Sequence determinants of C-terminal substrate recognition by the Tsp protease
Sequence determinants of C-terminal substrate recognition by the Tsp protease
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DOI：
10.1074/jbc.271.5.2589
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发表时间：
1996-02-02
影响因子：
4.8
通讯作者：
Sauer, RT
中科院分区：
文献类型：
--
作者：
Keiler, KC;Sauer, RT
细胞色素B（562）在体外或在大肠杆菌的周质中不被尾特异性蛋白酶Tsp切割，但当添加C-末端序列WVAAA时，细胞色素b（562）成为良好的底物。在该底物的最后三个残基位置随机化之后，回收了54个具有单残基取代的不同突变体，带有这些突变尾部的细胞色素变体的稳态表达水平在E.大肠杆菌菌株中缺失了tsp基因，但在含有Tsp的菌株中差异显著。纯化了野生型细胞色素B（562）和7种变体，显示出一系列细胞内表达水平。发现这些蛋白质在热变性实验中具有相同的T-m值，但被Tsp以相差多达30倍的速率切割。总的来说，这些蛋白质的体外Tsp切割速率与它们的体内切割速率相关，如通过脉冲追踪实验所确定的。这些结果表明细胞色素-b（562）变体的C-末端序列在决定它们在细胞中的蛋白水解命运方面是重要的，并且表明这种降解主要由Tsp介导。在三个C-末端位置中的每一个处存在不同的选择性规则。底物的C-末端残基（其中优选小的不带电荷的残基（Ala、Cys、Ser、Thr、瓦尔））的身份在确定Tsp切割底物的速率中是最重要的。在第二和第三位置处也优选非极性残基，但在良好底物中在这些位置处也可接受更大和更疏水的侧链。
Cytochrome b(562) is not cleaved by the tail-specific protease Tsp in vitro or in the periplasm of Escherichia coli but becomes a good substrate when the C-terminal sequence WVAAA is added, Following randomization of the final three residue positions of this substrate, 54 different mutants with single residue substitutions were recovered, The steady-state expression levels of cytochrome variants bearing these mutant tails were similar in an E. coli strain deleted for the tsp gene but differed markedly in a strain containing Tsp. Wild-type cytochrome b(562) and seven variants, displaying a range of intracellular expression levels, were purified. These proteins were found to have the same T-m values in thermal denaturation experiments but to be cleaved by Tsp at rates differing by as much as 30-fold. Overall, the rates of Tsp cleavage of these proteins in vitro correlate with their rates of cleavage in vivo as determined by pulse-chase experiments, These results indicate that the C-terminal sequence of the cytochrome-b(562) variants is important in determining their proteolytic fate in the cell and show that this degradation is mediated predominantly by Tsp, There are different selectivity rules at each of the three C-terminal positions. The identity of the C-terminal residue of the substrate, where small, uncharged residues (Ala, Cys, Ser, Thr, Val) are preferred, is most important in determining the rate of substrate cleavage by Tsp. Non-polar residues are also preferred at the second and third positions, but larger and more hydrophobic side chains are also acceptable at these positions in good substrates.