Mechanism of proton/substrate coupling in the heptahelical lysosomal transporter cystinosin
Mechanism of proton/substrate coupling in the heptahelical lysosomal transporter cystinosin
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DOI：
10.1073/pnas.1115581109
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发表时间：
2012-01-31
影响因子：
11.1
通讯作者：
Gasnier, Bruno
中科院分区：
文献类型：
--
作者：
Ruivo, Raquel;Bellenchi, Gian Carlo;Gasnier, Bruno
次级活性转运体利用初级离子泵提供的电化学梯度将代谢物或药物“上坡”地跨膜转运。在这里，我们报道了胱氨酸蛋白的离子偶联机制，胱氨酸蛋白是一种罕见的真核生物，质子驱动的转运体，与质子泵细菌视紫红质有远亲关系。在人类中，胱氨酸从溶酶体输出蛋白水解衍生的二聚体氨基酸胱氨酸，在胱氨酸病中受损。通过对胱氨酸引发的稳态和瞬态电流的电压依赖性分析和保守的可质子化残基的中和扫描诱变，我们发现胱氨酸结合与埋在膜中的临床相关的天冬氨酸的质子化偶联。氘同位素替代实验表明，这种天冬氨酸通过一个深质子通道从溶酶体腔进入。该天冬氨酸位于胱氨酸与一组功能未知的真核膜蛋白共享的两个PQ-loop基序之一，并且在其中约一半的PQ-loop基序中保守，因此表明其他PQ-loop蛋白可能会转运质子。
Secondary active transporters use electrochemical gradients provided by primary ion pumps to translocate metabolites or drugs "uphill" across membranes. Here we report the ion-coupling mechanism of cystinosin, an unusual eukaryotic, proton-driven transporter distantly related to the proton pump bacteriorhodopsin. In humans, cystinosin exports the proteolysis-derived dimeric amino acid cystine from lysosomes and is impaired in cystinosis. Using voltage-dependence analysis of steady-state and transient currents elicited by cystine and neutralization-scanning mutagenesis of conserved protonatable residues, we show that cystine binding is coupled to protonation of a clinically relevant aspartate buried in the membrane. Deuterium isotope substitution experiments are consistent with an access of this aspartate from the lysosomal lumen through a deep proton channel. This aspartate lies in one of the two PQ-loop motifs shared by cystinosin with a set of eukaryotic membrane proteins of unknown function and is conserved in about half of them, thus suggesting that other PQ-loop proteins may translocate protons.