Energy Requirements for Fatty Acid and Glycerolipid Biosynthesis from Acetate by Isolated Pea Root Plastids

Abstract

Fatty acid and glycerolipid biosynthesis from [14C]acetate by isolated pea root plastids is completely dependent on exogenously supplied ATP. CTP, GTP, and UTP are ineffective in supporting fatty acid biosynthesis, all resulting in <3% of the activity obtained with ATP. However, ADP alone or in combination with inorganic phosphate (Pi) or pyrophosphate (PPi) gave up to 28% of the ATP control activity, whereas AMP + PPi, PPi alone, or Pi alone were ineffective in promoting fatty acid biosynthesis. The components of the dihydroxyacetonephosphate (DHAP) shuttle (DHAP, oxaloacetate, and Pi), which promote intraplastidic ATP synthesis, restored 41% of the control ATP activity, whereas the omission of any of the shuttle components abolished this activity.When the DHAP shuttle components were supplemented with ADP, the rate of fatty acid biosynthesis was completely restored to that observed in the presence of ATP. Under the conditions of ADP + DHAP shuttle-driven fatty acid biosynthesis, exogenously supplied ATP gave only a 6% additional stimulation of activity. In general, variations in the energy source had only small effects on the proportions of radioactive fatty acids and glycerolipids synthesized. Most notably, higher amounts of radioactive oleic acid, free fatty acids, and diacylglycerol and lower amounts of phosphatidic acid were observed when ADP and/or the DHAP shuttle were substituted for ATP. The results presented here indicate that, although isolated pea root plastids readily utilize exogenously supplied ATP for fatty acid biosynthesis, these plastids can also synthesize sufficient ATP when provided with the appropriate cofactors.Fatty acid and glycerolipid biosynthesis from acetate are strictly energy dependent in all plastids. ATP is required for the synthesis of both acetyl-CoA and malonyl-CoA by acetylCoA synthetase and acetyl-CoA carboxylase, respectively (20). Similarly, the reduced nucleotides NADPH and NADH are required in the 13-ketoacyl-ACP2 reductase and 2-enoyl-ACP reductase steps ofde novo fatty acid biosynthesis, respectively, as well as the desaturation of stearoyl-ACP (20 tors are only indirectly required for plastidic glycerolipid biosynthesis in so far as this process is dependent on fatty acid synthesis. In chloroplasts, ATP and reduced nucleotides are supplied during photosynthesis and glycolytic metabolism, whereas glycolytic and oxidative pentose phosphate metabolism provides these cofactors in developing oilseed plastids (3,4). Similar metabolism may be involved in other nonphotosynthetic plastids (1,8,9, 1 1); however, the extent to which these metabolic pathways are involved remains to be fully defined.Fatty acid and glycerolipid biosynthesis from acetate in isolated pea root plastids is completely dependent on exogenously supplied ATP

Characterization of a novel eukaryotic ATP/ADP translocator located in the plastid envelope of Arabidopsis thaliana L.

“…For physiological reasons, knowledge of the nature of this important translocator protein is desired, since it has been demonstrated lately that several important metabolic pathways localized in plastids are strictly dependent on the exogenous supply of ATP. For example, both starch and fatty acid synthesis in isolated storage plastids are virtually abolished in the absence of exogenous ATP (Hill and Smith, 1991;Kang and Rawsthorne, 1994;Kleppinger-Sparace et al, 1992). The prominent physiological role of this protein is further demonstrated by the observation that the activity of the plastidic ATP/ADP translocator can actually be limiting for the rate of starch and fatty acid synthesis in isolated amyloplasts from cauliflower buds (MShlmann et aL, 1994).…”

The Utilization of Glycolytic Intermediates as Precursors for Fatty Acid Biosynthesis by Pea Root Plastids

“…Radiolabeled precursors were diluted with unlabeled substrates to a specific activity of 1 pCi/mmol. The in vitro incubation conditions for the assay and analysis of the incorporation of the labeled precursors into fatty acids and glycerolipids were as described elsewhere (Stahl and Sparace, 1991;Kleppinger-Sparace et al, 1992 …”

“…Plastids isolated from germinating pea roots (Kleppinger- Sparace et al, 1992) were purified by centrifugation through 10% Perco11 (Trimming and Emes, 1993) and washed and resuspended without BSA in 1.0 mM bis-tris-propane (pH 7.9) containing 0.33 M sorbitol to give approximately 1.0 mg mL-' plastid protein. Ontario).…”

“…Studies of the role of plastidic glycolytic metabolism in fatty acid biosynthesis in nonphotosynthetic plastids have emphasized the contribution of this pathway to the production of the ATP, and to a lesser extent, the NAD(P)H required for fatty acid biosynthesis (Kleinig and Liedvogel, 1980;Boyle et al, 1990;Kleppinger-Sparace et al, 1992;Qi et al, 1994). Relatively few studies, however, have described the complete flow of hexose carbon through the glycolytic pathway into fatty acid and glycerolipid biosynthesis in these plastids.…”

Molecular characterization of an Arabidopsis thaliana cDNA encoding a novel putative adenylate translocator of higher plants

“…Whereas the metabolic function of the chloroplast ATP/ADP translocator is not yet clear, non-green plastids strongly depend on a supply of cytosolic ATP via this translocator for biosynthesis of starch [3] and fatty acids [4]. The structure of the plastidic ATP/ADP translocator has not yet been elucidated.…”