Identification of a Chloroplast Coenzyme A-Binding Protein Related to the Peroxisomal Thiolases

Abstract

A 30-kD coenzyme A (COA)-binding protein was isolated from spinach (Spinacea oleracea) chloroplast soluble extracts using affinity chromatography under conditions in which 95% of the total protein was excluded. The 30-kD protein contains an eight-aminoacid sequence, DVRLYYCA, that is identical t o a region in a 36-kD protein of unknown function that i s encoded by a kiwifruit (Actinidia deliciosa) cDNA. Southern blotting also detected a spinach gene that is related to the kiwifruit cDNA. The kiwifruit 36-kD protein that was synthesized in Escherícbia coli was imported into chloroplasts and cleaved to a 30-kD form; it was processed to the same size in an organelle-free assay. Furthermore, the kiwifruit protein specifically bound to COA. The kiwifruit protein contains a single cysteine within a domain that is related to the peroxisomal 6-ketoacyCCoA thiolases, which catalyze the COA-dependent degradative step of fatty acid P-oxidation. Within 50 amino acids surrounding the cysteine, considered to be part of the thiolase active site, the kiwifruit protein shows approximately 26% sequence identity with the mango, cucumber, and rat peroxisomal thiolases. N-terminal alignment with these enzymes, relative to the cysteine, indicates that the 36-kD protein is cleaved after serine-58 during import, agreeing with the estimated size (approximately 6 kD) of a transit peptide. The 30-kD protein is also related to the E. coli and mitochondrial thiolases, as well as t o the acetoacetylSoA thiolases of prokaryotes. Features distinguish it from members o f the thiolase family, suggesting that it carries out a related but nove1 function. The protein i s more distantly related to chloroplast P-ketoacyl-acyl carrier protein synthase III, the initial condensing enzyme of fatty acid synthetase that utilizes acetyl-COA.Plastids are multifunctional organelles that carry out numerous biochemical reactions in different organs in addition to photosynthesis in green tissues. The role of plastids as the primary source of fatty acids in the plant cell has been well documented. The major products of fatty acid synthesis are palmitic acid and oleic acid. Fatty acids are either transferred to glycerol-3-phosphate via an acyltransferase, directing them along a "prokaryotic" pathway within the plastid, or released by a thioesterase for organel-