Evidence that isolated chloroplasts contain an integrated lipid-synthesizing assembly that channels acetate into long-chain fatty acids

Abstract

High rates of light-dependent fatty acid synthesis from acetate were measured in isolated chloroplasts that were permeabilized to varying extents by resuspension in hypotonic reaction medium. The reactions in hypotonic medium unsupplemented with cofactors were linear with time and were directly proportional to chlorophyll concentration, suggesting that the enzymes and cofactors of fatty acid synthesis remained tightly integrated and thylakoid associated within disrupted chloroplasts. Permeabilized chloroplasts expanded to at least twice the volume of intact chloroplasts, lost about 50% of their stromal proteins in the medium, and metabolized exogenous nucleotides. However, neither acetyl-coenzyme A (CoA) nor malonyl-CoA inhibited fatty acid synthesis from acetate; nor were [1-14C]acetyl-CoA and [14C]malonyl-CoA significantly incorporated into fatty acids. Fatty acid synthesis from acetate was independent of added cofactors but was totally light dependent. Changes in the products of fatty acid synthesis were consistent with the loss of endogenous glycerol-3-phosphate from permeabilized chloroplasts. However, in appropriately supplemented medium, the products of acetate incorporation by spinach (Spinacia oleracea) chloroplasts were similar when reactions were carried out in either isotonic or hypotonic medium. Taken together, the results of this study suggest that the enzymes of fatty acid synthesis with chloroplasts are organized into a multienzyme assembly that channels acetate into long-chain fatty acids, glycerides, and CoA esters.