Co- and post-translational modifications of the 26S proteasome in yeast
Co- and post-translational modifications of the 26S proteasome in yeast
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DOI：
10.1002/pmic.200900283
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发表时间：
2010-07-01
期刊：
影响因子：
3.4
通讯作者：
Hirano, Hisashi
中科院分区：
文献类型：
--
作者：
Kikuchi, Julia;Iwafune, Yuko;Hirano, Hisashi
酵母（酿酒酵母）26S 蛋白酶体由 19S 调节颗粒 (19S RP) 和 20S 蛋白酶体亚基组成。我们使用蛋白质组技术全面检测了这些亚基的共翻译和翻译后修饰。首先，我们使用MS/MS研究了Rpt1、Rpn13和Rpn15这三个19S RP亚基Rpt1、Rpn13和Rpn15的N端修饰，发现Rpt1的N端没有被修饰，而Rpn13和Rpn15的N端被乙酰化。其次，我们在蛋白酶体的 15 个亚基中总共鉴定了 33 个 Ser/Thr 磷酸化位点。我们和其他小组获得的数据表明，26S蛋白酶体含有至少88个磷酸氨基酸，包括63个pSer、23个pThr和2个pTyr残基。用 lambda 磷酸酶对 19S RP 进行去磷酸化处理，导致 ATP 酶活性降低 30%，这表明磷酸化参与了蛋白酶体中 ATP 酶活性的调节。第三，我们尝试检测 26S 蛋白酶体的糖基化亚基。然而，我们在 19S RP 和 20S 蛋白酶体亚基中既没有鉴定出 N-和 O-连接寡糖，也没有鉴定出 O-连接 β-N-乙酰葡糖胺。迄今为止，酵母 26S 蛋白酶体中总共有 110 个共翻译修饰和翻译后修饰，包括 N-α-乙酰化、N-α-肉豆蔻酰化和磷酸化。
The yeast (Saccharomyces cerevisiae) 26S proteasome consists of the 19S regulatory particle (19S RP) and 20S proteasome subunits. We detected comprehensively co- and post-translational modifications of these subunits using proteomic techniques. First, using MS/MS, we investigated the N-terminal modifications of three 19S RP subunits, Rpt1, Rpn13, and Rpn15, which had been unclear, and found that the N-terminus of Rpt1 is not modified, whereas that of Rpn13 and Rpn15 is acetylated. Second, we identified a total of 33 Ser/Thr phosphorylation sites in 15 subunits of the proteasome. The data obtained by us and other groups reveal that the 26S proteasome contains at least 88 phospho-amino acids including 63 pSer, 23 pThr, and 2 pTyr residues. Dephosphorylation treatment of the 19S RP with lambda phosphatase resulted in a 30% decrease in ATPase activity, demonstrating that phosphorylation is involved in the regulation of ATPase activity in the proteasome. Third, we tried to detect glycosylated subunits of the 26S proteasome. However, we identified neither N- and O-linked oligosaccharides nor O-linked beta-N-acetylglucosamine in the 19S RP and 20S proteasome subunits. To date, a total of 110 co- and post-translational modifications, induding N-alpha-acetylation, N-alpha-myristoylation, and phosphorylation, in the yeast 26S proteasome have been identified.