Assessment of uncoupling activity of the human uncoupling protein 3 short form and three mutants of the uncoupling protein gene using a yeast heterologous expression system
Assessment of uncoupling activity of the human uncoupling protein 3 short form and three mutants of the uncoupling protein gene using a yeast heterologous expression system
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DOI：
10.1016/s0014-5793(99)00811-x
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发表时间：
1999-07-09
期刊：
影响因子：
3.5
通讯作者：
Lowell, BB
中科院分区：
文献类型：
--
作者：
Hagen, T;Zhang, CY;Lowell, BB
人类解偶联蛋白3基因产生两个mRNA转录本，解偶联蛋白3L和解偶联蛋白3S，它们被预测分别编码解偶联蛋白3蛋白的长和短形式。解偶联蛋白3L在长度上与其他已知解偶联蛋白相似，但解偶联蛋白3S缺少最后37个C末端残基。人类解偶联蛋白3基因的剪接点突变导致解偶联蛋白3S的独占表达，以及解偶联蛋白3基因的一些点突变已被描述。本研究利用酵母表达系统对解偶联蛋白3S及其3个突变体(V9M、V102I、R282C)和解偶联蛋白3L的生化活性进行了比较。所有蛋白质的表达水平相似，并且对与解偶联功能相关的参数具有定性相似的影响。解偶联蛋白3S和解偶联蛋白3L使酵母的生长速度分别降低了35%和52%，全酵母的基础氧耗量分别增加了26%和48%，通过摄取荧光电位敏感染料3‘3-二己氧碳菁碘，全酵母的线粒体膜电位降低。在分离的线粒体中，解偶联蛋白3S和解偶联蛋白3L引起状态4呼吸的增加(分别为33%和35%)，但与解偶联蛋白1(102%)相比相对较小。解偶联蛋白3S的截短版本缺少最后三个C末端残基Tyr、Lys和Gly，这三个残基是在所有线粒体载体中高度保守的载体基序的一部分，解偶联活性大大降低。两个自然产生的解偶联蛋白3突变体V9M和V102I在对酵母生长和全酵母O-2消耗的影响方面与解偶联蛋白3L相似。与解偶联蛋白3L相比，R282C突变体的作用减弱。总之，解偶联蛋白3S和解偶联蛋白3的三个突变体似乎是具有类似解偶联蛋白3L的生化活性的功能蛋白，尽管解偶联蛋白3S和R282C突变体的功能略有降低。(C)1999年欧洲生化学会联合会。
The human uncoupling protein 3 gene generates two mRNA transcripts, uncoupling protein 3L and uncoupling protein 3S, which are predicted to encode long and short forms of the uncoupling protein 3 protein, respectively. While uncoupling protein 3L is similar in length to the other known uncoupling proteins, uncoupling protein 3S lacks the last 37 C-terminal residues. A splice site mutation in the human uncoupling protein 3 gene, resulting in the exclusive expression of uncoupling protein 3S, and a number of point mutations in the uncoupling protein 3 gene have been described. This study compares the biochemical activity of uncoupling protein 3S as well as three mutants of the uncoupling protein 3 gene (V9M, V102I, R282C) with that of uncoupling protein 3L utilizing a yeast expression system. All proteins were expressed at similar levels and had qualitatively similar effects on parameters related to the uncoupling function. Both uncoupling protein 3S and uncoupling protein 3L decreased the yeast growth rate by 35 and 52%, increased the whole yeast basal O-2 consumption by 26 and 48%, respectively, and decreased the mitochondrial membrane potential as measured in whole yeast by uptake of the fluorescent potential-sensitive dye 3'3-dihexyloxacarbocyanine iodide. In isolated mitochondria, uncoupling protein 3S and uncoupling protein 3L, caused a similar (33 and 35%, respectively) increase in state 4 respiration, which was relatively small compared to uncoupling protein 1 (102% increase). A truncated version of uncoupling protein 3S, lacking the last three C-terminal residues, Tyr, Lys and Gly, that are part of a carrier motif that is highly conserved among all mitochondrial carriers, had a greatly reduced uncoupling activity. The two naturally occurring uncoupling protein 3 mutants, V9M and V102I, mere similar to uncoupling protein 3L with respect to effects on the yeast growth and whole yeast O-2 consumption. The R282C mutant had a reduced effect compared to uncoupling protein 3L. In summary uncoupling protein 3S and the three mutants of uncoupling protein 3 appear to be functional proteins with biochemical activities similar to uncoupling protein 3L, although uncoupling protein 3S and the R282C mutant have a modestly reduced function. (C) 1999 Federation of European Biochemical Societies.