Institute of Molecular Biology and Biochemistry, Centre of Molecular Medicine, Medical University of Graz, Harrachgasse 21/III, 8010 Graz, Austria.
Cell Calcium. 2010 Nov;48(5):288-301. doi: 10.1016/j.ceca.2010.10.004. Epub 2010 Nov 2.
Uncoupling proteins 2 and 3 (UCP2/3) are essential for mitochondrial Ca(2+) uptake but both proteins exhibit distinct activities in regard to the source and mode of Ca(2+) mobilization. In the present work, structural determinants of their contribution to mitochondrial Ca(2+) uptake were explored. Previous findings indicate the importance of the intermembrane loop 2 (IML2) for the contribution of UCP2/3. Thus, the IML2 of UCP2/3 was substituted by that of UCP1. These chimeras had no activity in mitochondrial uptake of intracellularly released Ca(2+), while they mimicked the wild-type proteins by potentiating mitochondrial sequestration of entering Ca(2+). Alignment of the IML2 sequences revealed that UCP1, UCP2 and UCP3 share a basic amino acid in positions 163, 164 and 167, while only UCP2 and UCP3 contain a second basic residue in positions 168 and 171, respectively. Accordingly, mutants of UCP3 in positions 167 and 171/172 were made. In permeabilized cells, these mutants exhibited distinct Ca(2+) sensitivities in regard to mitochondrial Ca(2+) sequestration. In intact cells, these mutants established different activities in mitochondrial uptake of either intracellularly released (UCP3(R171,E172)) or entering (UCP3(R167)) Ca(2+). Our data demonstrate that distinct sites in the IML2 of UCP3 effect mitochondrial uptake of high and low Ca(2+) signals.
解偶联蛋白 2 和 3（UCP2/3）对于线粒体 Ca(2+)摄取是必不可少的，但这两种蛋白在 Ca(2+)动员的来源和方式方面表现出明显的活性。在本工作中，探讨了它们对线粒体 Ca(2+)摄取贡献的结构决定因素。先前的研究结果表明，跨膜环 2（IML2）对 UCP2/3 的贡献很重要。因此，UCP2/3 的 IML2 被 UCP1 的取代。这些嵌合体在细胞内释放的 Ca(2+)的线粒体摄取中没有活性，而它们通过增强进入的 Ca(2+)的线粒体隔离来模拟野生型蛋白。IML2 序列的比对表明，UCP1、UCP2 和 UCP3 在位置 163、164 和 167 共享一个碱性氨基酸，而只有 UCP2 和 UCP3 在位置 168 和 171 分别含有第二个碱性残基。因此，制作了 UCP3 在位置 167 和 171/172 的突变体。在通透细胞中，这些突变体在线粒体 Ca(2+)隔离方面表现出不同的 Ca(2+)敏感性。在完整细胞中，这些突变体在细胞内释放（UCP3(R171,E172))或进入（UCP3(R167)) Ca(2+)的线粒体摄取中建立了不同的活性。我们的数据表明，UCP3 的 IML2 中的不同位点影响高和低 Ca(2+)信号的线粒体摄取。
