrier protein import into chloroplasts does not require the phosphopan tetheine: evidence for a chloroplast holoACP synthase, Plant Cell 2, 1 95-206]. Here it is shown that exogenous addition of CoA to intact chl oroplasts in the import assay stimulates the conversion of apoACP to h oloACP. If adenosine 3',5'-bisphosphate [Ado(3',5')P-2], the byproduct of the transfer reaction, was also included the extent of conversion was greatly reduced. CoA has its effect after ACP precursor (preACP) i mport and proteolytic removal of the transit peptide, thus indicating that the chloroplast holoACP synthase resides in the stroma where fatt y acid synthase is found. When Ado(3',5')P-2 was added alone to the im port assay, it inhibited the synthesis of holoACP. Inhibition of the c onversion of apo- to holoACP with Ado(3',5')P-2 made it possible to ex amine whether the holoform of preACP could be imported into chloroplas ts. Pre-apoACP was synthesized in Escherichia coli and shown to be com petent for import in an ATP- and temperature-dependent manner. A parti ally 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')P-2 yielded > 60% holoACP, whereas the control reaction with pre-apoACP gave primarily apoACP. Hence the phosphopante theine prosthetic group of ACP does not block precursor movement throu gh the translocation apparatus of the chloroplast envelope.