Sobczak Iwona, Lolkema Juke S
Molecular Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Haren, The Netherlands.
Biochemistry. 2003 Aug 19;42(32):9789-96. doi: 10.1021/bi034683h.
The citrate transporter CitS of Klebsiella pneumoniae is a secondary transporter that transports citrate in symport with two sodium ions and one proton. Treatment of CitS with the alkylating agent N-ethylmaleimide resulted in a complete loss of transport activity. Treatment of mutant proteins in which the five endogenous cysteine residues were mutated into serines in different combinations revealed that two cysteine residues located in the C-terminal cytoplasmic loop, Cys-398 and Cys-414, were responsible for the inactivation. Labeling with the membrane impermeable methanethiosulfonate derivatives MTSET and MTSES in right-side-out membrane vesicles showed that the cytoplasmic loop was accessible from the periplasmic side of the membrane. The membrane impermeable but more bulky maleimide AmdiS did not inactivate the transporter in right-side-out membrane vesicles. Inactivation by N-ethylmaleimide, MTSES, and MTSET was prevented by the presence of the co-ion Na(+). Protection was obtained upon binding 2 Na(+), which equals the transport stoichiometry. In the absence of Na(+), the substrate citrate had no effect on the inactivation by permeable or impermeable thiol reagents. In contrast, when subsaturating concentrations of Na(+) were present, citrate significantly reduced inactivation suggesting ordered binding of the substrate and co-ion; citrate is bound after Na(+). In the presence of the proton motive force, the reactivity of the Cys residues was increased significantly for the membrane permeable N-ethylmaleimide, while no difference was observed for the membrane impermeable thiol reagents. The results are discussed in the context of a model for the opening and closing of the translocation pore during turnover of the transporter.
肺炎克雷伯菌的柠檬酸转运蛋白CitS是一种次级转运蛋白，它与两个钠离子和一个质子协同转运柠檬酸。用烷基化剂N-乙基马来酰亚胺处理CitS会导致转运活性完全丧失。对五个内源性半胱氨酸残基以不同组合突变为丝氨酸的突变蛋白进行处理后发现，位于C端细胞质环中的两个半胱氨酸残基Cys-398和Cys-414是失活的原因。用膜不透性的甲硫基磺酸酯衍生物MTSET和MTSES对外翻膜囊泡进行标记表明，细胞质环可从膜的周质侧接触到。膜不透性但体积更大的马来酰亚胺AmdiS不会使外翻膜囊泡中的转运蛋白失活。N-乙基马来酰亚胺、MTSES和MTSET的失活作用可被共离子Na⁺的存在所阻止。结合2个Na⁺时可获得保护，这与转运化学计量相等。在没有Na⁺的情况下，底物柠檬酸对可渗透或不可渗透的硫醇试剂的失活作用没有影响。相反，当存在亚饱和浓度的Na⁺时，柠檬酸显著降低失活作用，表明底物和共离子的结合是有序的；柠檬酸在Na⁺之后结合。在质子动力势存在的情况下，对于膜可渗透的N-乙基马来酰亚胺，半胱氨酸残基的反应性显著增加，而对于膜不可渗透的硫醇试剂则未观察到差异。本文在转运蛋白周转过程中转位孔开闭模型的背景下对结果进行了讨论。
