Phosphorylation of GENOMES UNCOUPLED 4 Alters Stimulation of Mg Chelatase Activity in Angiosperms

Abstract

) is a positive regulator of light-dependent chlorophyll biosynthesis. GUN4 activates Mg chelatase (MgCh) that catalyzes the insertion of an Mg 2+ ion into protoporphyrin IX. We show that Arabidopsis (Arabidopsis thaliana) GUN4 is phosphorylated at Ser 264 (S264), the penultimate amino acid residue at the C terminus. While GUN4 is preferentially phosphorylated in darkness, phosphorylation is reduced upon accumulation of Mg porphyrins. Expression of a phosphomimicking GUN4(S264D) results in an incomplete complementation of the white gun4-2 null mutant and a chlorotic phenotype comparable to gun4 knockdown mutants. Phosphorylated GUN4 has a reduced stimulatory effect on MgCh in vitro and in vivo but retains its protein stability and tetrapyrrole binding capacity. Analysis of GUN4 found in oxygenic photosynthetic organisms reveals the evolution of a C-terminal extension, which harbors the phosphorylation site of GUN4 expressed in angiosperms. Homologs of GUN4 from Synechocystis and Chlamydomonas lack the conserved phosphorylation site found in a C-terminal extension of angiosperm GUN4. Biochemical studies proved the importance of the C-terminal extension for MgCh stimulation and inactivation of GUN4 by phosphorylation in angiosperms. An additional mechanism regulating MgCh activity is proposed. In conjunction with the dark repression of 5-aminolevulinic acid synthesis, GUN4 phosphorylation minimizes the flow of intermediates into the Mg branch of the tetrapyrrole metabolic pathway for chlorophyll biosynthesis.

Post-translational coordination of chlorophyll biosynthesis and breakdown by BCMs maintains chlorophyll homeostasis during leaf development

“…The drastically reduced amounts of Mg-porphyrins and increased GUN4 and CHLH levels observed in bcm1 ( Fig. 2e, h) phenotypically resemble the effects of impaired Chl biosynthesis in MgCh mutants, such as missense or knockdown mutants of CHLH and GUN4 13,15,46 . Therefore, the genetic interaction between BCM1 and MgCh was investigated by combining bcm1-3 with gun5 and with two allelic gun4 mutants (Fig.…”

“…Thus, BCM1 is suggested to act posttranslationally on Chl biosynthesis. Strikingly, Chl synthesis mutants ( Supplementary Table 1), including gun5-1 and cch (two missense mutants of CHLH 46 ), gun4-1 and gun4-3 (two knockdown mutants of the MgCh regulator GUN4, with GUN4 being less stable in gun4-1 than in gun4-3 [13][14][15] ), and chl27 (a knockdown mutant of MgPMME cyclase), contained increased levels of BCM1, GUN4, and CHLH in comparison to WT, whereas ch1-2 (a missense mutant of CAO) accumulated WT-like level of BCM1 and reduced contents of GUN4 and CHLH ( Fig. 2i).…”

“…3). To determine whether this interaction is necessary for the stimulation of MgCh activity, the effect of BCM1 on MgCh activity was explored in vitro using recombinant MgCh components 15,47 . To exclude an effect of endogenous GUN4 on the stimulation of MgCh activity, we isolated thylakoid membranes from bcm1-3 gun4-1 and BCM1-OX3/gun4-1 seedlings.…”

“…Mg-chelatase (MgCh), which consists of the catalytic H subunit (CHLH) and the two AAA + proteins CHLD and CHLI, directs Proto into the Mg branch of TBS by catalysing the insertion of Mg 2+ into Proto to generate Mg-Proto (MgP) 11,12 . Remarkably, the GENOMES UNCOUPLED 4 (GUN4) is capable of binding Proto and MgP and stimulates MgCh activity [13][14][15] . MgP is further converted into MgP monomethylester (MgPMME) and protochlorophyllide (Pchlide) by MgP methyltransferase (CHLM) 16 and MgPMME cyclase 17 , respectively.…”

Molecular recognition of M1-linked ubiquitin chains by native and phosphorylated UBAN domains

“…To further characterize the Ub-binding properties of UBAN domains, we studied effects of UBAN phosphorylation on UBAN/Ub binding. It has been shown that phosphorylation of S473 in OPTN UBAN enhances its binding affinity towards Ub chains [27]. S473 in OPTN corresponds to the alanine (A303) residue in NEMO and a phospho-mimetic residue (E or D) in ABIN1-3 ( Fig.…”