Gain-of-function mutations identify amino acids within transmembrane domains of the yeast vacuolar transporter Zrc1 that determine metal specificity
Gain-of-function mutations identify amino acids within transmembrane domains of the yeast vacuolar transporter Zrc1 that determine metal specificity
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DOI：
10.1042/bj20090853
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发表时间：
2009-09-01
影响因子：
4.1
通讯作者：
Kaplan, Jerry
中科院分区：
文献类型：
--
作者：
Lin, Huilan;Burton, Damali;Kaplan, Jerry
阳离子扩散促进转运蛋白存在于生命的所有三个王国中，并参与将过渡金属转运出胞质溶胶。它们转运的金属包括Zn（2+）、Co（2+）、Fe（2+）、Cd（2+）、Ni（2+）和Mn（2+）;然而，没有一个转运蛋白可以转运所有金属。先前我们表明，酵母液泡锌转运蛋白Zrc 1中的单个氨基酸突变将其底物特异性从Zn（2+）改变为Fe（2+）和Mn（2+）[Lin，Kumanovics，纳尔逊，Warner，Ward和Kaplan（2008）J.Biol.Chem.283，33865-33873]。获得铁转运活性的突变体Zrc 1可以保护液泡铁转运蛋白（CCC 1）缺失的细胞免受高铁毒性。利用抑制高铁毒性和PCR诱变的ZRC 1，我们确定了其他氨基酸取代ZRC 1内改变其金属特异性。所有转运Fe（2+）的Zrc 1突变体都能转运Mn（2+）。一些Zrc 1突变体丧失了转运Zn（2+）的能力，但另一些则保留了转运Zn（2+）的能力。所有导致Fe（2+）转运活性增加的氨基酸取代都存在于跨膜结构域。除了在两个跨膜结构域中邻近推定的金属结合位点的残基的改变之外，远离结合位点的残基的改变影响底物特异性。这些结果表明，底物选择涉及跨膜结构域之间的协同性。
Cation diffusion facilitator transporters are found in all three Kingdoms of life and are involved in transporting transition metals out of the cytosol. The metals they transport include Zn(2+), Co(2+), Fe(2+), Cd(2+), Ni(2+) and Mn(2+); however, no single transporter transports all metals. Previously we showed that a single amino acid mutation in the yeast vacuolar zinc transporter Zrc1 changed its substrate specificity from Zn(2+) to Fe(2+) and Mn(2+) [Lin, Kumanovics, Nelson, Warner, Ward and Kaplan (2008) J. Biol. Chem. 283, 33865-33873]. Mutant Zrc1 that gained iron transport activity could protect cells with a deletion in the vacuolar iron transporter (CCC1) from high iron toxicity. Utilizing suppression of high iron toxicity and PCR mutagenesis of ZRC1, we identified other amino acid substitutions within ZRC1 that changed its metal specificity. All Zrc1 mutants that transported Fe(2+) could also transport Mn(2+). Some Zrc1 mutants lost the ability to transport Zn(2+), but others retained the ability to transport Zn(2+). All of the amino acid substitutions that resulted in a gain in Fe(2+) transport activity were found in transmembrane domains. In addition to alteration of residues adjacent to the putative metal-binding site in two transmembrane domains, alteration of residues distant from the binding site affected substrate specificity. These results suggest that substrate selection involves co-operativity between transmembrane domains.