Magnani M, Serafini G, Antonelli A, Malatesta M, Gazzanelli G
Instituto di Chimica Biologica G. Fornaini, Università degli Studi, Urbino, Italy.
J Biol Chem. 1991 Nov 5;266(31):21018-24.
Conjugate ubiquitin was previously found in the nucleus, cytoplasm, and membranes of eukaryotic cells while the enzymes of the ubiquitin-conjugating system appear to be cytoplasmic. We have prepared the mitochondrial fraction from rabbit brain by discontinuous density gradient ultracentrifugation and by Western blotting, using a specific antibody against conjugate ubiquitin, showing that it contains ubiquitin conjugates in a very wide molecular weight range. Electron microscopy and measurement of specific enzyme markers show that this fraction not only contains mitochondria but also some endoplasmic reticulum vesicles. Immunostaining with anti-ubiquitin IgG followed by immunodecoration with colloidal gold particles provides evidence for the presence of conjugate ubiquitin both in mitochondria and in the endoplasmic reticulum. Furthermore, this "mitochondrial fraction" shows a pronounced ATP-dependent ability to conjugate 125I-ubiquitin into a number of endogenous proteins as evidenced by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Addition of E1, E2, and E3, the enzymes of the ubiquitin conjugating system purified from rabbit reticulocytes, does not further increase this ubiquitination nor incorporate 125I-ubiquitin into additional protein bands. The same mitochondrial fraction is not able to carry out any ATP-dependent degradation of 125I-albumin; however, it contains an isopeptidase activity able to release the covalently incorporated 125I-ubiquitin and is also able to conjugate 125I-ubiquitin to exogenous proteins as oxidized RNase. By affinity chromatography on ubiquitin-agarose of fraction II of a crude Triton X-100 extract of the mitochondrial fraction, several proteins corresponding in Mr to the E1 and E2s enzymes were obtained. These proteins were also able to form specific ubiquitin-thiol ester bounds on sodium dodecyl sulfate-polyacrylamide gels and to support 125I-ubiquitin conjugation to oxidized RNase. Detergent fractionation of the mitochondrial fraction provided evidence for a possible localization of the ubiquitin conjugating activity in the mitochondrial external membrane and endoplasmic reticulum. The presence of an active ubiquitin protein conjugating system in mitochondria and endoplasmic reticulum may be related to the turnover of organelle proteins as well as to specific cell functions such as import of proteins into mitochondria and ubiquitination of externally oriented membrane-bound proteins.
之前在真核细胞的细胞核、细胞质和细胞膜中发现了缀合泛素，而泛素缀合系统的酶似乎位于细胞质中。我们通过不连续密度梯度超速离心法从兔脑中制备了线粒体组分，并使用针对缀合泛素的特异性抗体进行蛋白质印迹分析，结果表明该组分含有分子量范围非常广的泛素缀合物。电子显微镜检查和特定酶标志物的测定表明，该组分不仅含有线粒体，还含有一些内质网囊泡。用抗泛素IgG进行免疫染色，随后用胶体金颗粒进行免疫标记，为线粒体和内质网中存在缀合泛素提供了证据。此外，这种“线粒体组分”表现出明显的ATP依赖性能力，可将125I-泛素缀合到多种内源性蛋白质中，十二烷基硫酸钠-聚丙烯酰胺凝胶电泳和放射自显影证明了这一点。添加从兔网织红细胞中纯化的泛素缀合系统的酶E1、E2和E3，并不会进一步增加这种泛素化作用，也不会将125I-泛素掺入额外的蛋白条带中。相同的线粒体组分无法对125I-白蛋白进行任何ATP依赖性降解；然而，它含有一种异肽酶活性，能够释放共价结合的125I-泛素，并且还能够将125I-泛素缀合到外源性蛋白质如氧化核糖核酸酶上。通过对线粒体组分粗制Triton X-100提取物的组分II进行泛素-琼脂糖亲和层析，获得了几种分子量与E1和E2酶相对应的蛋白质。这些蛋白质在十二烷基硫酸钠-聚丙烯酰胺凝胶上也能够形成特定的泛素-硫酯键，并支持将125I-泛素缀合到氧化核糖核酸酶上。线粒体组分的去污剂分级分离为泛素缀合活性可能定位于线粒体外膜和内质网提供了证据。线粒体和内质网中存在活跃的泛素-蛋白质缀合系统可能与细胞器蛋白质的周转以及特定的细胞功能有关，如蛋白质导入线粒体和外向性膜结合蛋白的泛素化。
