Copper-dependent trafficking of the Ctr4-Ctr5 copper transporting complex.
Copper-dependent trafficking of the Ctr4-Ctr5 copper transporting complex.
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DOI：
10.1371/journal.pone.0011964
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发表时间：
2010-08-04
期刊：
影响因子：
3.7
通讯作者：
Labbé S
中科院分区：
文献类型：
--
作者：
Ioannoni R;Beaudoin J;Mercier A;Labbé S
在粟酒裂殖酵母中，铜的吸收是通过由Ctr 4和Ctr 5蛋白形成的异聚复合物进行的。铜诱导的差异亚细胞定位可能在微调细胞表面Ctr 4和Ctr 5分子的数量方面发挥关键作用。我们已经开发了一种双分子荧光互补（BiFC）分析蛋白质-蛋白质相互作用在体内的S。粟球。该测定是基于观察到的N-和C-末端亚片段的金星荧光蛋白可以重建一个功能性的荧光团，只有当它们被带到紧密接触。将ctr 4+和ctr 5+基因的野生型拷贝分别插入Venus的非荧光C-末端（VC）和N-末端（VN）编码片段的下游和框内。Ctr 4-VC和Ctr 5-VN融合蛋白的共表达允许它们在铜限制性细胞的质膜上被检测。类似地，在Ctr 5-Myc 12存在下共表达Ctr 4-VN和Ctr 4-VC的细胞在质膜处显示荧光信号。相比之下，在Ctr 4-Flag 2存在下共表达的Ctr 5-VN和Ctr 5-VC在质膜处不能被可视化，这表明需要两个Ctr 4分子与一个Ctr 5分子的组合。我们发现，当细胞暴露于高水平的铜时，位于质膜的Ctr 4-VC-Ctr 5-VN荧光复合物被内化。铜诱导的Ctr 4-VC-Ctr 5-VN复合物的内化不依赖于从头蛋白质合成。当细胞从高铜水平转移回低铜水平时，在质膜处再次出现BiFC荧光信号。这些发现揭示了异聚Ctr 4-Ctr 5复合物作为铜可用性的函数的铜依赖性内化和再循环。
In Schizosaccharomyces pombe, copper uptake is carried out by a heteromeric complex formed by the Ctr4 and Ctr5 proteins. Copper-induced differential subcellular localization may play a critical role with respect to fine tuning the number of Ctr4 and Ctr5 molecules at the cell surface. We have developed a bimolecular fluorescence complementation (BiFC) assay to analyze protein-protein interactions in vivo in S. pombe. The assay is based on the observation that N- and C-terminal subfragments of the Venus fluorescent protein can reconstitute a functional fluorophore only when they are brought into tight contact. Wild-type copies of the ctr4+ and ctr5+ genes were inserted downstream of and in-frame with the nonfluorescent C-terminal (VC) and N-terminal (VN) coding fragments of Venus, respectively. Co-expression of Ctr4-VC and Ctr5-VN fusion proteins allowed their detection at the plasma membrane of copper-limited cells. Similarly, cells co-expressing Ctr4-VN and Ctr4-VC in the presence of Ctr5-Myc12 displayed a fluorescence signal at the plasma membrane. In contrast, Ctr5-VN and Ctr5-VC co-expressed in the presence of Ctr4-Flag2 failed to be visualized at the plasma membrane, suggesting a requirement for a combination of two Ctr4 molecules with one Ctr5 molecule. We found that plasma membrane-located Ctr4-VC-Ctr5-VN fluorescent complexes were internalized when the cells were exposed to high levels of copper. The copper-induced internalization of Ctr4-VC-Ctr5-VN complexes was not dependent on de novo protein synthesis. When cells were transferred back from high to low copper levels, there was reappearance of the BiFC fluorescent signal at the plasma membrane. These findings reveal a copper-dependent internalization and recycling of the heteromeric Ctr4-Ctr5 complex as a function of copper availability.
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影响因子：
46.9
作者：
Nagai, T;Ibata, K;Miyawaki, A
通讯作者：
Miyawaki, A
DOI：
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