Role of protein kinase C-mediated protein phosphorylation in mitochondrial translocation of mouse CYP1A1, which contains a non-canonical targeting signal
Role of protein kinase C-mediated protein phosphorylation in mitochondrial translocation of mouse CYP1A1, which contains a non-canonical targeting signal
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DOI：
10.1074/jbc.m510725200
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发表时间：
2006-10-13
影响因子：
4.8
通讯作者：
Avadhani, Narayan G.
中科院分区：
文献类型：
--
作者：
Dasari, Venkata Ramesh;Anandatheerthavarada, Hindupur K.;Avadhani, Narayan G.
大量的线粒体蛋白缺乏典型的线粒体靶向信号。细胞色素P450（CYP）向内质网和线粒体（MT）的双峰运输，我们以前报道过，可能代表了一种模式的非经典蛋白质靶向MT。因此，我们研究了小鼠MT-CYP 1A 1的靶向机制，以了解双峰靶向机制的调控特征和进化保守性。小鼠MT-CYP 1A 1由两种NH 2-末端截短的分子种类组成，+91 A1和+331 A1。增加信号识别颗粒（SRP）结合的突变Pro-2 -> Leu和Tyr-5 -> Leu减少了完整细胞中蛋白质的MT靶向。相比之下，突变Leu-7 -> Asn和Leu-17 -> 3 Asn降低了SRP结合亲和力，增强了MT靶向，因此表明SRP结合是调节双峰靶向的重要调节步骤。蛋白激酶C（PKC）介导的磷酸化的新生链在Thr-35大大降低了SRP结合的亲和力，这表明一个重要的监管步骤。在支持这些结果，COS细胞转染实验表明，磷酸化模拟突变Thr-353 Asp或诱导细胞PKC引起增加的CYP 1A 1靶向MT和相应的较低水平的内质网。结果表明，进化保守的嵌合信号和双峰靶向CYP 1A 1在不同的物种。小鼠MT-CYP 1A 1是一种外源性膜蛋白，表现出大量三环类抗抑郁药、止痛药、抗精神病药和麻醉剂的高FDX 1 + FDXR介导的N-去甲基化，这些药物是微粒体CYP 1A 1的不良底物。
A large number of mitochondrial proteins lack canonical mitochondrial-targeting signals. The bimodal transport of cytochromes P450 (CYPs) to endoplasmic reticulum and mitochondria (MT), reported previously by us, likely represents one mode of non-canonical protein targeting to MT. Herein, we have studied the mechanism of mouse MT-CYP1A1 targeting to gain insight into the regulatory features and evolutionary conservation of bimodal targeting mechanism. Mouse MT-CYP1A1 consists of two NH2-terminal-truncated molecular species, + 91A1 and + 331A1. Mutations Pro-2 -> Leu and Tyr-5 -> Leu, which increase the signal recognition particle (SRP) binding, diminished MT targeting of the protein in intact cells. By contrast, mutations Leu-7 -> Asn and Leu-17 -> 3 Asn, which decreased SRP-binding affinity, enhanced MT targeting, thus suggesting that SRP binding is an important regulatory step that modulates bimodal targeting. Protein kinase C(PKC)-mediated phosphorylation of nascent chains at Thr-35 vastly decreased affinity for SRP binding suggesting an important regulatory step. In support of these results, COS cell transfection experiments show that phosphomimetic mutation Thr-353Asp or induced cellular PKC caused increased CYP1A1 targeting to MT and correspondingly lower levels to the endoplasmic reticulum. Results suggest evolutionary conservation of chimeric signals and bimodal targeting of CYP1A1 in different species. The mouse MT-CYP1A1 is an extrinsic membrane protein, which exhibited high FDX1 plus FDXR-mediated N-demethylation of a number of tricyclic antidepressants, pain killers, antipsychotics, and narcotics that are poor substrates for microsomal CYP1A1.