Kickhoefer V A, Siva A C, Kedersha N L, Inman E M, Ruland C, Streuli M, Rome L H
Department of Biological Chemistry, University of California, Los Angeles School of Medicine, Los Angeles, California 90095-1737, USA.
J Cell Biol. 1999 Sep 6;146(5):917-28. doi: 10.1083/jcb.146.5.917.
Mammalian vaults are ribonucleoprotein (RNP) complexes, composed of a small ribonucleic acid and three proteins of 100, 193, and 240 kD in size. The 100-kD major vault protein (MVP) accounts for >70% of the particle mass. We have identified the 193-kD vault protein by its interaction with the MVP in a yeast two-hybrid screen and confirmed its identity by peptide sequence analysis. Analysis of the protein sequence revealed a region of approximately 350 amino acids that shares 28% identity with the catalytic domain of poly(ADP-ribose) polymerase (PARP). PARP is a nuclear protein that catalyzes the formation of ADP-ribose polymers in response to DNA damage. The catalytic domain of p193 was expressed and purified from bacterial extracts. Like PARP, this domain is capable of catalyzing a poly(ADP-ribosyl)ation reaction; thus, the 193-kD protein is a new PARP. Purified vaults also contain the poly(ADP-ribosyl)ation activity, indicating that the assembled particle retains enzymatic activity. Furthermore, we show that one substrate for this vault-associated PARP activity is the MVP. Immunofluorescence and biochemical data reveal that p193 protein is not entirely associated with the vault particle, suggesting that it may interact with other protein(s). A portion of p193 is nuclear and localizes to the mitotic spindle.
哺乳动物穹窿体是核糖核蛋白（RNP）复合物，由一种小核糖核酸和三种大小分别为100、193和240千道尔顿的蛋白质组成。100千道尔顿的主要穹窿体蛋白（MVP）占颗粒质量的70%以上。我们通过酵母双杂交筛选中MVP与该蛋白的相互作用鉴定出了193千道尔顿的穹窿体蛋白，并通过肽序列分析证实了其身份。对该蛋白质序列的分析揭示了一个约350个氨基酸的区域，它与聚（ADP - 核糖）聚合酶（PARP）的催化结构域有28%的同源性。PARP是一种核蛋白，在DNA损伤时催化ADP - 核糖聚合物的形成。p193的催化结构域从细菌提取物中表达并纯化。与PARP一样，该结构域能够催化聚（ADP - 核糖基）化反应；因此，193千道尔顿的蛋白质是一种新的PARP。纯化的穹窿体也含有聚（ADP - 核糖基）化活性，表明组装好的颗粒保留了酶活性。此外，我们表明这种与穹窿体相关的PARP活性的一种底物是MVP。免疫荧光和生化数据显示，p193蛋白并不完全与穹窿体颗粒相关，这表明它可能与其他蛋白质相互作用。一部分p193存在于细胞核中并定位于有丝分裂纺锤体。
