Department of Biology, Indiana University, Bloomington, Indiana, USA.
Department of Biochemistry and Molecular Biology, University of Massachusetts Amherst, Amherst, Massachusetts, USA.
J Biol Chem. 2022 Jul;298(7):102045. doi: 10.1016/j.jbc.2022.102045. Epub 2022 May 18.
Regulatory proteolysis targets properly folded clients via a combination of cis-encoded degron sequences and trans-expressed specificity factors called adaptors. SmiA of Bacillus subtilis was identified as the first adaptor protein for the Lon family of proteases, but the mechanism of SmiA-dependent proteolysis is unknown. Here, we develop a fluorescence-based assay to measure the kinetics of SmiA-dependent degradation of its client SwrA and show that SmiA-SwrA interaction and the SwrA degron were both necessary, but not sufficient, for proteolysis. Consistent with a scaffolding adaptor mechanism, we found that stoichiometric excess of SmiA caused substrate-independent inhibition of LonA-dependent turnover. Furthermore, SmiA was strictly required even when SwrA levels were high suggesting that a local increase in substrate concentration mediated by the scaffold was not sufficient for proteolysis. Moreover, SmiA function could not be substituted by thermal denaturation of the substrate, consistent with a priming adaptor mechanism. Taken together, we conclude that SmiA functions via a mechanism that is a hybrid between scaffolding and priming models.
调控蛋白水解通过顺式编码降解序列和反式表达的特异性因子（称为衔接蛋白）的组合靶向正确折叠的客户。枯草芽孢杆菌的 SmiA 被确定为 Lon 蛋白酶家族的第一个衔接蛋白，但 SmiA 依赖性蛋白水解的机制尚不清楚。在这里，我们开发了一种基于荧光的测定法来测量 SmiA 依赖性降解其客户 SwrA 的动力学，并表明 SmiA-SwrA 相互作用和 SwrA 降解序列都是必需的，但不足以进行蛋白水解。与支架衔接蛋白机制一致，我们发现，SmiA 的化学计量过量会导致 LonA 依赖性周转率的底物非依赖性抑制。此外，即使 SwrA 水平较高，SmiA 也严格需要，这表明通过支架介导的底物浓度局部增加不足以进行蛋白水解。此外，即使底物水平较高，SmiA 的功能也不能被热变性替代，这与引发衔接蛋白机制一致。综上所述，我们得出结论，SmiA 通过一种介于支架和引发模型之间的机制发挥作用。
