A proteomic screen reveals the mitochondrial outer membrane protein Mdm34p as an essential target of the F-box protein Mdm30p
A proteomic screen reveals the mitochondrial outer membrane protein Mdm34p as an essential target of the F-box protein Mdm30p
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DOI：
10.1111/j.1365-2443.2008.01228.x
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发表时间：
2008-10-01
期刊：
影响因子：
2.1
通讯作者：
Ito, Takashi
中科院分区：
文献类型：
--
作者：
Ota, Kazuhisa;Kito, Keiji;Ito, Takashi
泛素化在真核细胞调控中起着重要的作用，并由包括泛素蛋白连接酶（E3）在内的一系列酶介导。Skp 1-Cullin-F-box蛋白复合物包括最大的E3家族，在每个成员中，独特的F-box蛋白结合其靶点以确定底物特异性。尽管基因组测序发现了越来越多的F盒蛋白，但由于难以鉴定其底物，其中大多数仍然是“孤儿”。为了解决这个问题，我们测试了一种定量蛋白质组学方法，结合稳定同位素标记的氨基酸在细胞培养（SILAC），平行亲和纯化（PAP），我们已经开发了有效的富集泛素化蛋白质，和质谱（MS）。我们应用这种SILAC-PAP-MS方法来比较具有和不具有过表达Mdm 30 p（一种与线粒体形态有关的F-box蛋白）的酵母细胞之间的泛素化蛋白。因此，我们确定了线粒体外膜蛋白Mdm 34 p作为Mdm 30 p的靶点。此外，我们发现，MDM 30的缺失引起的线粒体缺陷，不仅重演了突变体Mdm 34 p缺陷与Mdm 30 p的相互作用，但减轻了泛素化模拟形式的Mdm 34 p。这些结果表明，Mdm 34 p是Mdm 30 p的生理学重要靶标。
Ubiquitination plays various critical roles in eukaryotic cellular regulation and is medated by a cascade of enzymes including ubiquitin protein ligase (E3). The Skp1-Cullin-F-box protein complex comprises the largest E3 family, in each member of which a unique F-box protein binds its targets to define substrate specificity. Although genome sequencing uncovers a growing number of F-box proteins, most of them have remained as "orphans" because of the difficulties in identification of their substrates. To address this issue, we tested a quantitative proteomic approach by combining the stable isotope labeling by amino acids in cell culture (SILAC), parallel affinity purification (PAP) that we had developed for efficient enrichment of ubiquitinated proteins, and mass spectrometry (MS). We applied this SILAC-PAP-MS approach to compare ubiquitinated proteins between yeast cells with and without over-expressed Mdm30p, an F-box protein implicated in mitochondrial morphology. Consequently, we identified the mitochondrial outer membrane protein Mdm34p as a target of Mdm30p. Furthermore, we found that mitochondrial defects induced by deletion of MDM30 are not only recapitulated by a mutant Mdm34p defective in interaction with Mdm30p but alleviated by ubiquitination-mimicking forms of Mdm34p. These results indicate that Mdm34p is a physiologically important target of Mdm30p.