Cloning and functional analysis of the ubiquitin-specific protease gene UBP1 of Saccharomyces cerevisiae.
Cloning and functional analysis of the ubiquitin-specific protease gene UBP1 of Saccharomyces cerevisiae.
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DOI：
10.1016/s0021-9258(18)99059-9
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发表时间：
1991-06
期刊：
影响因子：
--
通讯作者：
J. Tobias;A. Varshavsky
中科院分区：
文献类型：
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作者：
J. Tobias;A. Varshavsky
在真核生物中，泛素与自身或其他蛋白质的天然和工程化融合体都通过在泛素的最后一个残基（Gly 76）之后加工蛋白酶来切割。使用同胞选择的方法，并利用细菌，如大肠杆菌缺乏泛素特异性酶的事实，我们已经克隆了一个基因，命名为UBP 1，酵母酿酒酵母编码的泛素特异性加工蛋白酶。除了多聚泛素之外，UBP 1蛋白酶在天然和工程融合物中的泛素部分的羧基末端切割，而不管它们的大小或氨基末端泛素延伸的存在。UBP 1的这些特性将其与之前克隆的酵母蛋白酶YUH 1区分开来，YUH 1使相对较短的遍在蛋白融合体去遍在蛋白，但对于较长的融合体（例如遍在蛋白-β-半乳糖苷酶）几乎没有活性。809个残基的UBP 1的氨基酸序列与其他已知蛋白质缺乏显著的相似性，包括236个残基的YUH 1蛋白酶。ubp 1空突变体是可行的，并且保留了去遍在蛋白-β-半乳糖苷酶的能力，这表明酵母中的遍在蛋白特异性蛋白酶家族并不限于UBP 1和YUH 1。
In eukaryotes, both natural and engineered fusions of ubiquitin to itself or other proteins are cleaved by processing proteases after the last (Gly76) residue of ubiquitin. Using the method of sib selection, and taking advantage of the fact that bacteria such as Escherichia coli lack ubiquitin-specific enzymes, we have cloned a gene, named UBP1, of the yeast Saccharomyces cerevisiae that encodes a ubiquitin-specific processing protease. With the exception of polyubiquitin, the UBP1 protease cleaves at the carboxyl terminus of the ubiquitin moiety in natural and engineered fusions irrespective of their size or the presence of an amino-terminal ubiquitin extension. These properties of UBP1 distinguish it from the previously cloned yeast protease YUH1, which deubiquitinates relatively short ubiquitin fusions but is virtually inactive with longer fusions such as ubiquitin-beta-galactosidase. The amino acid sequence of the 809-residue UBP1 lacks significant similarities to other known proteins, including the 236-residue YUH1 protease. Null ubp1 mutants are viable, and retain the ability to deubiquitinate ubiquitin-beta-galactosidase, indicating that the family of ubiquitin-specific proteases in yeast is not limited to UBP1 and YUH1.