In vivo dissection of functional domains of plant glutamyl-tRNA reductase, the regulatory key enzyme of tetrapyrrole biosynthesis

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Glutamyl-tRNA reductase (GluTR), the first committed enzyme of plant tetrapyrrole biosynthesis and considered to be the rate limiting enzyme for the supply of 5-aminolevulinic acid (ALA), is encoded in dicotyledonous plants by two HEMA genes. Posttranslational mechanisms control GluTR activity and interactions have been described with heme, FLU and GBP, a newly identified GluTR-binding protein with potential to recruit enzymes for ALA synthesis dedicated to heme biosynthesis. Previous in vitro and yeast analyses revealed domains for heme- and FLU-binding of plant GluTR. It is the aim of the proposal to dissect the functional domains of GluTR interacting with regulatory factors in Arabidopsis in vivo. Modified GluTRs are expressed in the hemA1 mutant and analysed for the degree of regulatory complementation of ALA and tetrapyrrole biosynthesis. It is proposed that the two GluTR isoforms differentially interact with their effectors, thereby forming different protein complexes for ALA synthesis which facilitate specific contributions to chlorophyll or heme biosynthesis. Structural features determining functional differences will be dissected by domain swapping experiments, while biochemical and genetic approaches aim at the characterization of ALA-synthesizing protein complexes containing GluTR1 and GluTR2, respectively. Evolution of posttranslational regulation in plant GluTR is addressed by starting interaction studies engaging further dicotyledonous plant species.