Functional analysis of an inosine-guanosine transporter from Leishmania donovani -: The role of conserved residues, aspartate 389 and arginine 393
Functional analysis of an inosine-guanosine transporter from Leishmania donovani -: The role of conserved residues, aspartate 389 and arginine 393
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DOI：
10.1074/jbc.m305141200
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发表时间：
2003-08-29
影响因子：
4.8
通讯作者：
Carter, NS
中科院分区：
文献类型：
--
作者：
Arastu-Kapur, S;Ford, E;Carter, NS
平衡核苷转运蛋白包括两个保守的，带电的残基，发生在预测的跨膜结构域8。为了评估这些“特征”残基在转运蛋白功能中的作用，使来自杜氏利什曼原虫的LdNT 2核苷转运蛋白内的Asp（389）和Arg（393）残基突变，并在转染到Deltaldnt 2寄生虫中后评价所得表型。而R393 K突变体保留类似于野生型LdNT 2的转运活性，R393 L，D389 E和D389 N突变导致转运能力的急剧损失。用绿色荧光蛋白标记野生型和突变型ldnt 2蛋白表明，D389 N和D389 E突变体正确地靶向寄生虫细胞表面和鞭毛，而R393 L在细胞表面的表达受到严重损害。为了测试Asp（389）和Arg（393）是否相互作用，在绿色荧光蛋白标记的LdNT 2构建体内产生一系列突变体D389 R/R393 R、D389 D/R393 D和D389 R/R393 D。虽然所有这些ldnt 2突变体的转运缺陷，D389 R/R393 D定位正确的质膜，而D389 R/R393 R和D389 D/R393 D都不能检测到。此外，一个不能转运的D389 N/R393 N双ldnt 2突变体也定位于寄生虫膜，而D389 L/R393 L ldnt 2突变体没有，这表明残基389和393之间的相互作用可能参与LdNT 2膜靶向。这些研究从遗传学上确定了Asp（389）对于最佳转运蛋白功能是关键的，并且Arg（393）处的带正电荷或极性残基对于LdNT 2在质膜上的适当表达是必需的。
Equilibrative nucleoside transporters encompass two conserved, charged residues that occur within predicted transmembrane domain 8. To assess the role of these "signature" residues in transporter function, the Asp(389) and Arg(393) residues within the LdNT2 nucleoside transporter from Leishmania donovani were mutated and the resultant phenotypes evaluated after transfection into Deltaldnt2 parasites. Whereas an R393K mutant retained transporter activity similar to that of wild type LdNT2, the R393L, D389E, and D389N mutations resulted in dramatic losses of transport capability. Tagging the wild type and mutant ldnt2 proteins with green fluorescent protein demonstrated that the D389N and D389E mutants targeted properly to the parasite cell surface and flagellum, whereas the expression of R393L at the cell surface was profoundly compromised. To test whether Asp(389) and Arg(393) interact, a series of mutants was generated, D389R/R393R, D389D/R393D, and D389R/ R393D, within the green fluorescent protein-tagged LdNT2 construct. Although all of these ldnt2 mutants were transport-deficient, D389R/ R393D localized properly to the plasma membrane, while neither D389R/ R393R nor D389D/R393D could be detected. Moreover, a transport-incompetent D389N/R393N double ldnt2 mutant also localized to the parasite membrane, whereas a D389L/R393L ldnt2 mutant did not, suggesting that an interaction between residues 389 and 393 may be involved in LdNT2 membrane targeting. These studies establish genetically that Asp(389) is critical for optimal transporter function and that a positively charged or polar residue at Arg(393) is essential for proper expression of LdNT2 at the plasma membrane.