STRUCTURAL-ANALYSIS, PLASTID LOCALIZATION, AND EXPRESSION OF THE BIOTIN CARBOXYLASE SUBUNIT OF ACETYL-COENZYME-A CARBOXYLASE FROM TOBACCO

Acetyl-coenzyme A carboxylase (ACCase, EC 6.4.1.2) catalyzes the synth esis of malonyl-coenzyme A, which is utilized in the plastid for de no vo fatty acid synthesis and outside the plastid for a variety of react ions, including the synthesis of very long chain fatty acids and flavo noids. Recent evidence for both multifunctional and multisubunit ACCas e isozymes in dicot plants has been obtained. We describe here the iso lation of a tobacco (Nicotiana tabacum L. cv bright yellow 2 [NT1]) cD NA clone (E3) that encodes a 58.4-kD protein that shares 80% sequence similarity and 65% identity with the Anabaena biotin carboxylase subun it of ACCase. Similar to other biotin carboxylase subunits of acetyl-C oA carboxylase, the E3-encoded protein contains a putative ATP-binding motif but lacks a biotin-binding site (methionine-lysine-methionine o r methionine-lysine-leucine). The deduced protein sequence contains a putative transit peptide whose function was confirmed by its ability t o direct in vitro chloroplast uptake. The subcellular localization of this biotin carboxylase has also been confirmed to be plastidial by we stern blot analysis of pea (Pisum sativum), alfalfa (Medicago sativa L .), and castor (Ricinus communis L.) plastid preparations. Northern bl ot analysis indicates that the plastid biotin carboxylase transcripts are expressed at severalfold higher levels in castor seeds than in lea ves.