Sácký Jan, Leonhardt Tereza, Kotrba Pavel
Department of Biochemistry and Microbiology, University of Chemistry and Technology, Prague, Technická 3, 166 28, Prague, Czech Republic.
Biometals. 2016 Apr;29(2):349-63. doi: 10.1007/s10534-016-9920-x. Epub 2016 Feb 23.
Russula atropurpurea can accumulate remarkably high concentrations of Zn in its sporocarps. We have previously demonstrated that 40 % of the intracellular Zn in this species is sequestered by MT-like RaZBP peptides. To see what other mechanisms for the handling of the accumulated Zn are available to R. atropurpurea, we searched its transcriptome for cDNAs coding for transporters of the cation diffusion facilitator (CDF) family. The transcriptome search enabled us to identify RaCDF1 and RaCDF2, which were further subjected to functional studies in metal sensitive Saccharomyces cerevisiae. The expression of RaCDF1 and its translational fusion with green fluorescent protein (GFP) protected the yeasts against Zn and Co, but not Cd or Mn, toxicity and led to increased Zn accumulation in the cells. The GFP fluorescence, observed in the RaCDF1::GFP-expressing yeasts on tonoplasts, indicated that the RaCDF1-mediated Zn and Co tolerance was a result of vacuolar sequestration of the metals. The expression of RaCDF2 supported Zn, but not Mn, tolerance in the yeasts and reduced the cellular uptake of Zn, which is congruent with the proposed idea of the Zn-efflux function of RaCDF2, supported by the localization of GFP-derived fluorescence on the plasma membrane of the yeasts expressing functional RaCDF2::GFP. Contrarily, RaCDF2 increased the sensitivity to Co and Cd in the yeasts and significantly promoted Cd uptake, which suggested that it can act as a bidirectional metal transporter. The notion that RaCDF1 and RaCDF2 are genuine CDF transporters in R. atropurputrea was further reinforced by the fact that the RaCDF-associated metal tolerance and uptake phenotypes were lost upon the replacement of histidyl (in RaCDF1) and aspartyl (in RaCDF2), which are highly conserved in the second transmembrane domain and known to be essential for the function of CDF proteins.
紫牛肝菌（Russula atropurpurea）的子实体能够积累高浓度的锌。我们之前已经证明，该物种细胞内40%的锌被类金属硫蛋白（MT）的RaZBP肽螯合。为了探究紫牛肝菌处理积累的锌还有哪些其他机制，我们在其转录组中搜索编码阳离子扩散促进因子（CDF）家族转运蛋白的cDNA。通过转录组搜索，我们鉴定出了RaCDF1和RaCDF2，并进一步在对金属敏感的酿酒酵母中对它们进行了功能研究。RaCDF1的表达及其与绿色荧光蛋白（GFP）的翻译融合保护酵母免受锌和钴的毒性影响，但对镉或锰的毒性无效，并导致细胞内锌积累增加。在表达RaCDF1::GFP的酵母液泡膜上观察到的GFP荧光表明，RaCDF1介导的锌和钴耐受性是金属液泡隔离的结果。RaCDF2的表达支持酵母对锌的耐受性，但对锰无效，并减少了细胞对锌的摄取，这与RaCDF2的锌外排功能的推测一致，表达功能性RaCDF2::GFP的酵母质膜上GFP衍生荧光的定位也支持了这一点。相反，RaCDF2增加了酵母对钴和镉的敏感性，并显著促进了镉的摄取，这表明它可以作为双向金属转运蛋白。在RaCDF1（组氨酸）和RaCDF2（天冬氨酸）的第二个跨膜结构域中高度保守且已知对CDF蛋白功能至关重要的氨基酸被替换后，与RaCDF相关的金属耐受性和摄取表型消失，这一事实进一步强化了RaCDF1和RaCDF2是紫牛肝菌中真正的CDF转运蛋白的观点。
