Artifactual uncoupling by uncoupling protein 3 in yeast mitochondria at the concentrations found in mouse and rat skeletal-muscle mitochondria
Artifactual uncoupling by uncoupling protein 3 in yeast mitochondria at the concentrations found in mouse and rat skeletal-muscle mitochondria
复制标题
DOI：
10.1042/0264-6021:3610049
复制
发表时间：
2002-01-01
影响因子：
4.1
通讯作者：
Brand, MD
中科院分区：
文献类型：
--
作者：
Harper, JA;Stuart, JA;Brand, MD
蛋白质印迹法检测到解偶联蛋白3（UCP 3）在野生型，但不是UCP 3基因敲除小鼠的睾丸肌线粒体。用纯化的重组UCP 3校准显示小鼠和大鼠骨骼肌含有0.14 μ g UCP 3/mg线粒体蛋白。这种非常低的UCP 3含量比来自暖适应仓鼠的棕色脂肪组织线粒体中的UCP 1浓度（24-84 μ g UCP 1/mg线粒体蛋白）低200-700倍。UCP 3存在于热适应大鼠的棕色脂肪组织线粒体中，但在大鼠心脏线粒体中检测不到。我们在酵母线粒体中表达了人类UCP 3，其水平与啮齿动物脑线粒体中的相似，是啮齿动物脑线粒体中的两倍和7倍。含有UCP 3的酵母线粒体比空载体对照更解偶联，特别是在7倍生理浓度下。然而，解偶联由UCP 3没有刺激已知的激活剂棕榈酸酯和超氧化物，也没有被GDP抑制，这表明所观察到的解偶联是非天然蛋白质的属性。作为对照，UCP 1在酵母线粒体中以与UCP 3相似的浓度表达，并且以高达UCP 1生理水平的50%表达。低水平的UCP 1棕榈酸依赖和GDP敏感的质子电导，但较高水平的UCP 1造成了额外的GDP不敏感的解偶联伪影。我们的结论是解偶联酵母线粒体的高水平的UCP 3表达完全是一个文物，并提供任何天然的解偶联活性的蛋白质没有证据。
Western blots detected uncoupling protein 3 (UCP3) in skeletal-muscle mitochondria from wild-type but not UCP3 knock-out mice. Calibration with purified recombinant UCP3 showed that mouse and rat skeletal muscle contained 0.14 mug of UCP3/mg of mitochondrial protein. This very low UCP3 content is 200-700-fold less than the concentration of UCP1 in brown-adipose-tissue mitochondria from warm-adapted hamster (24-84 mug of UCP1/mg of mitochondrial protein). UCP3 was present in brown-adipose-tissue mitochondria from warm-adapted rats but was undetectable in rat heart mitochondria. We expressed human UCP3 in yeast mitochondria at levels similar to, double and 7-fold those found in rodent skeletal-musele mitochondria. Yeast mitochondria containing UCP3 were more uncoupled than empty-vector controls, particularly at concentrations that were 7-fold physiological. However, uncoupling by UCP3 was not stimulated by the known activators palmitate and superoxide; neither were they inhibited by GDP, suggesting that the observed uncoupling was a property of non-native protein. As a control, UCP1 was expressed in yeast mitochondria at similar concentrations to that of UCP3 and at up to 50 % of the physiological level of UCP1. Low levels of UCP1 gave palmitate-dependent and GDP-sensitive proton conductance but higher levels of UCP1 caused an additional GDP-insensitive uncoupling artifact. We conclude that the uncoupling of yeast mitochondria by high levels of UCP3 expression is entirely an artifact and provides no evidence for any native uncoupling activity of the protein.