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-dosedependent 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.Arabidopsis thaliana | biotin carboxyl carrier protein | PII protein | organic acids | fatty acid metabolism

Feedback regulation of plastidic acetyl-CoA carboxylase by 18:1-acyl carrier protein in Brassica napus

“…5A). ACCase can be inactivated by dephosphorylation (18) or by interaction with PII protein in a 2-oxoglutarate-dependent manner (19). ACCase assays following phosphatase treatment of crude extracts or in the presence of 5 mM 2-oxoglutarate revealed no differences between control and Tween 80-fed cultures (Fig.…”

A Family of Negative Regulators Targets the Committed Step of de Novo Fatty Acid Biosynthesis

“…When mentioned in previous reports, these proteins were labeled as BCCP-like due to their sequence similarity to BCCP. The BADCs were observed to coelute with Arabidopsis hetACCase during size-exclusion chromatography (Olinares et al, 2010) and were also detected in pull-down experiments with the 2-oxoglutarate binding protein PII along with both BCCP isoforms (Feria Bourrellier et al, 2010). In both cases, a direct interaction between ACCase catalytic subunits and the BADCs was not established, and a functional role was neither determined nor proposed.…”

“…HetACCase activity is also suppressed by feedback inhibition through increased levels of acyl-acyl carrier protein (Davis and Cronan, 2001;Andre et al, 2012). Recently, an interaction between hetACCase and a 2-oxoglutarate binding protein PII was shown to reduce hetACCase activity (Feria Bourrellier et al, 2010). Recombinant PII protein was shown to pull down BCCP from chloroplast lysates in the absence of 2-oxoglutarate, likely through a direct interaction with the biotin cofactor.…”

Mechanism of 2-oxoglutarate signaling by the Synechococcus elongatus P II signal transduction protein

“…Moreover, P II affects gene expression in cyanobacteria through binding to the transcriptional coactivator of NtcA, PipX (10). In plants, acetyl-CoA carboxylase was recently shown to be regulated by P II , providing an additional link between carbon and nitrogen regulation (11). Although these P II targets share no common structural element, interaction with P II is inhibited by 2-OG.…”