The Compartmentation of Acetyl-Coenzyme a Carboxylase in Plants

Plant Physiol. (1 995) 108: 445-449

The Compartmentation of Acetyl-Coenzyme A Carboxylase in Plants

Yukiko Sasaki'*, Tomokazu Konishi, and Yukio Nagano Department of Food Science and Technology, Faculty of Agriculture, Kyoto University, Kyoto, Japan

Although the biochemical pathways for fatty acid syn- cellular and an evolutionary point of view. Dicots contain thesis are more or less similar in plants and animals both types of enzyme, a eukaryotic form in the cytosol and (Harwood, 1988), there is a major cell biological difference a prokaryotic one in the plastids, but the grasses have between these two groups of eukaryotes. In plants, the enzymes of the eukaryotic type in both compartments. major site of fatty acid synthesis is the plastid, an organelle absent from the animal cell. Many aspects of plastid biology, including fatty acid synthesis, reflect the organelle's UNANSWERED QUESTIONS origins as a prokaryotic symbiont. The synthesis of fatty acids, such as palmitic acid, the prototype 16-carbon fatty Early experiments indicated that spinach chloroplasts acid, requires one molecule of acetyl-COA and seven mol- have a prokaryotic form of ACCase (Kannangara and ecules of malonyl-COA, which are added sequentially with Stumpf, 1972), and it was speculated that this prokaryotic the addition of two carbons to the growing fatty acid chain form of ACCase was in the plastids and that the cytoplasm and the release of CO, at each step. These reactions are should have a eukaryotic form (Stumpf, 1980). However, catalyzed by fatty acid synthase, an enzyme complex evidence of compartmentation was never obtained and the known to exist in a prokaryotic and a eukaryotic form hypothesis was later rejected. Subsequently, all ACCases (Wakil et al., 1983; Harwood, 1988). The prokaryotic form purified from plants were shown to be of the eukaryotic (type 11) of fatty acid synthase is found in plants. The form and the prokaryotic form of the enzyme presumed to synthase is composed of severa1 dissociable proteins, be present in the chloroplast was never identified. The whereas the eukaryotic form (type I) found in animals and small polypeptides found in isolated chloroplasts were yeasts is composed of one or two large multifunctional, thought to be degradation products of the eukaryotic form nondissociable proteins. For either form, the synthesis re- (Harwood, 1988). The absence of a prokaryotic form of quires malonyl-COA, which is supplied by ACCase in the ACCase has always been an unexplained mystery. following reaction: Another question about ACCase arose because of the sensitivity of grasses to certain herbicides. Two different CH~COSCOA+ C02 + ATP + HOOCCH~COSCOA graminicides (herbicides that kill Gramineae), developed to improve crop production, have as their target the ACCase ADP Pi + + in the plastids (Lichtenthaler, 1990). These graminicides In plant cells, large amounts of malonyl-COA are needed were shown to kill Gramineae because they inhibit fatty in the plastids to sustain fatty acid synthesis, but malonyl- acid biosynthesis, but it remained unclear why the COA is also needed in the cytosol for the elongation of fatty ACCases of Gramineae, but not those of other plants, were acids exported from the plastids and for the synthesis of inhibited. flavonoids and phytoalexins. As with fatty acid synthase, These two mysteries were recently solved (Sasaki et al., ACCase also occurs in prokaryotic and eukaryotic forms in 1993; Alban et al., 1994; Konishi and Sasaki, 1994) and here nature. The prokaryotic form is composed of dissociable we describe recent progress in plant ACCase research. polypeptides, whereas the eukaryotic form is a homodimer of a multifunctional protein. But which form(s) of this enzyme occur(s) in plants? Both or only one? This biochem- TWO FORMS OF ACCASE ical mystery, which has been around since 1972, has finally been solved and the answer is intriguing, both from a The prokaryotic form of ACCase in Esckerickia coli consists of three dissociable components: a 17-kD BCCP (accB), a 49-kD biotin carboxylase (accC), and a 130-kD transcar- ' Present address: Laboratory of Plant Molecular Biology, School of Agricultura1 Sciences, Nagoya University, Nagoya boxylase complex made up of two pairs of (Y (accA) and 464 - O1 Japan. * Corresponding author; e-mail j45589aQnucc.cc.nagoya-u.ac.jp; Abbreviations: ACCase, acetyl-CoA carboxylase; BCCP, biotin fax 81-52-789-4296. carboxyl carrier protein; ORF, open reading frame. 445 446 Sasaki et al. Plant Physiol. Vol. 108, 1995

(accD) subunits (Li and Cronan, 1992a). This complex cat- chloroplasts. The antibodies inhibited ACCase activity in alyzes two different half-reactions to form malonyl-COA. extracts from isolated chloroplasts. The antibodies precipitated a protein complex that contained [accD] protein and Mg2+ at least two other proteins, one of which was biotinylated. ATP + HCO, + BCCP C0,-BCCP ADP Pi - + + ACCase is a biotinylated protein, and a biotinylated pro- biotin carboxylase tein that is a putative component of ACCase was immuno- C02-BCCP+ acetyl-COA BCCP + malonyl-COA precipitated together with the [accD] protein. These find- - ings indicated that the [accDl protein forms a multisubunit transcarboxylase complex and is a component of the prokaryotic form of The eukaryotic form of ACCase in rat liver consists of ACCase (Sasaki, et al., 1993). The molecular size of the multimers of a single multifunctional polypeptide of about prokaryotic form is about 700 kD; larger than that of the 260 kD, which has BCCP, biotin carboxylase, and transcar- eukaryotic form, which is about 500 kD (Sasaki et al., 1993; boxylase domains (Fig. 1); its cDNA was isolated in 1988. Alban et al., 1994; Konishi and Sasaki, 1994). The subunit The four genes encoding E. coli ACCase have been identi- composition is probably similar to that of the bacterial fied during the past 10 years. The gene identified most enzyme, although no protein or gene for the a-subunit of recently is accD. The amino acid sequence of the purified E. the transcarboxylase has yet been identified. coli P-subunit of transcarboxylase coincides with that deduced from a previously sequenced gene of then unknown function, dedB, which was renamed accD (Li and Cronan, LOCATION OF TWO FORMS IN DIFFERENT 1992a). COMPARTMENTS The identification of the prokaryotic form of ACCase in plastids raised two questions: where is the eukaryotic form IDENTIFICATION OF THE PROKARYOTIC FORM OF of the enzyme located and what type of enzyme is present ACCASE IN PEA PLASTIDS in wheat plastids, which lack the [accD] gene? The prokary- Starting in 1986, the complete sequences of plant chloro- otic form of ACCase has a biotinylated protein of about 35 plast genomes of liverwort (Ohyama et al., 1986), tobacco kD and the eukaryotic form has one of about 220 kD. These (Shinozaki et al., 1986), rice (Hiratsuka et al., 1989), beech- proteins can be separated by SDS-PAGE and probed with drop (Wolfe et al., 19921, and black pine (Wakasugi et al., streptavidin. Comparison of the sizes of biotinylated pro- 1994) have been reported, with effects on various fields of teins in an extract from isolated chloroplasts with those of plant science. The genes encoding proteins in plastids were a total leaf extract showed that in pea the eukaryotic form identified from the similarity of their deduced amino acid of ACCase was not associated with the chloroplasts but sequences with those previously reported, but about 30 was probably located in cytosol (Konishi and Sasaki, 1994). ORFs were not identified. The deduced protein from one Wheat chloroplasts and leaves did not contain the prokary- such ORF has sequence similarity with the bacterial accD otic form but only the eukaryotic form (Konishi and Sasaki, protein (Li and Cronan, 199213). Although this ORF was not 1994). Another member of the Gramineae family, maize, found in the plastid genomes of wheat (Ogihara et al., 1988) also contained isozymes of eukaryotic form both inside and and rice (Hiratsuka et al., 1989), the homology with accD outside of its plastids (Egli et al., 1993). These results suggested that this gene (also named accD but here desig- indicate that plants have at least two compartmentalized nated [accD] to differentiate from bacterial accD) might ACCases. A11 of the dicots studied contain the prokaryotic encode a functional subunit of ACCase. However, direct form in plastids and the eukaryotic form in the cytosol (Fig. biochemical evidence was needed to establish the function 1). In Gramineae, the prokaryotic form is absent and of the chloroplast gene product. isozymes of the eukaryotic form are found in both the For identification of the [accD] gene product in plastids, plastids and the cytosol (Fig. 1). a partia1 DNA fragment of the putative [accD] gene from Thus, the early hypothesis about compartmentation of pea was expressed in E. coli and the recombinant [RCCD] two forms (Stumpf, 1980), once rejected, was finally found protein was purified (Sasaki et al., 1993). Antibodies to be correct in 1994. Twenty years were needed before this against this recombinant protein were prepared and used hypothesis was verified because of the extreme lability of as probes for identification of the [accD] gene products in the prokaryotic form of ACCase and because the enzymes from Gramineae were mainly studied. These problems were resolved by use of recombinant DNA techniques and new information obtained from the complete sequence of chloroplast genomes.

in Gramineae plastids and cytosol ORlGlN OF PLANT TOLERANCE TOWARD G RAMlN ICI DES That Gramineae plants have the eukaryotic form but not Figure 1. Compartmentation of the two forms of ACCase. the prokaryotic form of ACCase in their plastids suggests a Acetyl-COA Carboxylase 447 possible explanation for the tolerance of these plants to- The [accD] gene is present in a11 plants so far examined ward the herbicides aryloxyphenoxypropionate (fenoxa- except for the Gramineae. The chain length of the [accD] prop) and cyclohexanedione (sethoxydim). Both herbi- protein varies from 316 amino acid residues in liverwort cides, developed around 1973, are used to control grass (Ohyama et al., 1986) to 590 residues in pea (Nagano et al., weeds in certain dicotyledonous crops (Lichtenthaler, 1990; 1991). The 300 or so amino acid residues at the COOH Golz et al., 1994). Fenoxaprop is a derivative of a specific terminus are conserved among various plant species, but medicine for unusual fatty acid metabolisms, and sethoxy- variability exists in the NH,-terminal domain of the pro- dim is a derivative of a pesticide. The structures of these tein. There are reiterated sequences in the chloroplast ge- herbicides are different, but the target molecule of both was nome upstream of pea [accD], and recombination may have shown in 1987 to be plastidic ACCase. The differences in occurred in that region (Nagano et al., 1991). In rice, there plastidic ACCases in Gramineae and other plants were not is an ORF with 106 amino acids, which is a remnant of the known at that time. [accD]. Gramineae plastid genomes have a divergent non- Recent findings (Sasaki et al., 1993; Konishi and Sasaki, coding region around the region corresponding to the 1994) about the differences in ACCases in wheat and pea [accD] gene: this is a hot spot with various insertions and indicate that the prokaryotic form of ACCase is resistant to deletions (Ogihara et al., 1988; Clegg et al., 1994). During graminicides but that the eukaryotic form is sensitive, and evolution of Gramineae, it is possible that the fortuitous such a difference has now been demonstrated (Alban et al., addition of a DNA sequence encoding a chloroplast-target- 1994; Konishi and Sasaki, 1994). ACCase activity in extracts ing domain to the eukaryotic form of ACCase may have from pea plastids (that is, the prokaryotic form) is not been followed by the loss of the [accDI gene from the inhibited by fenoxaprop or sethoxydim, but that from chloroplast genome and presumably genes of other wheat plastids (that is, the eukaryotic form) is inhibited ACCase subunits from the nuclear genome. The prokary- (Konishi and Sasaki, 1994). The activity of the eukaryotic otic form of ACCase in Gramineae has been replaced by a form from pea leaves is not inhibited by sethoxydim but is homolog of the eukaryotic form. Various systems in plas- inhibited by fenoxaprop. There may be a common structure tids, such as those for RNA synthesis, protein synthesis, susceptible to fenoxaprop in a11 eukaryotic forms of AC- and fatty acid synthesis, are of bacterial origin, but the Case. The wheat enzyme is more sensitive to fenoxaprop Gramineae system for fatty acid synthesis is a combination than is the eukaryotic form of pea. Even in susceptible of bacterial and eukaryotic origin. Gramineae species, there is variation in the herbicide re- The eukaryotic form of ACCase has been purified from sponse (Lichtenthaler, 1990; Egli et al., 1993). At present, various plants (Harwood, 1988). The molecular size is we do not know which site on the protein is responsible for about 500 kD, with multimers of a single polypeptide about the sensitivity to herbicides. Comparison of the amino acid 220 kD, like that in mammals. Recently, cDNAs of ACCases sequences deduced from various cDNAs of ACCase with from various photosynthetic eukaryotes were isolated. different herbicide sensitivities could lead to the identifi- These cDNAs do not have recognizable transit peptide cation of the structural requirements for resistance. The sequences and their gene products are probably expressed tolerance of intact dicotyledonous plants to these herbi- in the cytosol. cides is partly accounted for by the insensitivity of the To illustrate the relationships between the prokaryotic prokaryotic form of ACCase. The reason why ACCase in form and eukaryotic form of ACCase, we constructed a Gramineae only is inhibited is that Gramineae do not have phylogenetic tree for BCCP sequences (either as discrete the herbicide-resistant prokaryotic form of ACCase but genes or as regions of multifunctional ACCases) (Fig. 2). only the herbicide-sensitive eukaryotic form in their plas- Arabidopsis BCCP, a subunit of the prokaryotic form, is tids (Fig. 1). more closely related to propionyl-COA carboxylase than to eukaryotic ACCases. Most domains of the P-subunit of

STRUCTURAL FEATURES Wheat ACCase ACCase It is likely that the prokaryotic form of ACCase is com- Arabidopsis thaliana ACCase Cyclotella cryptfca ACCase posed of four polypeptides (Fig. 1). Apart from [accDl, no \ Yeast ACCase (FAS3) homologs of the E. coli genes have been identified in the Human ACCase completely sequenced chloroplast genomes. Instead, nuclear genes have been identified for putative biotin carbox- Human w ylase from tobacco (Shorrosh et al., 1995) and BCCP from Propionyl-COA Carboxylase Arabidopsis. To date, no gene corresponding to the E. coli Anabaena BCCP (accB) accA (a- subunit of transcarboxylase) has been identified. Plastids arose from photosynthetic bacteria, and these three E. coli BCCP (fabE or accs) genes of the original bacterium may have been transferred 3Arabidopsis thaliana BCCP to the nuclear genome as was already shown for plastid Figure 2. Amino acid sequence of E. coli BCCP (106-156 residues) ribosomal proteins. Thus, plastids have the bacterial type was aligned with several BCCPs, human propionyl-COA carboxylase, of machinery for fatty acid synthesis, but only one of the and several eukaryotic ACCases with the Clustal W program. The genes involved in fatty acid synthesis ([accD]) is encoded in unrooted evolutionary tree shown was based on this alignment (Sai- the plastid genome. tou and Nei, 1987). 448 Sasaki et al. Plant Physiol. Vol. 108, 1995 transcarboxylase ([accD] protein) have little sequence sim- Received December 14, 1994; accepted March 3, 1995. ilarity with eukaryotic ACCases and are also more closely Copyright Clearance Center: 0032-0889/95/10S/0445/05. related to propionyl-COA carboxylase (data not shown). These findings appear to reflect differences in the evolutionary origin of prokaryotic and eukaryotic ACCases. LITERATURE ClTED Alban C, Baldet P, Douce R(1994) Localization and characterization of two structurally different forms of acetyl-COA carboxy- ROLES OF THE TWO FORMS OF ACCASE lase in young pea leaves, of which one is sensitive to aryloxyphenoxypropionate herbicides. Biochem J 300: 557-565 Accumulated evidence shows that the components of Clegg MT, Gaut BS, Learn GH Jr, Morton BR (1994) Rates and fatty acid synthase are in the plastids and that their cDNAs patterns of chloroplast DNA evolution. Proc Natl Acad Sci USA 91: 6795-6801 have transit peptides that mediate targeting of the gene Ebel' J, Hahlbrock K (1977) Enzymes of flavone and flavonol- products to the chloroplast. One of the roles of the pro- glycoside biosynthesis. Coordinated and selective induction in karyotic form of ACCase in plastids is to provide the cell-suspension cultures of Petroselinum hortense. Eur J Biochem precursor, malonyl-COA, for fatty acid synthesis. The syn- 75 201-209 thesis of fatty acids in chloroplasts is feasible because ATP Egli MA, Gengenbach BG, Gronwald JW, Somers DA, Wyse DL (1993) Characterization of maize acetyl-coenzyme A carboxy- and NADPH produced by the photosynthetic electron- lase. Plant Physiol 101: 499-506 transfer reaction are abundantly available. The fatty acids Golz A, Focke M, Lichtenthaler HK (1994) Inhibitors of de novo synthesized in plastids are widely used not only for the fatty acid biosynthesis in higher plants. J Plant Physiol 143: synthesis of thylakoid membrane lipids but also for the 426-433 Hardie DG, MacKintosh RW (1992) AMP-activated protein kinase: synthesis of other acyl lipids outside plastids. Membrane an archetypal protein kinase cascade? Bioessays 14 699-704 lipids are synthesized abundantly in developing young Harwood JL (1988) Fatty acid metabolism. Annu Rev Plant Physiol leaves, and there is more plastidic ACCase in such young Plant Mo1 Biol 39: 101-138 leaves than in mature ones. A nonphotosynthetic parasitic Hiratsuka J, Shimada H, Whittier R, Ishibashi T, Sakamoto M, plant, beechdrop, which has the smallest plastid genome Mori M, Kondo C, Honji Y, Sun C-R,Meng B-Y, Li Y-Q, Kanno A, Nishizawa Y, Hirai A, Shinozaki K, Sugiura M (1989) The identified so far because of a loss of photosynthetic and complete sequence of the rice (Oryza sativa) chloroplast genome: chlororespiratory genes, still has the [accD] gene (Wolfe et intermolecular recombination between distinct tRNA genes ac- al., 1992), suggesting that fatty acid synthesis in plastids is counts for a major plastid DNA inversion during the evolution essential. of the cereals. Mo1 Gen Genet 217: 185-194 Kannangara CG, Stumpf PK (1972) Fat metabolism in higher At present, there is no evidence that de novo fatty acid plants. LIV. A procaryotic type acetyl COAcarboxylase in spin- synthesis occurs outside of plastids. The roles of the eu- ach chloroplasts. Arch Biochem Biophys 152 83-91 karyotic form of ACCase in the cytosol are at least to Konishi T, Sasaki Y (1994) Compartmentalization of two forms of provide malonyl-COA for chain elongation of C,,-C,, fatty acetyl-COA carboxylase in plants and the origin of their toler- acids and to provide malonyl-COA for the synthesis of ante toward herbicides. Proc Natl Acad Sci USA 91: 3598-3601 Li S-J, Cronan JE Jr (1992a) The genes encoding the two carboxy- flavonoids. Indeed, the eukaryotic form of ACCase is abun- ltransferase subunits of Escherichiu coli acetyl-COA carboxylase. J dant in epidermal tissue (Alban et al., 1994), where most Biol Chem 267: 16841-16847 cuticular waxes and flavonoids are synthesized. Cuticular Li S-J, Cronan JE Jr (199213) Putative zinc finger protein encoded wax and flavonoids are important in the interaction of by a conserved chloroplast gene is very likely a subunit of a plants with their environment, for example, for protection biotin-dependent carboxylase. Plant Mo1 Biol 20: 759-761 Lichtenthaler HK (1990) Mode of action of herbicides affecting against UV light and pathogens. The findings that total acetyl-COA carboxylase and fatty acid biosynthesis. 2 Naturfor- ACCase activity increases with UV irradiation (Ebel and sch 45~:521-528 Hahlbrock, 1977) and that the transcript of the eukaryotic Nagano Y, Matsuno R, Sasaki Y (1991) Sequence and transcrip- form of alfalfa ACCase is induced by an elicitor (Shorrosh tional analysis of the gene cluster trnQ-z~A-psal-ORF231-petAin pea chloroplasts. Curr Genet 20 431-436 et al., 1994) suggest that the eukaryotic form of ACCase can Ogihara Y, Terachi T, Sasakuma T (1988) Intramolecular recom- help to control the synthesis of protective compounds bination of chloroplast genome mediated by short direct-repeat when necessary. sequences in wheat species. Proc Natl Acad Sci USA 85 8573- Plastidic ACCase activity increases with light and de- 8577 creases in the dark. This change in activity is rapid and not Ohyama K, Fukuzawa H, Kohchi T, Shirai H, Sano T, Sano S, Umesono K, Shiki Y, Takeuchi M, Chang Z, Aota S, Inokuchi caused by a change in the expression of the [accDl gene H, Ozeki H (1986) Chloroplast gene organization deduced from (Sasaki et al., 1993). The activation can be partly explained complete sequence of liverwort Mavchantia polymorpha chloro- by photosynthesis-dependent changes in pH, Mg2+, and plast DNA. Nature 322 572-574 adenine nucleotide levels in the chloroplast stroma (Har- Saitou N, Nei M (1987) The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mo1 Biol Evol 4 wood, 1988), but the other factors may be involved as well. 406-425 In animals and yeasts, ACCase is a key enzyme in carbon Sasaki Y, Hakamada K, Suama Y, Nagano Y, Furusawa I, Mat- metabolism and is regulated by AMP-activated protein suno R (1993) Chloroplast-encoded protein as a subunit of kinase (Hardie and MacKintosh, 1992). We do not yet have acetyl-COA carboxylase in pea plant. J Biol Chem 268: evidence that the eukaryotic form of ACCase in plants is 25118-25123 Shinozaki K, Ohme M, Tanaka M, Wakasugi T, Hayashida N, regulated in this manner. The regulatory role of the two Matsubayashi T, Zaita N, Chunwongse J, Obokata J, Yamagu- plant ACCases in carbon metabolism is an important ques- chi-Shinozaki K, Ohto C, Torazawa K, Meng BY, Sugita M, tion to be explored in the future. Deno H, Kamogashira T, Yamada K, Kusuda J, Takaiwa F, Acetyl-COA Carboxylase 449

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