An Arabidopsis GluTR Binding Protein Mediates Spatial Separation of 5-Aminolevulinic Acid Synthesis in Chloroplasts

Abstract

5-Aminolevulinic acid (ALA) is the universal precursor for tetrapyrrole biosynthesis and is synthesized in plants in three enzymatic steps: ligation of glutamate (Glu) to tRNAGlu by glutamyl-tRNA synthetase, reduction of activated Glu to Glu-1-semialdehyde by glutamyl-tRNA reductase (GluTR), and transamination to ALA by Glu 1-semialdehyde aminotransferase. ALA formation controls the metabolic flow into the tetrapyrrole biosynthetic pathway. GluTR is proposed to be the key regulatory enzyme that is tightly controlled at transcriptional and posttranslational levels. We identified a GluTR binding protein (GluTRBP; previously called PROTON GRADIENT REGULATION7) that is localized in chloroplasts and part of a 300-kD protein complex in the thylakoid membrane. Although the protein does not modulate activity of ALA synthesis, the knockout of GluTRBP is lethal in Arabidopsis thaliana, whereas mutants expressing reduced levels of GluTRBP contain less heme. GluTRBP expression correlates with a function in heme biosynthesis. It is postulated that GluTRBP contributes to subcompartmentalized ALA biosynthesis by maintaining a portion of GluTR at the plastid membrane that funnels ALA into the heme biosynthetic pathway. These results regarding GluTRBP support a model of plant ALA synthesis that is organized in two separate ALA pools in the chloroplast to provide appropriate substrate amounts for balanced synthesis of heme and chlorophyll.

Crystal structure of Arabidopsis glutamyl-tRNA reductase in complex with its stimulator protein

“…1C). This explains the loss of GluTR-binding ability of the defective pgr7 mutant, whose Cterminal 84 residues are truncated (8,9).…”

“…GluBP was proposed to be involved in heme-mediated metabolism (8). To examine the effect of heme on GluBP activity, we measured ALA formation in the presence of GluBP and heme by using the coupled enzyme assay described earlier.…”

“…The NADPH-dependent reduction of glutamyl-tRNA catalyzed by GluTR is rate-limiting for ALA formation, and GluTR is the hub of a multilayered regulation for all of the tetrapyrrole biosynthetic pathway in photosynthetic organisms at the transcriptional and posttranslational levels (3,4). Fine tuning of GluTR activity in chloroplasts is achieved by a combination of three known factors: (i) the feedback inhibitor heme (5), (ii) the negative regulator of chlorophyll biosynthesis FLU (6,7), and (iii) the spatial organizer GluTR binding protein (GluBP) (8). GluBP was previously identified as proton gradient regulation 7 (PGR7) because its truncated mutant is defective in photosynthetic electron transport (9).…”

“…GluBP was previously identified as proton gradient regulation 7 (PGR7) because its truncated mutant is defective in photosynthetic electron transport (9). This pgr7 phenotype presumably results from a deficiency in heme for the cytochrome b 6 f complex in the photosynthetic electron transport chain (8). It has been suggested that GluBP ensures heme biosynthesis during dark periods by preventing GluTR from binding to FLU, and thus distinguishes chlorophyll and heme-synthesizing pathways (10).…”

“…The fact that GluBP is found in all chloroplast-containing organisms indicates that GluBP is a common regulator of GluTR (8). Despite the insight gained from these studies, the molecular basis underlying GluTR regulation by GluBP remains to be explored.…”