Porphyrins Promote the Association of GENOMES UNCOUPLED 4 and a Mg-chelatase Subunit with Chloroplast Membranes

Abstract

In plants, chlorophylls and other tetrapyrroles are synthesized from a branched pathway that is located within chloroplasts. GUN4 (GENOMES UNCOUPLED 4) stimulates chlorophyll biosynthesis by activating Mg-chelatase, the enzyme that commits porphyrins to the chlorophyll branch. GUN4 stimulates Mg-chelatase by a mechanism that involves binding the ChlH subunit of Mg-chelatase, as well as a substrate (protoporphyrin IX) and product (Mg-protoporphyrin IX) of Mg-chelatase. We chose to test whether GUN4 might also affect interactions between Mg-chelatase and chloroplast membranes, the site of chlorophyll biosynthesis. To test this idea, we induced chlorophyll precursor levels in purified pea chloroplasts by feeding these chloroplasts with 5-aminolevulinic acid, determined the relative levels of GUN4 and Mg-chelatase subunits in soluble and membrane-containing fractions derived from these chloroplasts, and quantitated Mg-chelatase activity in membranes isolated from these chloroplasts. We also monitored GUN4 levels in the soluble and membrane-containing fractions derived from chloroplasts fed with various porphyrins. Our results indicate that 5-aminolevulinic acid feeding stimulates Mg-chelatase activity in chloroplast membranes and that the porphyrin-bound forms of GUN4 and possibly ChlH associate most stably with chloroplast membranes. These findings are consistent with GUN4 stimulating chlorophyll biosynthesis not only by activating Mg-chelatase but also by promoting interactions between ChlH and chloroplast membranes.

Porphyrin Binding to Gun4 Protein, Facilitated by a Flexible Loop, Controls Metabolite Flow through the Chlorophyll Biosynthetic Pathway

“…It also appears that Gun4 proteins from virtually all sources stimulate MgCh activity via an interaction with the ChlH subunit and bind porphyrins with the highest affinity to MgP (14,16,17). The essential role of porphyrin binding for Gun4 function can be suggested from amino acid alignments (13) because residues in the proposed MgP binding site (␣6/␣7 loop residues, Arg-214, and Asn-211) are conserved through evolution from cyanobacteria to plants.…”

“…According to Ref. 16, the homologue Arg-214 residue in Arabidopsis Gun4 shows a defect in MgP binding, although it is not essential, unlike in the Synechocystis counterpart.…”

“…The Role of Gun4 in Regulation of Tetrapyrrole BiosynthesisAlthough Gun4 proteins bind a broad spectrum of porphyrins including heme b or pheophorbide (16) (13). These results suggest that Gun4 is implicated in release of the product (MgP) from ChlH.…”

“…Gun4 was shown to bind various porphyrins, exhibiting the highest affinity to MgP, the product of MgCh enzyme, and a weaker affinity to P IX (13,16). There is evidence that Gun4 physically interacts with ChlH (11,14,17), which is the porphyrin-binding MgCh subunit that presumably also contains the active site for chelation (18).…”

TNO1 Is Involved in Salt Tolerance and Vacuolar Trafficking in Arabidopsis

“…ICF was collected in the bottom of the tube, and ICF-depleted leaves after centrifugation were ground in 50 mM sodium citrate for the control. ICF and ICF-depleted protein extract were analyzed by immunoblotting using anti-AALP (1:1,000; Ahmed et al, 2000), anti-RD21 (1:1,000; Hayashi et al, 2001), anti-invertase (1:3,000; Rojo et al, 2003), anti-ChlI (1:2,000; Adhikari et al, 2009), and anti-PEX11d (1:1,000; Orth et al, 2007).…”