University of Minnesota

Plastid localization of a multifunctional acetyl-CoA carboxylase
--Egli, M and Gengenbach, B

Acetyl-CoA carboxylase (ACCase; E.C. catalyzes synthesis of the malonyl-CoA required for subsequent synthesis of fatty acids and secondary metabolites in plants. Its activity is positively correlated with rates of fatty acid synthesis in both leaves and developing oil seeds, and thus it may be important in regulating plant lipid synthesis. Current information indicates that plastidic ACCase activity in dicots is due to a multisubunit ACCase enzyme similar to that in bacteria but which is absent in the Poaceae (Konishi and Sasaki, Proc. Natl. Acad. Sci. 91:3598-3601, 1994). In contrast, most ACCase activity in leaves and oil-storing embryos of maize is associated with a high-molecular weight, multifunctional plastid-localized polypeptide (Egli et al., Plant Physiol. 101:499-506, 1993; Somers et al., Plant Physiol. 101:1097-1101, 1993). Complete coding sequences for higher plant MF ACCase polypeptides from wheat (Gornicki et al., Proc. Natl. Acad. Sci. 91:6860-6864, 1994), and several dicots (Anderson et al., Plant Physiol. 109:338, 1995; Roesler et al., Plant Physiol. 105:611-617, 1994; Schulte et al., Plant Physiol. 106:793-794, 1994; Shorrosh et al., Proc. Natl. Acad. Sci. 91:4323-4327, 1994) have been described. Although de novo FA synthesis occurs in plastids, these genes appear to encode cytosolic isoforms or their cellular location is unclear (Schulte et al.).

We recently published the complete coding sequence of a multifunctional maize ACCase that corresponds to one of four distinct types of ACCase genomic clones (Egli et al., Plant Physiol. 108:1299-1300, 1995; Lutz et al., 37th. Ann. Maize Genetics Conf., poster 34, 1995). The N-terminus of the predicted maize ACCase polypeptide is longer than that of predicted cytosolic ACCase isoforms and it appears to have several properties typical of chloroplast transit peptides: (1) no acidic residues within aa# 1-49, (2) high S content within aa# 23-35, and (3) an R-rich region between S- and D-rich regions (aa# 36-49) (Von Heijne and Nishikawa, FEBS Lett. 278:1-3, 1991). In vitro chloroplast import assays were used to demonstrate that this putative transit peptide is indeed functional.

Truncated ACCase cDNAs encoding the first structural domain of biotin carboxylase (Waldrop et al., Biochemistry 33:10249-10256, 1994) plus (BCN1; nt 1-833) or minus the putative cTP (-pBCN1; nt 278-833) were synthesized by RT-PCR and cloned into the EcoRV site of PCR-script (Stratagene). Linearized, capped transcripts were translated in vitro in a wheat germ system (Ambion) to produce 35S-polypeptides. In vitro import of 35S polypeptides by mesophyll chloroplasts of 7-d old leaves of maize (A188) and pea ("Little Marvel") was tested as described by Cline et al. (J. Biol. Chem. 260:3691-3696, 1985). Aliquots of the import supernatants from lysed chloroplasts and of the original in vitro-translated proteins were analysed by SDS-PAGE in 8-25% Phast gels (Pharmacia) and 35S-proteins were detected by autoradiography.

Both pea and maize chloroplasts imported 35S-BCN1 polypeptides but neither imported -pBCN1, which begins at ACCase aa#83 (V -> M mutation) and lacks a transit peptide. As estimated by SDS-PAGE, 30-min import converted the original 32-kD BCN1 polypeptide to a doublet of 27.2 and 27.5 kD in maize and produced an additional 30-kD band in pea. Formation of the 27.2-kD polypeptide could result from cleavage after ~aa #47, a likely cleavage site because it lies between S- and D-rich regions, and R residues are located at -2, -7,and -8 (Gavel and Von Heijne, FEBS Lett. 261:455-458, 1990). Time-dependence of BCN1 import was further examined (1-30 min) to determine if any imported polypeptides were a result of incomplete processing or proteolysis. Import was maximal after 15 min, but import time had no effect on the relative amounts of different-sized import products. The data suggest that, in maize, efficient cleavage of BCN1 occurs at two closely adjacent sites and that partially-processed products are also formed during BCN1 import by pea chloroplasts.

The maize ACCase gene described here (Genbank accession # U19183 ) encodes a protein that contains a chloroplast transit peptide which functions in both monocots and dicots; this is the first plastidic multifunctional ACCase to be identified in a higher plant. 

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