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In Vivo Quantitative Monitoring of Subunit Stoichiometry for Metabolic Complexes

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posted on 2018-03-27, 00:00 authored by Rashaun S. Wilson, Jay J. Thelen
Metabolic pathways often employ assemblies of individual enzymes to facilitate substrate channeling to improve thermodynamic efficiency and confer pathway directionality. It is often assumed that subunits to multienzyme complexes are coregulated and accumulate at fixed levels in vivo, reflecting complex stoichiometry. Such assumptions can be experimentally tested using modern tandem mass spectrometry, and herein we describe such an approach applied toward an important metabolic complex. The committed step of de novo fatty acid synthesis in the plastids of most plants is catalyzed by the multienzyme, heteromeric acetyl-CoA carboxylase (hetACCase). This complex is composed of four catalytic subunits and a recently discovered regulatory subunit resembling the biotin carboxyl carrier protein but lacking the biotinylation motif necessary for activity. To better understand this novel form of regulation, a targeted tandem mass-spectrometry-based assay was developed to absolutely quantify all subunits to the Arabidopsis thaliana hetACCase. After validation against pure, recombinant protein, this multiplexed assay was used to quantify hetACCase subunits in siliques in various stages of development. Quantitation provided a developmental profile of hetACCase and BADC protein expression that supports a recently proposed regulatory mechanism for hetACCase and demonstrates a promising application of targeted mass spectrometry for in vivo analysis of protein complexes.