On the Export of Fatty Acids from the Chloroplast

Abstract

The model for export of fatty acids from plastids proposes that the acyl-ACP (acyl carrier protein) product of de novo fatty acid synthesis is hydrolyzed in the stroma by acyl-ACP thioesterases and the free fatty acid (FFA) released is then transferred to the outer envelope of the plastid where it is reactivated to acyl-CoA for utilization in cytosolic glycerolipid synthesis. Experiments were performed to assess whether the delivery of nascent FFA from the stroma for long chain acyl-CoA synthesis (LACS) occurs via simple diffusion or a more complex mechanism. The flux through the in vivo FFA pool was estimated using kinetic labeling experiments with spinach and pea leaves. The maximum half-life for FFA in the export pool was ≤1 s. Isolated pea chloroplasts incubated in the light with [14C]acetate gave a linear accumulation of FFA. When CoASH and ATP were present there was also a linear accumulation of acyl-CoA thioesters (plus derived polar lipids), with no measurable lag phase (<30 s), indicating that the FFA pool supplying LACS rapidly reached steady state. The LACS reaction was also measured independently in the dark after in situ generated FFA had accumulated yielding estimates of LACS substrate-velocity relationships. Based on these experiments the LACS reaction with in situ generated FFA as substrate is only about 3% of the LACS activity required in vivo at the very low concentrations of the FFA export pool calculated from the in vivo experiment. Furthermore, bovine serum albumin rapidly removed in situ generated FFA from chloroplasts, but could not compete effectively for "nascent" FFA substrates of LACS. Together the data suggest a locally channeled pool of exported FFA that is closely linked to LACS.