Reevaluation of the role of the Pam18:Pam16 interaction in translocation of proteins by the mitochondrial Hsp70-based import motor.
Reevaluation of the role of the Pam18:Pam16 interaction in translocation of proteins by the mitochondrial Hsp70-based import motor.
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DOI：
10.1091/mbc.e11-08-0715
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发表时间：
2011-12
影响因子：
3.3
通讯作者：
Craig EA
中科院分区：
文献类型：
--
作者：
Pais JE;Schilke B;Craig EA
Pam 18是基于Hsp 70的线粒体输入马达的J蛋白辅伴侣，与结构相关的蛋白Pam 16形成异二聚体。遗传和生物化学研究表明，这种相互作用在维持Pam 18与易位子的关联中起着关键作用，而不是其先前提出的调节作用。基于热休克蛋白70（Hsp 70）的输入马达，与线粒体内膜基质侧的易位子相关，通过结合和释放的循环驱动蛋白质的易位。由translocon相关的J-蛋白Pam 18刺激Hsp 70的ATP酶活性对于该过程是关键的。Pam 18通过其J型结构域与结构相关的蛋白质Pam 16形成异二聚体。这种相互作用已被提出执行一个关键的调节功能，抑制ATP酶的刺激活性的Pam 18。使用生化和遗传分析，我们测试了这一假设，通过评估在体内功能的Pam 18变体具有改变的能力，刺激热休克蛋白70的ATP酶活性。所观察到的遗传相互作用的模式是相反的预测，如果异二聚体提供抑制功能，相反的模式是一致的突变，已知引起异二聚体的稳定性降低。对Pam 16的一个以前未表征的区域的分析揭示了其形成活性Pam 18：Pam 16复合物的需要，该复合物能够刺激Hsp 70的ATP酶活性。总之，我们的数据与Pam 18和Pam 16形成稳定的异源二聚体的想法一致，并且Pam 18：Pam 16相互作用的关键作用是Pam 18通过其与Pam 16的相互作用与易位子的物理束缚。
Pam18, the J-protein cochaperone of the Hsp70-based mitochondrial import motor, forms a heterodimer with the structurally related protein Pam16. Genetic and biochemical studies suggest a critical role of this interaction in maintaining Pam18's association with the translocon rather than its previously proposed regulatory role. The heat-shock protein 70 (Hsp70)–based import motor, associated with the translocon on the matrix side of the mitochondrial inner membrane, drives translocation of proteins via cycles of binding and release. Stimulation of Hsp70's ATPase activity by the translocon-associated J-protein Pam18 is critical for this process. Pam18 forms a heterodimer with the structurally related protein Pam16, via their J-type domains. This interaction has been proposed to perform a critical regulatory function, inhibiting the ATPase stimulatory activity of Pam18. Using biochemical and genetic assays, we tested this hypothesis by assessing the in vivo function of Pam18 variants having altered abilities to stimulate Hsp70's ATPase activity. The observed pattern of genetic interactions was opposite from that predicted if the heterodimer serves an inhibitory function; instead the pattern was consistent with that of mutations known to cause reduction in the stability of the heterodimer. Analysis of a previously uncharacterized region of Pam16 revealed its requirement for formation of an active Pam18:Pam16 complex able to stimulate Hsp70's ATPase activity. Together, our data are consistent with the idea that Pam18 and Pam16 form a stable heterodimer and that the critical role of the Pam18:Pam16 interaction is the physical tethering of Pam18 to the translocon via its interaction with Pam16.