Chloroplast acetyl-CoA carboxylase activity is 2-oxoglutarate-regulated by interaction of PII with the biotin carboxyl carrier subunit

Abstract

The PII protein is a signal integrator involved in the regulation of nitrogen metabolism in bacteria and plants. Upon sensing of cellular carbon and energy availability, PII conveys the signal by interacting with target proteins, thereby modulating their biological activity. Plant PII is located to plastids; therefore, to identify new PII target proteins, PII-affinity chromatography of soluble extracts from Arabidopsis leaf chloroplasts was performed. Several proteins were retained only when Mg-ATP was present in the binding medium and they were specifically released from the resin by application of a 2-oxoglutarate-containing elution buffer. Mass spectroscopy of SDS/PAGE-resolved protein bands identified the biotin carboxyl carrier protein subunits of the plastidial acetyl-CoA carboxylase (ACCase) and three other proteins containing a similar biotin/lipoyl-binding motif as putative PII targets. ACCase is a key enzyme initiating the synthesis of fatty acids in plastids. In in vitro reconstituted assays supplemented with exogenous ATP, recombinant Arabidopsis PII inhibited chloroplastic ACCase activity, and this was completely reversed in the presence of 2-oxoglutarate, pyruvate, or oxaloacetate. The inhibitory effect was PII-dose-dependent and appeared to be PII-specific because ACCase activity was not altered in the presence of other tested proteins. PII decreased the V(max) of the ACCase reaction without altering the K(m) for acetyl-CoA. These data show that PII function has evolved between bacterial and plant systems to control the carbon metabolism pathway of fatty acid synthesis in plastids.

Fig. 1.

Identification of PII-interacting proteins in leaf chloroplast extracts. Soluble proteins from purified chloroplasts were loaded onto a PII-affinity resin. Unbound proteins were removed by washing with 50 mL of buffer A. Lane W shows the absence of protein at the end of the wash process. Bound proteins were eluted with 5 mM 2-OG (8 × 0.5 mL fractions corresponding to lanes E1–E8). In control experiments omitting Mg-ATP or with NAGK fixed to the affinity resin, these proteins were not bound. (A) Eluted proteins were TCA-precipitated and subjected to SDS/PAGE (12% acrylamide) and silver staining. (B) Bound proteins were eluted with 0.5 mM 2-OG and, after SDS/PAGE, submitted to Western blotting with BCCP antibodies. (C) In a reverse approach, soluble biotin-containing proteins from intact chloroplasts were fixed to immobilized avidin and their interaction with recombinant PII was tested in the presence of Mg-ATP. PII was eluted with 5 mM 2-OG and fractions were evaluated for PII content by Western blotting using PII antibodies. For explanation of a and b, see Table 1 and Fig. S1.

Fig. 2.

Characterization of chloroplastic ACCase activity. The ACCase activity was assayed by using a total chloroplast extract from the Arabidopsis PIIV1 mutant in the presence, or absence, of 0.5 mM acetyl-CoA. Avidin (0.2 units), a general ACCase inhibitor, and fenoxaprop (100 mM), a specific inhibitor of the cytosolic ACCase, were tested to validate the measured chloroplastic ACCase activity. The data represent the average value (±SE) from 23 (+/−acetyl-CoA), 5 (avidin), and 4 (fenoxaprop) experiments carried out on different chloroplast preparations.

Fig. 3.

PII inhibits chloroplastic ACCase activity. (A) ACCase activity was assayed in the absence (−PII) and presence of 0.36 μM PII (+PII). The specificity of PII inhibition was tested by addition of recombinant NAGK (1.51 μM) or CPO (1.35 μM) (plastidial proteins), or BSA (0.72 μM), a nonplant protein. (B) The response curve of ACCase activity to increasing concentrations of trimeric PII showed that the inhibition was PII-dose-dependent. The data represent the average value (±SE) from 5 (PII, NAGK, CPO, BSA) and 4 (PII-dose-dependence) experiments carried out on different chloroplast preparations.

Fig. 4.

Effect of PII on the kinetic parameters of the ACCase reaction as determined from acetyl-CoA saturation curves. ACCase activity was assayed in the absence (open circles) and the presence (filled circles) of 0.36 μM PII. Kinetic parameters were calculated from Lineweaver–Burke analyses. PII did not alter the K_m for acetyl-CoA when the V_max was reduced by 50%. The data represent the average values (±SEs) from three experiments carried out on different chloroplast preparations.

Fig. 5.

Effect of metabolites on PII-inhibited chloroplastic ACCase activity. (A) ACCase activity was assayed in the presence of 0.36 μM PII and selected metabolites (5 mM). In control assays omitting PII, the metabolites did not alter the ACCase activity. (B) Recovery of the ACCase activity by increasing concentrations of 2-OG. The data in A represent the average values (±SEs) from nine (+/−PII), eight (2-OG), and three (other metabolites) experiments carried out on different chloroplast preparations. The data in B represent the average values (±SEs) from three experiments carried out on different chloroplast preparations.