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Abstract：
MIRO (mitochondrial Rho GTPase) consists of two GTPase domains flanking two Ca2+-binding EF-hand domains. A C-terminal transmembrane helix anchors MIRO to the outer mitochondrial membrane, where it functions as a general adaptor for the recruitment of cytoskeletal proteins that control mitochondrial dynamics. One protein recruited by MIRO is TRAK (trafficking kinesin-binding protein), which in turn recruits the microtubule-based motors kinesin-1 and dynein-dynactin. The mechanism by which MIRO interacts with TRAK is not well understood. Here, we map and quantitatively characterize the interaction of human MIRO1 and TRAK1 and test its potential regulation by Ca2+ and/or GTP binding. TRAK1 binds MIRO1 with low micromolar affinity. The interaction was mapped to a fragment comprising MIRO1\'s EF-hands and C-terminal GTPase domain and to a conserved sequence motif within TRAK1 residues 394 to 431, immediately C-terminal to the Spindly motif. This sequence is sufficient for MIRO1 binding in vitro and is necessary for MIRO1-dependent localization of TRAK1 to mitochondria in cells. MIRO1\'s EF-hands bind Ca2+ with dissociation constants (KD) of 3.9 μM and 300 nM. This suggests that under cellular conditions one EF-hand may be constitutively bound to Ca2+ whereas the other EF-hand binds Ca2+ in a regulated manner, depending on its local concentration. Yet, the MIRO1-TRAK1 interaction is independent of Ca2+ binding to the EF-hands and of the nucleotide state (GDP or GTP) of the C-terminal GTPase. The interaction is also independent of TRAK1 dimerization, such that a TRAK1 dimer can be expected to bind two MIRO1 molecules on the mitochondrial surface.
摘要：
MIRO（线粒体RhoGTPase）由两个GTPase结构域组成，两个结合Ca2的EF手结构域侧翼。C端跨膜螺旋将MIRO锚定到线粒体外膜，在这里，它可以作为一个通用的适配器，用于募集控制线粒体动力学的细胞骨架蛋白。MIRO招募的一种蛋白质是TRAK（运输驱动蛋白结合蛋白），反过来招募基于微管的运动驱动蛋白1和动力蛋白-动力蛋白。MIRO与线粒体膜上的TRAK相互作用的机制尚不清楚。这里,我们绘制并定量表征了人MIRO1和TRAK1的相互作用，并测试了Ca2和/或GTP与MIRO1结合的潜在调节。TRAK1以低微摩尔亲和力结合MIRO1。相互作用被映射到包含MIRO1的EF手和C端GTP酶结构域的片段，并映射到TRAK1残基394-431内的保守序列基序，紧邻Spindly基序的C端。该序列足以在体外结合MIRO1，并且对于MIRO1依赖性TRAK1定位到细胞中的线粒体是必需的。MIRO1的EF-手结合Ca2+,解离常数(KD)为3.9μM和300nM。这表明，在细胞条件下，一个EF-手可能组成性地与Ca2结合，而另一个EF-手以调节的方式与Ca2结合。取决于当地的浓度。然而，MIRO1-TRAK1相互作用独立于Ca2与EF手的结合以及C末端GTP酶的核苷酸状态（GDP或GTP）。这种相互作用也独立于TRAK1二聚化，这样可以预期TRAK1二聚体结合线粒体表面上的两个MIRO1分子。
