The BADC and BCCP subunits of chloroplast acetyl-CoA carboxylase sense the pH changes of the light–dark cycle
2020; Elsevier BV; Volume: 295; Issue: 29 Linguagem: Inglês
10.1074/jbc.ra120.012877
ISSN1083-351X
AutoresYajin Ye, Yan G. Fulcher, David J. Sliman, Mizani T. Day, Mark J. Schroeder, Rama K. Koppisetti, Philip D. Bates, Jay J. Thelen, Steven R. Van Doren,
Tópico(s)Microbial Metabolic Engineering and Bioproduction
ResumoAcetyl-CoA carboxylase (ACCase) catalyzes the first committed step in the de novo synthesis of fatty acids. The multisubunit ACCase in the chloroplast is activated by a shift to pH 8 upon light adaptation and is inhibited by a shift to pH 7 upon dark adaptation. Here, titrations with the purified ACCase biotin attachment domain-containing (BADC) and biotin carboxyl carrier protein (BCCP) subunits from Arabidopsis indicated that they can competently and independently bind biotin carboxylase (BC) but differ in responses to pH changes representing those in the plastid stroma during light or dark conditions. At pH 7 in phosphate buffer, BADC1 and BADC2 gain an advantage over BCCP1 and BCCP2 in affinity for BC. At pH 8 in KCl solution, however, BCCP1 and BCCP2 had more than 10-fold higher affinity for BC than did BADC1. The pH-modulated shifts in BC preferences for BCCP and BADC partners suggest they contribute to light-dependent regulation of heteromeric ACCase. Using NMR spectroscopy, we found evidence for increased intrinsic disorder of the BADC and BCCP subunits at pH 7. We propose that this intrinsic disorder potentially promotes fast association with BC through a "fly-casting mechanism." We hypothesize that the pH effects on the BADC and BCCP subunits attenuate ACCase activity by night and enhance it by day. Consistent with this hypothesis, Arabidopsis badc1 badc3 mutant lines grown in a light–dark cycle synthesized more fatty acids in their seeds. In summary, our findings provide evidence that the BADC and BCCP subunits function as pH sensors required for light-dependent switching of heteromeric ACCase activity. Acetyl-CoA carboxylase (ACCase) catalyzes the first committed step in the de novo synthesis of fatty acids. The multisubunit ACCase in the chloroplast is activated by a shift to pH 8 upon light adaptation and is inhibited by a shift to pH 7 upon dark adaptation. Here, titrations with the purified ACCase biotin attachment domain-containing (BADC) and biotin carboxyl carrier protein (BCCP) subunits from Arabidopsis indicated that they can competently and independently bind biotin carboxylase (BC) but differ in responses to pH changes representing those in the plastid stroma during light or dark conditions. At pH 7 in phosphate buffer, BADC1 and BADC2 gain an advantage over BCCP1 and BCCP2 in affinity for BC. At pH 8 in KCl solution, however, BCCP1 and BCCP2 had more than 10-fold higher affinity for BC than did BADC1. The pH-modulated shifts in BC preferences for BCCP and BADC partners suggest they contribute to light-dependent regulation of heteromeric ACCase. Using NMR spectroscopy, we found evidence for increased intrinsic disorder of the BADC and BCCP subunits at pH 7. We propose that this intrinsic disorder potentially promotes fast association with BC through a "fly-casting mechanism." We hypothesize that the pH effects on the BADC and BCCP subunits attenuate ACCase activity by night and enhance it by day. Consistent with this hypothesis, Arabidopsis badc1 badc3 mutant lines grown in a light–dark cycle synthesized more fatty acids in their seeds. In summary, our findings provide evidence that the BADC and BCCP subunits function as pH sensors required for light-dependent switching of heteromeric ACCase activity. In most plants (dicots and nongrass monocots), a multisubunit, heteromeric acetyl-CoA carboxylase (hetACCase) resides in plastids to generate the malonyl-CoA required for de novo fatty acid (FA) synthesis (1Ohlrogge J.B. Jaworski J.G. Regulation of fatty acid synthesis.Annu. Rev. Plant Physiol. Plant Mol. Biol. 1997; 48 (15012259): 109-13610.1146/annurev.arplant.48.1.109Crossref PubMed Scopus (526) Google Scholar, 2Sasaki Y. Nagano Y. Plant acetyl-CoA carboxylase: structure, biosynthesis, regulation, and gene manipulation for plant breeding.Biosci. Biotechnol. Biochem. 2004; 68 (15215578): 1175-118410.1271/bbb.68.1175Crossref PubMed Scopus (288) Google Scholar) (Fig. 1A). This first committed step of fatty acid synthesis controls carbon flow into the pathway and, thus, is highly regulated (1Ohlrogge J.B. Jaworski J.G. Regulation of fatty acid synthesis.Annu. Rev. Plant Physiol. Plant Mol. Biol. 1997; 48 (15012259): 109-13610.1146/annurev.arplant.48.1.109Crossref PubMed Scopus (526) Google Scholar, 2Sasaki Y. Nagano Y. Plant acetyl-CoA carboxylase: structure, biosynthesis, regulation, and gene manipulation for plant breeding.Biosci. Biotechnol. Biochem. 2004; 68 (15215578): 1175-118410.1271/bbb.68.1175Crossref PubMed Scopus (288) Google Scholar). Newly synthesized FAs are processed into glycerolipids for plant cell membranes or storage triacylglycerols (oils) within the seed or mesocarp. The varied markets for vegetable oils, including cooking and dietary oils, biodiesel, and chemical feedstocks, motivates ongoing interest in the engineering of oilseeds (3Thelen J.J. Ohlrogge J.B. Metabolic engineering of fatty acid biosynthesis in plants.Metab. Eng. 2002; 4 (11800570): 12-2110.1006/mben.2001.0204Crossref PubMed Scopus (328) Google Scholar, 4Cahoon E.B. Shockey J.M. Dietrich C.R. Gidda S.K. Mullen R.T. Dyer J.M. Engineering oilseeds for sustainable production of industrial and nutritional feedstocks: solving bottlenecks in fatty acid flux.Curr. Opin. Plant Biol. 2007; 10 (17434788): 236-24410.1016/j.pbi.2007.04.005Crossref PubMed Scopus (178) Google Scholar) and the regulatory properties of hetACCase (5Salie M.J. Thelen J.J. Regulation and structure of the heteromeric acetyl-CoA carboxylase.Biochim. Biophys. Acta. 2016; 1861: 1207-121310.1016/j.bbalip.2016.04.004Crossref PubMed Scopus (71) Google Scholar, 6Salie M.J. Zhang N. Lancikova V. Xu D. Thelen J.J. A family of negative regulators targets the committed step of de novo fatty acid biosynthesis.Plant Cell. 2016; 28 (27559025): 2312-232510.1105/tpc.16.00317Crossref PubMed Scopus (34) Google Scholar, 7Keereetaweep J. Liu H. Zhai Z. Shanklin J. Biotin attachment domain-containing proteins irreversibly inhibit acetyl CoA carboxylase.Plant Physiol. 2018; 177 (29626162): 208-21510.1104/pp.18.00216Crossref PubMed Scopus (32) Google Scholar). The formation of malonyl-CoA by ACCase was reported to be the only light-regulated step for de novo FA synthesis in plants (8Nakamura Y. Yamada M. The light-dependent step of de novo synthesis of long chain fatty acids in spinach chloroplasts.Plant Sci. Lett. 1979; 14: 291-29510.1016/S0304-4211(79)90173-1Crossref Scopus (18) Google Scholar). The activity of hetACCase in chloroplasts is increased in the light by the conditions created by the photosynthetic light reactions, namely, high ATP levels, a shift to pH 8 in the stroma, and a pool of reduced thioredoxin that enhances hetACCase by reducing disulfide bonds within the carboxyltransferase (CT) subunits (9Harwood J.L. Fatty acid metabolism.Annu. Rev. Plant Physiol. Plant Mol. Biol. 1988; 39: 101-13810.1146/annurev.pp.39.060188.000533Crossref Google Scholar, 10Sauer A. Heise K.-P. On the light dependence of fatty acid synthesis in spinach chloroplasts.Plant Physiol. 1983; 73 (16663156): 11-1510.1104/pp.73.1.11Crossref PubMed Google Scholar, 11Sauer A. Heise K.-P. Regulation of acetyl-coenzyme A carboxylase and acetyl-coenzyme A synthetase in spinach chloroplasts.Zeitschrift. Naturforsch. C. 1984; 39: 268-27510.1515/znc-1984-3-412Crossref Scopus (41) Google Scholar, 12Sasaki Y. Kozaki A. Hatano M. Link between light and fatty acid synthesis: thioredoxin-linked reductive activation of plastidic acetyl-CoA carboxylase.Proc. Natl. Acad. Sci. U S A. 1997; 94 (9380765): 11096-1110110.1073/pnas.94.20.11096Crossref PubMed Scopus (133) Google Scholar). The first half-reaction of hetACCase is catalyzed by the biotin carboxylase (BC) subcomplex, which uses bicarbonate and ATP to carboxylate the biotin cofactor of a biotin carboxyl carrier protein (BCCP; Fig. 1A) (2Sasaki Y. Nagano Y. Plant acetyl-CoA carboxylase: structure, biosynthesis, regulation, and gene manipulation for plant breeding.Biosci. Biotechnol. Biochem. 2004; 68 (15215578): 1175-118410.1271/bbb.68.1175Crossref PubMed Scopus (288) Google Scholar, 5Salie M.J. Thelen J.J. Regulation and structure of the heteromeric acetyl-CoA carboxylase.Biochim. Biophys. Acta. 2016; 1861: 1207-121310.1016/j.bbalip.2016.04.004Crossref PubMed Scopus (71) Google Scholar, 13Cronan Jr, J.E. Waldrop G.L. Multi-subunit acetyl-CoA carboxylases.Prog. Lipid Res. 2002; 41 (12121720): 407-43510.1016/S0163-7827(02)00007-3Crossref PubMed Scopus (330) Google Scholar, 14Reverdatto S. Beilinson V. Nielsen N.C. a multisubunit acetyl coenzyme A carboxylase from soybean.Plant Physiol. 1999; 119 (10069834): 961-97810.1104/pp.119.3.961Crossref PubMed Scopus (55) Google Scholar, 15Waldrop G.L. Holden H.M. Maurice M.S. The enzymes of biotin dependent CO2 metabolism: what structures reveal about their reaction mechanisms.Protein Sci. 2012; 21 (22969052): 1597-161910.1002/pro.2156Crossref PubMed Scopus (64) Google Scholar). The carboxyltransferase subcomplex of α-CT and β-CT subunits catalyzes the second half-reaction that transfers the carboxyl group from biotin to acetyl-CoA to form malonyl-CoA (2Sasaki Y. Nagano Y. Plant acetyl-CoA carboxylase: structure, biosynthesis, regulation, and gene manipulation for plant breeding.Biosci. Biotechnol. Biochem. 2004; 68 (15215578): 1175-118410.1271/bbb.68.1175Crossref PubMed Scopus (288) Google Scholar, 5Salie M.J. Thelen J.J. Regulation and structure of the heteromeric acetyl-CoA carboxylase.Biochim. Biophys. Acta. 2016; 1861: 1207-121310.1016/j.bbalip.2016.04.004Crossref PubMed Scopus (71) Google Scholar, 13Cronan Jr, J.E. Waldrop G.L. Multi-subunit acetyl-CoA carboxylases.Prog. Lipid Res. 2002; 41 (12121720): 407-43510.1016/S0163-7827(02)00007-3Crossref PubMed Scopus (330) Google Scholar, 14Reverdatto S. Beilinson V. Nielsen N.C. a multisubunit acetyl coenzyme A carboxylase from soybean.Plant Physiol. 1999; 119 (10069834): 961-97810.1104/pp.119.3.961Crossref PubMed Scopus (55) Google Scholar, 15Waldrop G.L. Holden H.M. Maurice M.S. The enzymes of biotin dependent CO2 metabolism: what structures reveal about their reaction mechanisms.Protein Sci. 2012; 21 (22969052): 1597-161910.1002/pro.2156Crossref PubMed Scopus (64) Google Scholar). The structural bases of the functions of the BC, BCCP, and CT subunits were reviewed (15Waldrop G.L. Holden H.M. Maurice M.S. The enzymes of biotin dependent CO2 metabolism: what structures reveal about their reaction mechanisms.Protein Sci. 2012; 21 (22969052): 1597-161910.1002/pro.2156Crossref PubMed Scopus (64) Google Scholar). An oligomeric complex of the folded region of BCCP and BC subunits from the ACCase from Escherichia coli was elucidated by crystallography (16Broussard T.C. Kobe M.J. Pakhomova S. Neau D.B. Price A.E. Champion T.S. Waldrop G.L. The three-dimensional structure of the biotin carboxylase-biotin carboxyl carrier protein complex of E. coli acetyl-CoA carboxylase.Structure. 2013; 21 (23499019): 650-65710.1016/j.str.2013.02.001Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). In plants, the malonyl-CoA pool in the plastid for de novo FA synthesis is distinct from the cytosolic malonyl-CoA pool used for FA elongation, which is generated by a separate homomeric ACCase (2Sasaki Y. Nagano Y. Plant acetyl-CoA carboxylase: structure, biosynthesis, regulation, and gene manipulation for plant breeding.Biosci. Biotechnol. Biochem. 2004; 68 (15215578): 1175-118410.1271/bbb.68.1175Crossref PubMed Scopus (288) Google Scholar). A BCCP-like biotin attachment domain-containing protein (At1g52670) was first described in 2009 (17Chen M. Mooney B.P. Hajduch M. Joshi T. Zhou M. Xu D. Thelen J.J. System analysis of an arabidopsis mutant altered in de novo fatty acid synthesis reveals diverse changes in seed composition and metabolism.Plant Physiol. 2009; 150 (19279196): 27-4110.1104/pp.108.134882Crossref PubMed Scopus (58) Google Scholar) and subsequently named biotin attachment domain-containing 2 (BADC2) (6Salie M.J. Zhang N. Lancikova V. Xu D. Thelen J.J. A family of negative regulators targets the committed step of de novo fatty acid biosynthesis.Plant Cell. 2016; 28 (27559025): 2312-232510.1105/tpc.16.00317Crossref PubMed Scopus (34) Google Scholar). The BADC1 and BADC3 members of this gene family were identified and were demonstrated to associate with the subcomplex containing BCCP1, BCCP2, and BC subunits of Arabidopsis (6Salie M.J. Zhang N. Lancikova V. Xu D. Thelen J.J. A family of negative regulators targets the committed step of de novo fatty acid biosynthesis.Plant Cell. 2016; 28 (27559025): 2312-232510.1105/tpc.16.00317Crossref PubMed Scopus (34) Google Scholar). The mature BADC subunits at 22 to 24 kDa in mass (6Salie M.J. Zhang N. Lancikova V. Xu D. Thelen J.J. A family of negative regulators targets the committed step of de novo fatty acid biosynthesis.Plant Cell. 2016; 28 (27559025): 2312-232510.1105/tpc.16.00317Crossref PubMed Scopus (34) Google Scholar) are slightly larger than the BCCP subunits of 18 to 21 kDa and less than half the mass of the BC and CT subunits (Table S1). Although BADC subunits retain the sequence similarity of the biotin/lipoyl attachment domain, BADC1, BADC2, and BADC3 specifically lack the canonical four-amino-acid sequence motif for biotinylation in BCCPs and were experimentally shown to lack a covalently bound biotin prosthetic group (6Salie M.J. Zhang N. Lancikova V. Xu D. Thelen J.J. A family of negative regulators targets the committed step of de novo fatty acid biosynthesis.Plant Cell. 2016; 28 (27559025): 2312-232510.1105/tpc.16.00317Crossref PubMed Scopus (34) Google Scholar). The addition of excess BADC1, BADC2, or BADC3 to extracts from developing Arabidopsis siliques inhibited hetACCase activity by about 37%, 33%, and 24%, respectively (6Salie M.J. Zhang N. Lancikova V. Xu D. Thelen J.J. A family of negative regulators targets the committed step of de novo fatty acid biosynthesis.Plant Cell. 2016; 28 (27559025): 2312-232510.1105/tpc.16.00317Crossref PubMed Scopus (34) Google Scholar). The addition of BADC1 to leaf extracts inhibited ACCase activity up to ∼27% in a concentration-dependent fashion (6Salie M.J. Zhang N. Lancikova V. Xu D. Thelen J.J. A family of negative regulators targets the committed step of de novo fatty acid biosynthesis.Plant Cell. 2016; 28 (27559025): 2312-232510.1105/tpc.16.00317Crossref PubMed Scopus (34) Google Scholar). BADC subunits' lack of biotinylation and partial inhibition of hetACCase support the hypothesis that BADC subunits inhibit by competing with BCCP subunits for access to BC (6Salie M.J. Zhang N. Lancikova V. Xu D. Thelen J.J. A family of negative regulators targets the committed step of de novo fatty acid biosynthesis.Plant Cell. 2016; 28 (27559025): 2312-232510.1105/tpc.16.00317Crossref PubMed Scopus (34) Google Scholar). Partial RNAi silencing of BADC1 in Arabidopsis seeds increased seed oil content (6Salie M.J. Zhang N. Lancikova V. Xu D. Thelen J.J. A family of negative regulators targets the committed step of de novo fatty acid biosynthesis.Plant Cell. 2016; 28 (27559025): 2312-232510.1105/tpc.16.00317Crossref PubMed Scopus (34) Google Scholar). Comparison of T-DNA knockout badc1 badc3 Arabidopsis with the WT found ACCase activity to be higher and seeds to contain 33% more triacylglycerols in badc1 badc3 lines (7Keereetaweep J. Liu H. Zhai Z. Shanklin J. Biotin attachment domain-containing proteins irreversibly inhibit acetyl CoA carboxylase.Plant Physiol. 2018; 177 (29626162): 208-21510.1104/pp.18.00216Crossref PubMed Scopus (32) Google Scholar). This suggests that the relief of inhibition by BADC1 and BADC3 results in higher hetACCase activity and carbon flux through de novo FA synthesis (7Keereetaweep J. Liu H. Zhai Z. Shanklin J. Biotin attachment domain-containing proteins irreversibly inhibit acetyl CoA carboxylase.Plant Physiol. 2018; 177 (29626162): 208-21510.1104/pp.18.00216Crossref PubMed Scopus (32) Google Scholar). Potential mechanisms for BADC competition with BCCP subunits remain unclear. Results in Arabidopsis suggest that BCCP and BADC are capable of binding to BC somewhat independently (6Salie M.J. Zhang N. Lancikova V. Xu D. Thelen J.J. A family of negative regulators targets the committed step of de novo fatty acid biosynthesis.Plant Cell. 2016; 28 (27559025): 2312-232510.1105/tpc.16.00317Crossref PubMed Scopus (34) Google Scholar, 7Keereetaweep J. Liu H. Zhai Z. Shanklin J. Biotin attachment domain-containing proteins irreversibly inhibit acetyl CoA carboxylase.Plant Physiol. 2018; 177 (29626162): 208-21510.1104/pp.18.00216Crossref PubMed Scopus (32) Google Scholar). However, this has yet to be demonstrated in quantitative fashion. More roles for the BADC subunits in Arabidopsis have emerged recently. An important but unidentified role in seed development is indicated by the inability to obtain badc2 badc3 mutant seeds as well as by the smaller seeds, roots, and rosettes of badc1 badc2 mutants (7Keereetaweep J. Liu H. Zhai Z. Shanklin J. Biotin attachment domain-containing proteins irreversibly inhibit acetyl CoA carboxylase.Plant Physiol. 2018; 177 (29626162): 208-21510.1104/pp.18.00216Crossref PubMed Scopus (32) Google Scholar, 18Shivaiah K.K. Ding G. Upton B. Nikolau B.J. Non-catalytic subunits facilitate quaternary organization of plastidic acetyl-CoA carboxylase.Plant Physiol. 2020; 182 (31792149): 756-77510.1104/pp.19.01246Crossref PubMed Scopus (10) Google Scholar). The BADC1 and BADC3 subunits were implicated in long-term response to oversupply of FAs by feedback inhibition (7Keereetaweep J. Liu H. Zhai Z. Shanklin J. Biotin attachment domain-containing proteins irreversibly inhibit acetyl CoA carboxylase.Plant Physiol. 2018; 177 (29626162): 208-21510.1104/pp.18.00216Crossref PubMed Scopus (32) Google Scholar). When Arabidopsis BC was coexpressed in E. coli with Arabidopsis BCCP1 or BCCP2, a high level of coexpression with a BADC subunit boosted BCCP recruitment of BC by more than 10-fold to nearly stoichiometric ratios in each of the BCCP-BADC-BC subcomplexes (18Shivaiah K.K. Ding G. Upton B. Nikolau B.J. Non-catalytic subunits facilitate quaternary organization of plastidic acetyl-CoA carboxylase.Plant Physiol. 2020; 182 (31792149): 756-77510.1104/pp.19.01246Crossref PubMed Scopus (10) Google Scholar). The in vitro catalytic efficiencies and maximum catalytic velocities of the BCCP-BADC-BC subcomplexes reconstituted in E. coli were similarly elevated over those of their BCCP-BC counterparts, with BADC2 and BADC3 providing the biggest enhancement (18Shivaiah K.K. Ding G. Upton B. Nikolau B.J. Non-catalytic subunits facilitate quaternary organization of plastidic acetyl-CoA carboxylase.Plant Physiol. 2020; 182 (31792149): 756-77510.1104/pp.19.01246Crossref PubMed Scopus (10) Google Scholar). Assembly and stoichiometry of macromolecular assemblies, such as hetACCase, is complex and often difficult to ascertain, given the need for absolute quantitation in vivo. The MS approach known as absolute quantitation of multiplexed reaction monitoring (AQUA-MRM) provided the molar protein quantities of hetACCase subunits in Arabidopsis siliques when the seeds are actively filling with oil. The molar quantities of the subunits rank in the order β-CT > BC > BADC1 > BADC3 ≈ BCCP1 > α-CT ≈ BCCP2 > BADC2 (19Wilson R.S. Thelen J.J. In vivo quantitative monitoring of subunit stoichiometry for metabolic complexes.J. Proteome Res. 2018; 17 (29582652): 1773-178310.1021/acs.jproteome.7b00756Crossref PubMed Scopus (5) Google Scholar). During oilseed development, BADC1 and BCCP1, being the most highly expressed of the nonbiotinylated and biotinylated smaller subunits, respectively, merit study of their behaviors. Illumination and the photosynthetic light reactions pump protons out of the chloroplast stroma, raising its pH from 7 to 8 (20Werdan K. Heldt H.W. Milovancev M. The role of pH in the regulation of carbon fixation in the chloroplast stroma. Studies on CO2 fixation in the light and dark.Biochim. Biophys. Acta. 1975; 396: 276-29210.1016/0005-2728(75)90041-9Crossref PubMed Scopus (260) Google Scholar). We focus here on investigation of the hypothesis that the light-dependent swings in pH of the plastid stroma regulate association of BCCP and BADC subunits with the biotin carboxylase subunit, which forms and alters the subcomplexes catalyzing carboxylation of biotinylated BCCP1 or BCCP2. We considered the possibility that the physiological pH range affects the BCCP and BADC subunits in terms of (i) their affinities for BC and (ii) their structural scaffolds in solution. The BADC and BCCP subunits associate with BC directly and independently with KD of <10 μm under all solution conditions evaluated. The affinities are greater at pH 7 than at pH 8. The intrinsic disorder detected by NMR in pH titrations of BCCP1, BCCP2, and BADC1 is also increased at pH 7. At pH 7, enhanced BADC occupation of BC predicts that biotin carboxylation should be slower at pH 7. At pH 8, enhanced BCCP subunit occupation of BC predicts enhanced biotin-mediated transfer of carboxyl groups between the active sites of BC and CT subunits. Thus, the BCCP and BADC subunits sense pH and respond in a differential manner that may help to (i) inhibit the biotin carboxylase half-reaction in the neutral pH of dark and low-light conditions and (ii) accelerate biotin carboxylation in pH 8 light adaptation and thereby enhance the light-dependent regulation of hetACCase in plastids. Consistent with these predictions and with the abundance of the BADC1 and BADC3 subunits, the seeds produced by Arabidopsis badc1 badc3 lines accumulate more total lipid when the lines are grown with a daily light/dark cycle. The ∼90 residues at the C-terminal end of the BCCP1, BCCP2, BADC1, BADC2, and BADC3 sequences from A. thaliana exhibit homology to structures in the Protein Data Bank. Homology models, using crystallographic coordinates of BCCP from E. coli, contain two anti-parallel β-sheets in each of these subunits of acetyl-CoA carboxylase (Fig. 1, B and C). Homology models of this region from each BADC subunit comprise eight β-strands, seven strands in BCCP2, and six strands in BCCP1, plus a potential but ambiguous seventh strand at its C terminus. At the N-terminal end of this region, the BADC subunits add a β-strand to one β-sheet (Fig. S1 and Fig. 1C, purple), while the corresponding segment of the BCCP1 and BCCP2 models is similar but diverges (Fig. S2B). The exposed "thumb" loop is much longer and enriched in basic residues in the models of the BADC subunits (Fig. 1C and Fig. S2B). The lysine in the β-hairpin loop that becomes biotinylated in BCCP subunits is replaced by glycine in the BADC subunits. Apart from localized differences at the termini and thumb, the backbone coordinates of the homology models are highly similar (Fig. S2). Due to the small size of the folded regions, we examined the sequences of the mature BCCP1, BCCP2, BADC1, BADC2, and BADC3 subunits from A. thaliana for disorder. The unusual structural, sequence, and functional properties of intrinsic disorder were reviewed (21Uversky V.N. Dunker A.K. Understanding protein non-folding.Biochim. Biophys. Acta. 2010; 1804 (20117254): 1231-126410.1016/j.bbapap.2010.01.017Crossref PubMed Scopus (932) Google Scholar). Intrinsically disordered regions (IDRs) exhibit elevated mean charge, low mean hydrophobicity, depletion of bulky hydrophobic side chains, and enrichment in disorder-promoting Pro, Glu, Ser, Lys, and Gln residues (21Uversky V.N. Dunker A.K. Understanding protein non-folding.Biochim. Biophys. Acta. 2010; 1804 (20117254): 1231-126410.1016/j.bbapap.2010.01.017Crossref PubMed Scopus (932) Google Scholar, 22Uversky V.N. Gillespie J.R. Fink A.L. Why are "natively unfolded" proteins unstructured under physiologic conditions?.Proteins Struct. Funct. 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MetaDisorder: a meta-server for the prediction of intrinsic disorder in proteins.BMC Bioinformatics. 2012; 13 (22624656): 11110.1186/1471-2105-13-111Crossref PubMed Scopus (256) Google Scholar). These predictions suggest that a large IDR lies in the middle of each sequence, preceding the structured domain (Fig. 1, D–H). The predictions also feature an N-terminal IDR followed by an ordered region of 20 to 30 residues. The thumb region of BADC1 is predicted to be an IDR (Fig. 1, D–H). MetaDisorderMD2 suggests disorder for 59% of the residues of BCCP1, 56% of BCCP2, 62% of BADC1, 55% of BADC2, and 51% of BADC3. The BCCP subunits average a proline content of 14%, while the BADC subunits average 7% proline. The proline residues, which promote extended structure, are concentrated in the large central IDR predicted as well as in the thumb (Fig. S1). Glu and Asp residues comprise 11 to 12% of the amino acids of the BCCP and BADC subunits. The enrichment in negative charge provides electrostatic repulsion opposed to hydrophobic collapse (21Uversky V.N. Dunker A.K. Understanding protein non-folding.Biochim. Biophys. Acta. 2010; 1804 (20117254): 1231-126410.1016/j.bbapap.2010.01.017Crossref PubMed Scopus (932) Google Scholar). The question of the pH dependence of the BADC and BCCP subunits' affinities for the BC subunit arose from the pH dependence of ACCase activity (10Sauer A. Heise K.-P. On the light dependence of fatty acid synthesis in spinach chloroplasts.Plant Physiol. 1983; 73 (16663156): 11-1510.1104/pp.73.1.11Crossref PubMed Google Scholar, 12Sasaki Y. Kozaki A. Hatano M. Link between light and fatty acid synthesis: thioredoxin-linked reductive activation of plastidic acetyl-CoA carboxylase.Proc. Natl. Acad. Sci. U S A. 1997; 94 (9380765): 11096-1110110.1073/pnas.94.20.11096Crossref PubMed Scopus (133) Google Scholar) and from the sensitivity of the folds of BCCP1, BCCP2, and BADC1 to pH (see below). 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The changes in thermal molecular movement are detected by fluorescence, which is sensitive to modest amounts of the binding partners. We compared pH conditions representative of daylight (pH 8) and dark (pH 7). Good's buffers have been popular for assays of hetACCase activity (6Salie M.J. Zhang N. Lancikova V. Xu D. Thelen J.J. A family of negative regulators targets the committed step of de novo fatty acid biosynthesis.Plant Cell. 2016; 28 (27559025): 2312-232510.1105/tpc.16.00317Crossref PubMed Scopus (34) Google Scholar, 18Shivaiah K.K. Ding G. Upton B. Nikolau B.J. Non-catalytic subunits facilitate quaternary organization of plastidic acetyl-CoA carboxylase.Plant Physiol. 2020; 182 (31792149): 756-77510.1104/pp.19.01246Crossref PubMed Scopus (10) Google Scholar). However, the 1.2 to 2 mm phosphate (Pi) present in the stroma of chloroplasts fully active in photophosphorylation (28Robinson S.P. Giersch C. 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Influence of potassium deficiency on photosynthesis, chlorophyll content, and chloroplast ultrastructure of cotton plants.Photosynthetica. 2001; 39: 103-10910.1023/A:1012404204910Crossref Scopus (318) Google Scholar). Titrations detected by MST suggest that the affinities for BC strongly depend on the ionic milieu (Fig. 2). Comparisons of the Gibbs free energies of association with BC clarify the effects of pH and choice of buffer (Fig. 3).Figure 3Dependence of affinities for BC on pH, buffer, cation, and partner subunit. The Gibbs free ene
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