Significance of zinc in a regulatory protein, CCM1, which regulates the carbon-concentrating mechanism in Chlamydomonas reinhardtii
Significance of zinc in a regulatory protein, CCM1, which regulates the carbon-concentrating mechanism in Chlamydomonas reinhardtii
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DOI：
10.1093/pcp/pcn003
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发表时间：
2008-02-01
影响因子：
4.9
通讯作者：
Fukuzawa, Hideya
中科院分区：
文献类型：
--
作者：
Kohinata, Tsutomu;Nishino, Haruku;Fukuzawa, Hideya
在无机碳（CO2和HCO 3-：Ci）用于光合作用的可用性差的条件下，水生光合生物诱导活性Ci吸收系统，其允许Ci在细胞内积累，即所谓的碳浓缩机制（CCM）。在一个单细胞的绿色的衣藻，莱茵衣藻，一个调节因子CCM 1是必不可少的CCM通过传感CO2的可用性的调节。CCM 1具有两个推定的锌结合结构域，在其N-末端区域具有几个保守的半胱氨酸和组氨酸残基。为了确定结构域是否实际上结合锌原子，CCM 1的N-末端部分表达为谷胱甘肽S-转移酶融合蛋白，并进行原子吸收光谱法。发现1摩尔锌分别结合到CCM 1的1摩尔氨基酸区域171和72101。在定点突变蛋白H54 Y、C77 V和C80 V的情况下，锌结合能力丧失。对携带突变的Ccm 1基因的转基因衣原体细胞的生理分析表明，氨基酸残基如C36、C41、H54、C77、C80、H90和C93对于响应于Ci限制性胁迫条件的CCM的诱导是必不可少的。大小排阻色谱法，然后免疫印迹分析表明，CCM 1是目前作为一个蛋白质复合物的约290580 kDa的独立的Ci可用性。
In conditions with the poor availability of inorganic carbon (CO2 and HCO3-: 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 CO2 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 171 and 72101 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 290580 kDa independent of Ci availability.