Subcellular localization of acyl carrier protein in leaf protoplasts of Spinacia oleracea.

Abstract

This communication demonstrates that all de novo fatty acid biosynthesis in spinach leaf cells requires acyl carrier protein (ACP) and occurs specifically in the chloroplasts. Antibodies raised to purified spinach ACP inhibited at least 98% of malonyl CoA-ependent fatty acid synthesis by spinach leaf homogenates. Therefore, the presence of ACP in a compartment of the spinach leaf cell would serve as a marker for de novo fatty acid biosynthesis. A radioimmunoassa capable of detecting 10-'S mol (10-" g) of spinach ACP was deve oped to measure the levels of ACP in leaf cell components isolated by sucrose gradient centrifugation of a gentle lysate of spinach leaf protoplasts. All of the ACP of the leaf cell could be attributed to the chloroplast. Less than 1% of the ACP associated with chloroplasts resulted from binding of free ACP to chloro lasts. Of interest, ACP from Escherichia coli, soybean, and sunflower showed only partial crossreactivity with spinach ACP by the radioimmunoassay. These results strongly suggest that, in the leaf cell, chloroplasts are the sole site for the degnovo synthesis of C16 and C18 fatty acids. These fatty acids are then transported into the cytoplasm for further modification and are either inserted into extrachloroplastic membrane lipids or returned to the chloroplast for insertion into lamellar membrane lipids. In plants, acyl carrier protein (ACP) plays an essential role in both the synthesis and the subsequent metabolism of the C16 and C18 fatty acids (1). Whereas de novo fatty acid synthesis in isolated chloroplasts has long been known (1), the site of synthesis of fatty acids that are required for the formation of plasma, mitochondrial, and other extrachloroplast membranes in the leaf cell is not clear. In sharp contrast, in both animal and yeast cells, synthesis occurs in the cytoplasm (2). In Escherichi colA cells, it has been shown that ACP is localized on or near the inner face of the plasma membrane and this implies that the nonassociated fatty acid synthetase enzymes may be organized in the same area in vivo (3).Although earlier studies have provided evidence that fatty acid biosynthesis occurs in chloroplasts (1) and in proplastids (4-8), until recently the methods used for the isolation of these organelles led to substantial breakage and release of enzymes into the cytoplasmic fraction. Consequently, it has been difficult to assign a precise site for an enzyme in the leaf cell. In addition, after cell disruption, attempts to localize the complex set of reactions comprising fatty acid synthesis can be further complicated by cofactor dilution (particularly ACP), enzyme dilution, and inactivation. Hence, it has been difficult to conclude from these earlier studies whether de novo fatty acid synthesis takes place only in the chloroplast (or plastid) or in the cytoplasm or other organelles as well. Damage to organelles can be greatly reduced in the isolation procedure by using protoplasts as the starting material (9). Gentle lysis of isolated protoplasts gives an incre...

Testing Models of Fatty Acid Transfer and Lipid Synthesis in Spinach Leaf Using in Vivo Oxygen-18 Labeling

“…The chemical principle of this model was first proposed by Shine et al (1976) as the basis of switching from ACP-thioester metabolism to acyl-CoA thioester metabolism in plant fatty acid synthesis. Subsequent experiments showed that ACP-dependent fatty acid synthesis was entirely a chloroplast function (Ohlrogge et al, 1979), implying that the acyl-ACP thioesterase was a stromal enzyme activity. Acyl-CoA synthetase was demonstrated as an activity of the chloroplast envelope (Roughan and Slack, 1977;Joyard and Stumpf, 1981), and later more specifically of the outer envelope (Andrews and Keegstra, 1983;Block et al, 1983).…”

Metabolic Responses to the Reduction in Palmitate Caused by Disruption of the FATB Gene in Arabidopsis

“…The net loss of palmitate from phosphatidylcholine plus phosphatidylethanolamine is similar for wild type and mutant, suggesting that mechanisms are not present that can preferentially preserve the saturated fatty acids. In summary, the leaf cell responds to the loss of saturated fatty acid production in the fatb-ko mutant by increasing both fatty acid synthesis and degradation, but in doing so the mechanisms for increased fatty acid turnover contribute to the lowering of the percentage of saturated fatty acids found in eukaryotic lipids.In plants, the major site for de novo fatty acid synthesis (FAS) occurs in the plastid (Ohlrogge et al, 1979). Fatty acids are either utilized in this organelle or exported to supply diverse cytoplasmic biosynthetic pathways and cellular processes.…”

“…In plants, the major site for de novo fatty acid synthesis (FAS) occurs in the plastid (Ohlrogge et al, 1979). Fatty acids are either utilized in this organelle or exported to supply diverse cytoplasmic biosynthetic pathways and cellular processes.…”

Isolation and Characterization of Two Safflower Oleoyl-Acyl Carrier Protein Thioesterase cDNA Clones

“…Fatty acid biosynthesis in plants is catalyzed by a series of soluble, discrete enzymes (23) that are localized in the chloroplasts of leaves (16) and the proplastids of developing seed tissue (14, 25,31). Fatty acids up to 18 carbon atoms in length are synthesized in twocarbon increments as ACP2 thioesters (15).…”