Molecular biology and biochemical assessment of acetolactate synthase (ALS)-mediated protein-protein interactions in arabidopsis thaliana (L.) heynh
Date
2001-01-01
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Doctoral
Abstract
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 AtALS-csr1-1 transgene for generation of transgenic 'A. thaliana' lines enabled the capture and purification of His₆-tagged-AtALS-csr1-1 using an IMAC system, and the identification of coenriched proteins that interacted with ALS. Specifically, IMAC Co²⁺ 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 (α2β2) 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', α2β 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.
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Degree
Doctor of Philosophy (Ph.D.)
Department
Applied Microbiology and Food Science
Program
Applied Microbiology and Food Science