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 t o 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 [l -'4C]acetyl-CoA and ['4C]malonyl-CoA significantly incorporated into fatty acids. Fatty acid synthesis from acetate was independent of added cofactors but was totally light dependent. Changes i n 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 longchain fatty acids, glycerides, and COA esters.We have previously noted (Roughan et al., 1979a(Roughan et al., , 1980Roughan and Slack, 1981) that isolated spinach (Spinacia oleracea) chloroplasts synthesize fatty acids from acetate at high rates in hypotonic medium (66 mM sorbitol) usually containing ATP and COA. This medium, however, causes the organelles to swell and the envelopes to rupture, as indicated by a number of parameters, including the total loss of bicarbonate-dependent oxygen-evolving activity (Roughan et al., 1980). On the other hand, isolation of intact chloroplasts has long been known to be a prerequisite for demonstrating high rates of fatty acid synthesis by chloroplasts in vitro (Stumpf, 1972).

Toward a Functional Catalog of the Plant Genome. A Survey of Genes for Lipid Biosynthesis

“…In microarray analysis of leaves, flowers, and roots, two plastidial ACP genes were found to be expressed at a similar level compared with stearoyl-ACP desaturase. This observation of a similar concentration of substrates and enzymes is consistent with recent suggestions of channeling of acyl-ACP intermediates in the plastidial FAS machinery (Roughan and Ohlrogge, 1996).…”

“…Although easily dissociable in vitro, it has been suggested that these enzymes are associated in vivo in some more organized form (Roughan and Ohlrogge, 1996). However, almost no information is available regarding the stoichiometric ratios of the component enzymes.…”

Metabolic, Genomic, and Biochemical Analyses of Glandular Trichomes from the Wild Tomato SpeciesLycopersicon hirsutumIdentify a Key Enzyme in the Biosynthesis of Methylketones

“…There are some indications that the plastidic enzymes involved in fatty acid biosynthesis are part of a complex (Roughan and Ohlrogge, 1996). It is also likely that MKS1 is part of this protein complex in the L. hirsutum glands and that its kinetic parameters are quite different when it acts as part of such a complex.…”

Biochemical and Molecular Biological Characterization of CAC2, the Arabidopsis thaliana Gene Coding for the Biotin Carboxylase Subunit of the Plastidic Acetyl-Coenzyme A Carboxylase

“…5), these effects occur in the range of 0.1 to 1 mM, below the range of the stromal concentrations of these effectors (1-3 mM). However, recent findings indicate that the chloroplastic ACCase along with acetyl-COA synthetase and the fatty acid synthase enzymes may be in a multienzyme complex (Roughan and Ohlrogge, 1996). This complex appears to sequester and recycle a small pool of ATP/ADP nucleotides it requires for catalysis.…”