Importance of the Cyanobacterial Gun4 Protein for Chlorophyll Metabolism and Assembly of Photosynthetic Complexes

Abstract

Gun4 is a porphyrin-binding protein that activates magnesium chelatase, a multimeric enzyme catalyzing the first committed step in chlorophyll biosynthesis. In plants, GUN4 has been implicated in plastid-to-nucleus retrograde signaling processes that coordinate both photosystem II and photosystem I nuclear gene expression with chloroplast function. In this work we present the functional analysis of Gun4 from the cyanobacterium Synechocystis sp. PCC 6803. Affinity co-purification of the FLAG-tagged Gun4 with the ChlH subunit of the magnesium chelatase confirmed the association of Gun4 with the enzyme in cyanobacteria. Inactivation of the gun4 gene abolished photoautotrophic growth of the resulting gun4 mutant strain that exhibited a decreased activity of magnesium chelatase. Consequently, the cellular content of chlorophyll-binding proteins was highly inadequate, especially that of proteins of photosystem II. Immunoblot analyses, blue native polyacrylamide gel electrophoresis, and radiolabeling of the membrane protein complexes suggested that the availability of the photosystem II antenna protein CP47 is a limiting factor for the photosystem II assembly in the gun4 mutant.

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

“…In the green alga Chlamydomonas reinhardtii, overexpression of Gun4 also increases Chl content per cell (38), whereas a C. reinhardtii strain possessing a low Gun4 content has reduced both Chl and P IX levels (15,20). Only after the complete elimination of Gun4 do cyanobacteria, algae, and plants accumulate aberrant concentrations of P IX (14,20,39).…”

“…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.…”

“…Available in vitro kinetic data imply that Gun4 stimulates MgCh by substantially reducing the P IX and Mg 2ϩ concentrations required for catalysis (12). Inactivation of the gun4 gene perturbs Chl biosynthesis and accumulation of Chl-binding proteins in cyanobacteria (14), green algae (15), and plants (11,19). However, phenotypes of available Gun4 mutants are difficult to explain simply by a defect in MgCh activity, and the in vivo activities of Gun4 appear to be complex (15,19,20).…”

“…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). Available in vitro kinetic data imply that Gun4 stimulates MgCh by substantially reducing the P IX and Mg 2ϩ concentrations required for catalysis (12).…”

“…In bacteriochlorophyll-producing bacteria, these three subunits are sufficient for a highly active MgCh enzyme when assayed in vitro (10). In contrast, MgCh from cyanobacteria, algae, and plants requires a protein called Gun4 that strongly enhances MgCh activity in vitro (11)(12)(13) and very likely also in vivo (14,15).…”