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首页> 外文期刊>Biochemistry >Transmembrane Domain 5 of the LdNTl.l Nucleoside Transporter Is an Amphipathic Helix That Forms Part of the Nucleoside Translocation Pathway
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Transmembrane Domain 5 of the LdNTl.l Nucleoside Transporter Is an Amphipathic Helix That Forms Part of the Nucleoside Translocation Pathway

机译：LDNT1.L核苷转运蛋白的跨膜结构域5是构成核苷易位途径的一部分的两亲性螺旋

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摘要

Transporters of the equilibrative nucleoside transporter (ENT) family promote the uptake of nucleosides,nucleobases,and a variety of therapeutic drugs in eukaryotes from protozoa to mammals.Despite its importance,the translocation pathway that mediates the internalization of these substrates has not been identified yet in any of the ENT carriers.Previous genetic studies on the LdNTl.l nucleoside transporter from Leishmania donovani defined two amino acid residues in predicted transmembrane domains (TMD) 5 and 7 that may line this translocation pathway.The role of TMD5 in forming a portion of the aqueous channel was investigated using the substituted-cysteine accessibility method.A series of 22 cysteine substitution mutants spanning predicted TMD5 were created from a fully functional,cysteine-less,parental LdNTl.l.Cysteine replacement at six positions (M_(176)C,T_(186)C,S_(187)C,Q_(190)C,V_(193)C,and K_(194)C) produced permeases that were inhibited by incubation with sulfhydryl-specific methanethiosulfonate reagents,denoting their solvent accessibility to the translocation pathway.Adenosine was able to block this thiol modification,implying that access to the domain becomes restricted as a consequence of the substrate binding.Strikingly,the Q_(190)C substitution interacted differentially with the substrates adenosine and uridine,suggesting that binding of adenosine but not uridine might directly occlude this position.When superimposed on a helical model,all six mutants clustered along one face of the amphipathic alpha-helix predicted for TMD5,strongly suggesting its involvement in the translocation pathway through LdNT1.1.
机译：平衡核苷转运蛋白（ENT）的转运蛋白促进了从原生动物到哺乳动物的真核生物中核苷，核碱基和各种治疗药物的摄取。尚未确定介绍这些基材内化的易位途径尚未确定在任何ENT载体中。来自Leishmania Donovani的Ldntl.L核苷转运蛋白的另一种遗传研究确定了预测跨膜结构域（TMD）5和7中的两个氨基酸残基，其可以在这种易位途径上线。TMD5在形成一部分时的作用使用取代的半胱氨酸可接近度研究了水通道。一系列22种半胱氨酸取代突变体，跨越预测的TMD5，从六个位置处的全功能性，半胱氨酸，父母的LDNT1.L.Lysteine替换产生（M_（176） C，T_（186）C，S_（187）C，Q_（190）C，V_（193）C和K_（194）C）产生的介质，其通过与巯基特异性孵育而抑制将其甲基磺酸磺酸盐试剂表示对易位途径的溶剂可替代性。腺苷能够阻断这种硫醇改性，这意味着由于对畴的接近而被限制为基板结合。Q_（190）C替换与差异相互作用依物腺苷和尿苷，表明腺苷但不是尿苷的结合可能直接遮挡该位置。当呈螺旋模型上叠加时，所有六个突变体都沿着对TMD5预测的两性α-螺旋的一面聚集，强烈建议其参与易位通路通过LDNT1.1。

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  • 来源
    《Biochemistry》 |2004年第21期|共10页
  • 作者

    Raquel Valdes; Gayatri Vasudevan; David Conklin; Scott M.Landfear;

  • 作者单位

    Department of Molecular Microbiology and Immunology Oregon Health &

    Science University Portland Oregon 97239;

    Department of Molecular Microbiology and Immunology Oregon Health &

    Science University Portland Oregon 97239;

    Department of Molecular Microbiology and Immunology Oregon Health &

    Science University Portland Oregon 97239;

    Department of Molecular Microbiology and Immunology Oregon Health &

    Science University Portland Oregon 97239;

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  • 正文语种 eng
  • 中图分类 生物化学;
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