Interfaculty Institute of Biochemistry, University of Tuebingen, Tuebingen, Germany.
Mol Cell Biol. 2013 Oct;33(20):4083-94. doi: 10.1128/MCB.00227-13. Epub 2013 Aug 19.
A special group of mitochondrial outer membrane proteins spans the membrane once, exposing soluble domains to both sides of the membrane. These proteins are synthesized in the cytosol and then inserted into the membrane by an unknown mechanism. To identify proteins that are involved in the biogenesis of the single-span model protein Mim1, we performed a high-throughput screen in yeast. Two interesting candidates were the cytosolic cochaperone Djp1 and the mitochondrial import receptor Tom70. Our results indeed demonstrate a direct interaction of newly synthesized Mim1 molecules with Tom70. We further observed lower steady-state levels of Mim1 in mitochondria from djp1Δ and tom70 tom71Δ cells and massive mislocalization of overexpressed GFP-Mim1 to the endoplasmic reticulum in the absence of Djp1. Importantly, these phenotypes were observed specifically for the deletion of DJP1 and were not detected in mutant cells lacking any of the other cytosolic cochaperones of the Hsp40 family. Furthermore, the djp1Δ tom70Δ tom71Δ triple deletion resulted in a severe synthetic sick/lethal growth phenotype. Taking our results together, we identified Tom70 and Djp1 as crucial players in the biogenesis of Mim1. Moreover, the involvement of Djp1 provides a unique case of specificity between a cochaperone and its substrate protein.
一组特殊的线粒体外膜蛋白单次跨膜，将可溶性结构域暴露于膜的两侧。这些蛋白质在细胞质中合成，然后通过未知机制插入膜中。为了鉴定参与单跨模型蛋白 Mim1 生物发生的蛋白质，我们在酵母中进行了高通量筛选。两个有趣的候选者是细胞质伴侣 Djp1 和线粒体输入受体 Tom70。我们的结果确实表明新合成的 Mim1 分子与 Tom70 之间存在直接相互作用。我们进一步观察到，djp1Δ 和 tom70 tom71Δ 细胞中线粒体中 Mim1 的稳态水平较低，并且在没有 Djp1 的情况下，过量表达的 GFP-Mim1 大量错误定位到内质网。重要的是，这些表型仅在 DJP1 的缺失中观察到，而在缺乏 HSP40 家族任何其他细胞质伴侣的突变细胞中未检测到。此外，djp1Δ tom70Δ tom71Δ 三重缺失导致严重的合成病/致死生长表型。综合我们的结果，我们确定 Tom70 和 Djp1 是 Mim1 生物发生的关键参与者。此外，Djp1 的参与提供了伴侣蛋白与其底物蛋白之间特异性的独特案例。
