Cysteine-scanning mutagenesis of transmembrane segments 4 and 5 of the Tn10-encoded metal tetracycline/H+ antiporter reveals a permeability barrier in the middle of a transmembrane water-filled channel
Cysteine-scanning mutagenesis of transmembrane segments 4 and 5 of the Tn10-encoded metal tetracycline/H+ antiporter reveals a permeability barrier in the middle of a transmembrane water-filled channel
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DOI：
10.1074/jbc.m910354199
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发表时间：
2000-07-28
影响因子：
4.8
通讯作者：
Yamaguchi, A
中科院分区：
文献类型：
--
作者：
Iwaki, S;Tamura, N;Yamaguchi, A
构建了Tn 10编码的金属四环素/H+反向转运蛋白（TetA（B））的跨膜片段4和5及侧翼区的半胱氨酸扫描突变体。所有突变体均正常表达。在57个突变体（L99 C至I155 C）中，9个保守的精氨酸、天冬氨酸和甘氨酸取代突变体的四环素抗性大大降低，几乎没有转运活性，而5个保守的甘氨酸和脯氨酸取代突变体尽管具有高或中度耐药性，但在倒置膜囊泡中的四环素转运活性大大降低。除L142 C和I143 C突变体外，所有其他半胱氨酸扫描突变体均保持正常的耐药性和正常的四环素转运活性。基于N-[C-14]乙基马来酰亚胺（[C-14]NEM）反应性，确定跨膜（TM）区TM 4和TM 5分别包含20个氨基酸残基（Leu-99至Ile-118）和17个氨基酸残基（Ala-136至Ala-152）。TM 4可以分为两半，即，NEM非反应性周质的一半和周期性反应性的细胞质的一半，表明TM 4是倾斜的充满水的跨膜通道，只有它的细胞质的一半面临的通道。另一方面，周期性地观察到NEM反应性突变（每两个残基）沿着TM 5的整个长度，在TM 5的中间Leu-142和Gly-145之间存在膜不可渗透的巯基试剂4-乙酰氨基-4 ′-马来酰亚胺基芪-2，2 ′-二磺酸的渗透屏障，而对于TM 4的所有NEM反应性突变体都不能接近4-乙酰氨基-4 '-马来酰亚胺基芪-2，2'-二磺酸，表明TM 4的面向通道侧位于渗透屏障内。四环素保护G141 C突变体不受NEM结合，而TM 4和TM 5中的其他突变体不受四环素保护。
Cysteine-scanning mutants as to putative transmembrane segments 4 and 5 and the flanking regions of Tn10-encoded metal-tetracycline/H+ antiporter (TetA (B)) were constructed. All mutants were normally expressed, Among the 57 mutants (L99C to I155C), nine conserved arginine-, aspartate-, and glycine-replaced ones exhibited greatly reduced tetracycline resistance and almost no transport activity, and five conserved glycine- and proline-replaced mutants exhibited greatly reduced tetracycline transport activity in inverted membrane vesicles despite their high or moderate drug resistance. All other cysteine-scanning mutants retained normal drug resistance and normal tetracycline transport activity except for the L142C and I143C mutants. The transmembrane (TM) regions TM4 and TM5 were determined to comprise 20 amino acid residues, Leu-99 to Ile-118, and 17 amino acid residues, Ala-136 to Ala-152, respectively, on the basis of N-[C-14]ethylmaleimide ([C-14]NEM) reactivity, The NEM reactivity patterns of the TM4 and TM5 mutants were quite different from each other. TM4 could be divided into two halves, that is, a NEM nonreactive periplasmic half and a periodically reactive cytoplasmic half, indicating that TM4 is tilted toward a water-filled transmembrane channel and that only its cytoplasmic half faces the channel. On the other hand, NEM-reactive mutations were observed periodically (every two residues) along the whole length of TM5, A permeability barrier for a membrane-impermeable sulfhydryl reagent, 4-acetamido-4'-maleimidylstilbene-2,2'-disulfonic acid, was present in the middle of TM5 between Leu-142 and Gly-145, whereas all the NEM-reactive mutants as to TM4 were not accessible to 4-acetamido-4'-maleimidylstilbene-2,2'-disulfonic acid, indicating that the channel-facing side of TM4 is located inside the permeability barrier. Tetracycline protected the G141C mutant from the NEM binding, whereas the other mutants in TM4 and TM5 were not protected by tetracycline.