Identification of essential amino acid residues of the NorM Na+/multidrug antiporter in Vibrio parahaemolyticus
Identification of essential amino acid residues of the NorM Na+/multidrug antiporter in Vibrio parahaemolyticus
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DOI：
10.1128/jb.187.5.1552-1558.2005
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发表时间：
2005-03-01
影响因子：
3.2
通讯作者：
Moriyama, Y
中科院分区：
文献类型：
--
作者：
Otsuka, M;Yasuda, M;Moriyama, Y
NorM是多药和毒性化合物排出（MATE）家族的成员，在副溶血性弧菌中作为Na +/多药反向转运蛋白发挥作用，尽管Na +/多药反向转运的潜在机制尚不清楚。NorM跨膜区的酸性氨基酸残基Asp32、Glu251和Asp367在MATE家族的一个簇中是保守的。在这项研究中，我们调查的作用（S）的酸性氨基酸残基Asp32，Glu251和Asp367在跨膜区的NorM的定点突变。野生型NorM和具有氨基酸替换D32E（D32至E）、D32N、D32K、E251D、E251Q、D367A、D367E、D367N和D367K的突变蛋白在大肠杆菌KAM 32细胞的内膜中表达和定位，而具有D32A、E251A和E251K的突变蛋白则不表达和定位。与具有野生型NorM的细胞相比，具有突变NorM蛋白的细胞对卡那霉素、诺氟沙星和溴化乙锭的抗性降低，但NorM D367E突变体对溴化乙锭的抗性更强。NorM突变体D32 E、D32 N、D32 K、D367 A和D367 K细胞丧失了Na+依赖性的排出乙锭离子的能力，以及由溴化乙锭诱发的移动Na+的能力。E251D和D367N突变体都降低了Na+依赖的乙锭离子挤出，但溴化乙锭诱发的Na+运动保留。相反，D367E引起的增加运输的乙锭离子和Na+。这些结果表明，Asp32，Glu251和Asp367参与Na+依赖性药物转运过程。
NorM is a member of the multidrug and toxic compound extrusion (MATE) family and functions as a Na+/multidrug antiporter in Vibrio parahaemolyticus, although the underlying mechanism of the Na+/multidrug antiport is unknown. Acidic amino acid residues Asp32, Glu251, and Asp367 in the transmembrane region of NorM are conserved in one of the clusters of the MATE family. In this study, we investigated the role(s) of acidic amino acid residues Asp32, Glu251, and Asp367 in the transmembrane region of NorM by site-directed mutagenesis. Wild-type NorM and mutant proteins with amino acid replacements D32E (D32 to E), D32N, D32K, E251D, E251Q, D367A, D367E, D367N, and D367K were expressed and localized in the inner membrane of Escherichia coli KAM32 cells, while the mutant proteins with D32A, E251A, and E251K were not. Compared to cells with wild-type NorM, cells with the mutant NorM protein exhibited reduced resistance to kanamycin, norfloxacin, and ethidium bromide, but the NorM D367E mutant was more resistant to ethidium bromide. The NorM mutant D32E, D32N, D32K, D367A, and D367K cells lost the ability to extrude ethidium ions, which was Na+ dependent, and the ability to move Na+, which was evoked by ethidium bromide. Both E251D and D367N mutants decreased Na+-dependent extrusion of ethidium ions, but ethidium bromide-evoked movement of Na+ retained. In contrast, D367E caused increased transport of ethidium ions and Na+. These results suggest that Asp32, Glu251, and Asp367 are involved in the Na+-dependent drug transport process.