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首页> 外文期刊>American Journal of Physiology >Identification of a functionally critical GXXG motif and its relationship to the folate binding site of the proton-coupled folate transporter (PCFT-SLC46A1)
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Identification of a functionally critical GXXG motif and its relationship to the folate binding site of the proton-coupled folate transporter (PCFT-SLC46A1)

机译：鉴定功能关键的GXXG基序及其与质子偶联叶酸转运蛋白（PCFT-SLC46A1）的叶酸结合位点的关系

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摘要

The proton-coupled folate transporter (PCFT) mediates intestinal folate absorption, and loss-of-function mutations in this gene result in the autosomal recessive disorder hereditary folate malabsorption. The current study, focused on a structure-functional analysis of this transporter, identified Gly-189 and Gly-192 (a GxxG motif) located in the fifth transmembrane domain as residues that could not be replaced with alanine without a loss of function. In contrast, function was preserved when Gly-56 and Gly-59 (the other conservative GXXG motif in human PCFT) were replaced with alanine. Similarly, Gly-93 and Gly-97, which constitute the only conserved GXXXG dimerization motif in human PCFT, tolerated alanine substitution. To explore the role of this region in folate binding, the residues around Gly-189 and Gly-192 were analyzed by the substituted cysteine accessibility method. Both I188C and M193C mutants were functional and were inhibited by membrane-impermeable sulfhydryl-reactive reagents; this could be prevented with PCFT substrate, but the protection was sustained at 0°C only for the I188C mutant, consistent with localization of Ile-188 in the PCFT folate binding pocket. The functional role of residues around Gly-189 and Gly-192 is consistent with a molecular structural model in which these two residues along with Ieu-188 are accessible to the PCFT aqueous translocation pathway.
机译：质子偶联的叶酸转运蛋白（PCFT）介导肠道叶酸吸收，该基因中的功能丧失突变导致常染色体隐性遗传性叶酸吸收不良。当前的研究侧重于对该转运蛋白的结构功能分析，确定了位于第五跨膜结构域的Gly-189和Gly-192（GxxG基序）是无法被丙氨酸取代而不会丧失功能的残基。相反，当Gly-56和Gly-59（人PCFT中另一个保守的GXXG基序）替换为丙氨酸时，功能得以保留。同样，构成人PCFT中唯一保守的GXXXG二聚化基元的Gly-93和Gly-97可以耐受丙氨酸取代。为了探索该区域在叶酸结合中的作用，通过取代的半胱氨酸可及性方法分析了Gly-189和Gly-192周围的残基。 I188C和M193C突变体均具有功能，并被不可透过膜的巯基反应试剂抑制。这可以用PCFT底物来预防，但是仅对I188C突变体才在0°C保持保护，这与PCFT叶酸结合口袋中Ile-188的定位一致。 Gly-189和Gly-192周围残基的功能作用与分子结构模型一致，在该分子结构模型中，这两个残基以及Ieu-188均可通过PCFT水溶液转运途径进入。

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