Dix D, Bridgham J, Broderius M, Eide D
Department of Biochemistry and Molecular Biology, University of Minnesota, Duluth, Minnesota 55812, USA.
J Biol Chem. 1997 May 2;272(18):11770-7. doi: 10.1074/jbc.272.18.11770.
The low affinity Fe2+ uptake system of Saccharomyces cerevisiae requires the FET4 gene. In this report, we present evidence that FET4 encodes the Fe2+ transporter protein of this system. Antibodies prepared against FET4 detected two distinct proteins with molecular masses of 63 and 68 kDa. In vitro synthesis of FET4 suggested that the 68-kDa form is the primary translation product, and the 63-kDa form may be generated by proteolytic cleavage of the full-length protein. Consistent with its role as an Fe2+ transporter, FET4 is an integral membrane protein present in the plasma membrane. The level of FET4 closely correlated with uptake activity over a broad range of expression levels and is itself regulated by iron. Furthermore, mutations in FET4 can alter the kinetic properties of the low affinity uptake system, suggesting a direct interaction between FET4 and its Fe2+ substrate. Mutations affecting potential Fe2+ ligands located in the predicted transmembrane domains of FET4 significantly altered the apparent Km and/or Vmax of the low affinity system. These mutations may identify residues involved in Fe2+ binding during transport.
酿酒酵母的低亲和力Fe2+摄取系统需要FET4基因。在本报告中，我们提供证据表明FET4编码该系统的Fe2+转运蛋白。针对FET4制备的抗体检测到两种不同的蛋白质，分子量分别为63 kDa和68 kDa。FET4的体外合成表明，68 kDa的形式是主要的翻译产物，而63 kDa的形式可能是全长蛋白质经蛋白水解切割产生的。与其作为Fe2+转运蛋白的作用一致，FET4是一种存在于质膜中的整合膜蛋白。在广泛的表达水平范围内，FET4的水平与摄取活性密切相关，并且其本身受铁的调节。此外，FET4中的突变可以改变低亲和力摄取系统的动力学特性，表明FET4与其Fe2+底物之间存在直接相互作用。影响位于FET4预测跨膜结构域中的潜在Fe2+配体的突变显著改变了低亲和力系统的表观Km和/或Vmax。这些突变可能确定了转运过程中参与Fe2+结合的残基。
