Laboratoire de Biologie Physico-Chimique des Protéines Membranaires, LBPC-PM, CNRS, UMR7099, Université de Paris, France.
Institut de Biologie Physico-Chimique, Fondation Edmond de Rothschild pour le Développement de la Recherche Scientifique, Paris, France.
FEBS J. 2021 May;288(9):3024-3033. doi: 10.1111/febs.15629. Epub 2020 Dec 7.
Uncoupling protein 1 (UCP1) is found in the inner mitochondrial membrane of brown adipocytes. In the presence of long-chain fatty acids (LCFAs), UCP1 increases the proton conductance, which, in turn, increases fatty acid oxidation and energy release as heat. Atomic models of UCP1 and UCP2 have been generated based on the NMR backbone structure of UCP2 in dodecylphosphocholine (DPC), a detergent known to inactivate UCP1. Based on NMR titration experiments on UCP1 with LCFA, it has been proposed that K56 and K269 are crucial for LCFA binding and UCP1 activation. Given the numerous controversies on the use of DPC for structure-function analyses of membrane proteins, we revisited those UCP1 mutants in a more physiological context by expressing them in the mitochondria of Saccharomyces cerevisiae. Mitochondrial respiration, assayed on permeabilized spheroplasts, enables the determination of UCP1 activation and inhibition. The K56S, K269S, and K56S/K269S mutants did not display any default in activation, which shows that the NMR titration experiments in DPC detergent are not relevant to UCP1 function.
解偶联蛋白 1（UCP1）存在于棕色脂肪细胞的线粒体内膜中。在长链脂肪酸（LCFAs）存在的情况下，UCP1 增加质子通透性，从而增加脂肪酸氧化和以热量形式释放能量。基于 NMR 背靠背结构的 UCP2 在十二烷基磷酸胆碱（DPC）中的原子模型，DPC 是一种已知使 UCP1 失活的去污剂。基于与 LCFAs 的 NMR 滴定实验，提出 K56 和 K269 对于 LCFAs 结合和 UCP1 激活至关重要。鉴于 DPC 在膜蛋白结构功能分析中的使用存在许多争议，我们通过在酿酒酵母的线粒体中表达这些 UCP1 突变体，在更生理的环境中重新研究了这些 UCP1 突变体。在通透化的原生质球上进行的线粒体呼吸测定可确定 UCP1 的激活和抑制。K56S、K269S 和 K56S/K269S 突变体没有显示任何激活缺陷，这表明 DPC 去污剂中的 NMR 滴定实验与 UCP1 功能无关。
