Membrane topology and mitochondrial targeting of mitofusins, ubiquitous mammalian homologs of the transmembrane GTPase Fzo.
Membrane topology and mitochondrial targeting of mitofusins, ubiquitous mammalian homologs of the transmembrane GTPase Fzo.
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发表时间：
2002-04
影响因子：
4
通讯作者：
M. Rojo;Frédéric Legros;D. Château;A. Lombès
中科院分区：
文献类型：
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作者：
M. Rojo;Frédéric Legros;D. Château;A. Lombès
两个人FZO同源物，线粒体融合蛋白Mfn 1和Mfn 2，通过RT-PCR和蛋白质印迹显示是普遍存在的线粒体蛋白。蛋白酶消化实验表明，Mfn 2是一种外膜蛋白，其N-末端和C-末端结构域暴露于胞质溶胶中。Mfn 2（Mfn 2-TMCT）的跨膜和C-末端结构域靶向线粒体，并且这些结构域的缺失导致截短的Mfn 2（Mfn 2-NT）的胞质定位。当C-末端结构域被短的中性/疏水（Mfn 2-IYFFT）或极性/碱性（Mfn 2-RRD）氨基酸取代时，Mfn 2靶向内质网或线粒体。Mfn 2的卷曲螺旋结构域（跨膜结构域的上游和下游）对于线粒体靶向也很重要：缺失任何卷曲螺旋结构域的Mfn 2突变体仅部分靶向线粒体，并且大量蛋白质保留在胞质中。我们发现这些卷曲螺旋结构域相互作用：如果与Mfn 2-TMCT共表达，则错误的Mfn 2-NT或Mfn 2-IYFFT定位于线粒体。当卷曲螺旋结构域在任何共转染的分子中缺失时，这种重新定位被消除。我们还发现Mfn 2可以独立于细胞骨架将活性线粒体聚集在核周区域，使线粒体膜紧密接触并修改线粒体结构，而不会干扰内外膜的完整性。
Two human Fzo-homologs, mitofusins Mfn1 and Mfn2, are shown by RT-PCR and western blot to be ubiquitous mitochondrial proteins. Protease digestion experiments reveal that Mfn2 is an outer membrane protein with N-terminal and C-terminal domains exposed towards the cytosol. The transmembrane and C-terminal domains of Mfn2 (Mfn2-TMCT) are targeted to mitochondria and deletion of these domains leads to the cytosolic localization of truncated Mfn2 (Mfn2-NT). Mfn2 is targeted to the endoplasmic reticulum or to mitochondria when the C-terminal domain is replaced by short stretches of neutral/hydrophobic (Mfn2-IYFFT) or polar/basic (Mfn2-RRD) amino acids. The coiled-coil domains of Mfn2, upstream and downstream of the transmembrane domain, are also important for mitochondrial targeting: Mfn2-mutants deleted of any of its coiled-coil domains are only partially targeted to mitochondria and significant protein amounts remain cytosolic. We show that these coiled-coil domains interact with each other: mistargeted Mfn2-NT or Mfn2-IYFFT localize to mitochondria if co-expressed with Mfn2-TMCT. This relocalization is abolished when the coiled-coil domain is deleted in any of the co-transfected molecules. We also found that Mfn2 can cluster active mitochondria in the perinuclear region independently of the cytoskeleton, bring mitochondrial membranes into close contact and modify mitochondrial structure, without disturbing the integrity of the inner and outer membrane.