Acyl carrier protein import into chloroplasts. Both the precursor and mature forms are substrates for phosphopantetheine attachment by a soluble chloroplast holo-acyl carrier protein synthase.
Recently a chloroplast holo-acyl carrier protein (holoACP) synthase activity was identified which attached the phosphopantetheine prosthetic group to acyl carrier protein, producing holoACP (Fernandez and Lamppa (1990) Plant Cell 2, 195-206). Here we show that the mature form of ACP (apoACP), after entry into the chloroplast and removal of the transit peptide, is a substrate for modification by the holoACP synthase. Modification occurs optimally at 37 degrees C and is inhibited by 5 mM 3',5'-ADP and 2 mM EDTA. An ACP construct (matACP) lacking the transit peptide was also converted to the holoACP form in an organelle-free assay, independent of precursor cleavage. The matACP construct was used to monitor the chromatographic separation of the holoACP synthase from the transit peptidase. Superose 12 gel filtration analysis indicates that the holoACP synthase has an apparent Mr of approximately 50,000. Using fractions enriched for the holoACP synthase it was demonstrated that the precursor of ACP is also modified in the presence of CoA and subsequently can be proteolytically processed directly to holoACP. Kinetic analysis, however, indicates that removal of the transit peptide is a much faster reaction than phosphopantetheine addition, suggesting that apoACP is the primary substrate for the chloroplast holoACP synthase in vivo.
Fernandez
M D
MD
Department of Biochemistry and Molecular Biology, University of Chicago, Illinois 60637.
Lamppa
G K
GK
D000213:Acyl Carrier Protein / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D002736:Chloroplasts / Q000378:metabolism*; D007700:Kinetics; D008954:Models, Biological; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D015345:Oligonucleotide Probes; D010204:Pantetheine / Q000031:analogs & derivatives* / Q000378:metabolism; D010770:Phosphotransferases / Q000378:metabolism*; D010944:Plants / Q000378:metabolism*; D011498:Protein Precursors / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D013379:Substrate Specificity; D017855:Transferases (Other Substituted Phosphate Groups)
D000213:Acyl Carrier Protein; D015345:Oligonucleotide Probes; D011498:Protein Precursors; D010204:Pantetheine; D010770:Phosphotransferases; D017855:Transferases (Other Substituted Phosphate Groups); C021364:holo-(acyl-carrier-protein) synthase; C003129:4'-phosphopantetheine