Significance of zinc in a regulatory protein, CCM1, which regulates the carbon-concentrating mechanism in Chlamydomonas reinhardtii.
In conditions with the poor availability of inorganic carbon (CO(2) and HCO(3) (-): Ci) for photosynthesis, aquatic photosynthetic organisms induce active Ci uptake systems that allow accumulation of Ci within the cell, the so-called carbon-concentrating mechanism (CCM). In a unicellular green alga, Chlamydomonas reinhardtii, a regulatory factor CCM1 is indispensable for the regulation of the CCM by sensing CO(2) availability. CCM1 has two putative zinc-binding domains with several conserved cysteine and histidine residues in its N-terminal region. To determine whether the domains actually bind zinc atoms, the N-terminal parts of CCM1 were expressed as glutathione S-transferase fusion proteins and subjected to atomic absorption spectrometry. It was found that 1 mol of zinc is bound to 1 mol of amino acid regions 1-71 and 72-101 of CCM1, respectively. In the case of the site-directed mutant proteins, H54Y, C77V and C80V, the zinc-binding ability was lost. Physiological analyses of the transgenic Chlamydomonas cells harboring a mutated Ccm1 gene revealed that amino acid residues such as C36, C41, H54, C77, C80, H90 and C93 were indispensable for induction of the CCM in response to Ci-limiting stress conditions. Size exclusion chromatography followed by immunoblot analyses indicated that CCM1 is present as a protein complex of approximately 290-580 kDa independent of Ci availability.
Kohinata
Tsutomu
T
Division of Integrated Life Science, Graduate School of Biostudies, Kyoto University, Kyoto 606-8502, Japan.
Nishino
Haruku
H
Fukuzawa
Hideya
H
D048868:Adaptor Proteins, Signal Transducing; D020418:Algal Proteins / Q000737:chemistry* / Q000378:metabolism*; D000818:Animals; D002244:Carbon / Q000378:metabolism*; D016825:Chlamydomonas reinhardtii / Q000378:metabolism*; D005786:Gene Expression Regulation; D016297:Mutagenesis, Site-Directed; D010940:Plant Proteins; D011485:Protein Binding; D011994:Recombinant Proteins; D015534:Trans-Activators / Q000737:chemistry* / Q000378:metabolism*; D015032:Zinc / Q000378:metabolism*
D048868:Adaptor Proteins, Signal Transducing; D020418:Algal Proteins; D010940:Plant Proteins; D011994:Recombinant Proteins; D015534:Trans-Activators; C425934:ccm1 protein, Chlamydomonas reinhardtii; D002244:Carbon; D015032:Zinc