Molecular biology and biochemical assessment of acetolactate synthase (ALS)-mediated protein-protein interactions in arabidopsis thaliana (L.) heynh
Pokatong, Wilbur Donald Raymond
The biosynthesis of the branched-chain amino acids (BCAAs) isoleucine, leucine and valine in microorganisms and plants is accomplished by a series of enzymatic reactions, with acetolactate synthase (ALS) common to both pathways. This study was aimed at molecular and cell biological analyses of protein-protein interactions involving the ALS catalytic subunit and ALS-interacting proteins (AIPx) in 'Arabidopsis thaliana', and characterization of genes encoding AlPx. The study was conducted primarily to biochemically validate the persistence of 'in vivo' protein-protein interactions between the ALS and AIP1p and/or AIP3p which were selected on the basis of their structural similarity to bacterial ALS regulatory subunits. Characterization of ' AIP1' and 'AIP3' genomic DNAs, cDNAs and their deduced polypeptides revealed both similarities and differences. The similarity between AIP1p/AIP3p and their prokaryotic counterparts suggests that domains of prokaryotic origin may serve as the common ancestral core for bothpolypeptides. The N- or C-terminal fusion of 'His6' tags to the At'ALS-csr'1-1 transgene for generation of transgenic ' A. thaliana' lines enabled the capture and purification of ' His6'-tagged-At'ALS-csr1'-1 using an IMAC system, and the identification of coenriched proteins that interacted with ALS. Specifically, IMAC Co+2 batch chromatography was used successfully to determine protein-protein interactions between ALS and AIP1p and/or AIP3p. The results constitute direct biochemical evidence in support of the existence of ALS:AIPx protein-protein interactions in plant extracts. Unlike the bacterial ALS with a tetrameric (α2β 2) structure, the plant ALS appears to exhibit a different conformation consisting of αββ' (a single catalytic subunit plus a heterodimer of two regulatory subunits), α2ββ ', αβ, αβ', α 2β and/or α2β' heteromers. The postulate that AIP1p (β) and AIP3p (β') are ALS regulatory subunits was supported by the study of transgenic lines which over-expressed 'AIP1' and 'AIP3'. The introduction of 'AIP1' and 'AIP3' transgenes disrupted the regulation of BCAA biosynthesis with a total net increase of free amino acids, regardless of whether the endo- and trans-genes were down- or up-regulated via ectopic expression or co-suppression. These results suggest that the disruption of regulation of BCAA metabolism may be perceived as a direct consequence of AIP1p and AIP3p being regulatory subunits that mediate negative allosteric regulation of BCAA biosynthesis.