Abstract
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.
Highlights
BOTH THE PRECURSOR AND MATURE FORMS ARE SUBSTRATES FOR PHOSPHOPANTETHEINE ATTACHMENT BY A SOLUBLE CHLOROPLAST HOLO-ACYL CARRIER PROTEIN SYNTHASE*
We show that the mature form ofACP, after entry into the chloroplast and removal of the transit peptide, is a substrate for modification by the holo-acyl carrier protein (holoACP) synthase
The major form in spinach leaves, ACP I, is nuclear encoded, synthesized as a precursor with an N-terminal transit peptide, and post-translationaly imported into the chloroplast, where it is proteolytically processsed to its mature form (Kuo and Ohlrogge, 1984; Scherer and Knauf, 1987)
Summary
BOTH THE PRECURSOR AND MATURE FORMS ARE SUBSTRATES FOR PHOSPHOPANTETHEINE ATTACHMENT BY A SOLUBLE CHLOROPLAST HOLO-ACYL CARRIER PROTEIN SYNTHASE*. Incontrast, the addition ofFAD to that removal of the transit peptide is amuch faster ferredoxin-NADP+oxidoreductase has been reported to occur reaction than phosphopantetheineaddition, suggesting in the cytoplasm before its import and localization to the that apoACP is the primary substrate for the chloro- chloroplast thylakoid membranes (Carillo, 1985) It has been plast holoACP synthase in vivo. Out a number of essential metabolic processes in addition to It had been suggestedthat thephosphopantetheine is transphotosynthesis Among these is de novo fatty acid biosyn- ferred from CoA to ACP by a cytoplasmicallylocated holoACP thesis, which in higher plant leaves takes place primarily in synthase (Elhussein et al, 1988)which might confer upon the the chloroplast stroma (Ohlrogge et al, 1979; Stumpf, 1980) protein aconformation essential for proper translocation into a recent report suggests that fattyacid biosynthesis the organelle.
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