Site-directed mutagenesis identifies residues in uncoupling protein (UCP1) involved in three different functions
Site-directed mutagenesis identifies residues in uncoupling protein (UCP1) involved in three different functions
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DOI：
10.1021/bi992448m
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发表时间：
2000-03-28
期刊：
影响因子：
2.9
通讯作者：
Klingenberg, M
中科院分区：
文献类型：
--
作者：
Echtay, KS;Winkler, E;Klingenberg, M
利用位点特异性诱变技术，我们从棕色脂肪组织中构建了几个解偶联蛋白（UCP1）突变体，以研究27、167、209和210位酸性侧链在H+和Cl-转运以及核苷酸结合中的功能。用线粒体和重组囊泡测定H+转运活性。这些突变UCPs （D27N、D27E、E167Q、D209N、D210N和D209N + D210N）在酵母中以接近wt水平表达。除了D27N（螺旋内）外，它们在线粒体内的Hf转运活性与重组蛋白相关良好，重组蛋白对H+转运有很强的抑制作用，线粒体内的非偶联呼吸仅减少50%。在双相邻酸性残基（螺旋4和螺旋5之间）中，D210和D209的突变分别使H+转运减少了80%和20%。这些突变体保留完整的Cl-转运活性。结果表明，D210参与胞质侧的H+摄取，D27参与H+通过细胞膜的转运。不同的是，E167Q失去了Cl-转运活性，但保留了转运H+的能力。H+和Cl-输运分别失活，反驳了UCP输运H+的脂肪酸阴离子输运机制。双相邻酸性残基（D209, D210）的突变只降低了三磷酸核苷（NTP）的pH依赖性，而对二磷酸核苷（NDP）的结合没有影响。结果发现D209和D210是控制H214在结合口袋中的位置的残基，从而有助于NTP的pH控制，而NDP结合的螺母。
Using site-specific mutagenesis, we have constructed several mutants of uncoupling protein (UCP1) from brown adipose tissue to investigate the function of acidic side chains at positions 27, 167, 209, and 210 in H+ and Cl- transport as well as in nucleotide binding. The H+ transport activity was measured with mitochondria and with reconstituted vesicles. These mutant UCPs (D27N, D27E, E167Q, D209N, D210N, and D209N + D210N) are expressed at near wt levels in yeast. Their Hf transport activity in mitochondria correlates well with the reconstituted protein except for D27N (intrahelical), which shows strong inhibition of H+ transport in the reconstituted system and only 50% decrease of uncoupled respiration in mitochondria. In the double adjacent acidic residues (between helix 4 and helix 5), mutation of D210 and of D209 decreases H+ transport 80% and only 20%, respectively. These mutants retain full Cl- transport activity. The results indicate that D210 participates in H+ uptake at the cytosolic side and D27 in H+ translocation through the membrane. Differently, E167Q has lost Cl- transport activity but retains the ability to transport H+. The separate inactivation of H+ and Cl- transport argues against the fatty acid anion transport mechanism of H+ transport by UCP. The mutation of the double adjacent acidic residues (D209, D210) decreases pH dependency for only nucleoside triphosphate (NTP) but not diphosphate (NDP) binding. The results identify D209 and D210 in accordance with the previous model as those residues which control the location of H214 in the binding pocket, and thus contribute to the pH control of NTP but nut of NDP binding.