Parrell Daniel, Zhang Yang, Olenic Sandra, Kroos Lee
Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, Michigan, USA.
Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, Michigan, USA
J Bacteriol. 2017 Sep 5;199(19). doi: 10.1128/JB.00381-17. Print 2017 Oct 1.
RasP is a predicted intramembrane metalloprotease of that has been proposed to cleave the stress response anti-sigma factors RsiW and RsiV, the cell division protein FtsL, and remnant signal peptides within their transmembrane segments. To provide evidence for direct effects of RasP on putative substrates, we developed a heterologous coexpression system. Since expression of catalytically inactive RasP E21A inhibited expression of other membrane proteins in , we added extra transmembrane segments to RasP E21A, which allowed accumulation of most other membrane proteins. A corresponding active version of RasP appeared to promiscuously cleave coexpressed membrane proteins, except those with a large periplasmic domain. However, stable cleavage products were not observed, even in mutant Fusions of transmembrane segment-containing parts of FtsL and RsiW to maltose-binding protein (MBP) also resulted in proteins that appeared to be RasP substrates upon coexpression in , including FtsL with a full-length C-terminal domain (suggesting that prior cleavage by a site 1 protease is unnecessary) and RsiW designed to mimic the PrsW site 1 cleavage product (suggesting that further trimming by extracytoplasmic protease is unnecessary). Purified RasP cleaved His-MBP-RsiW(73-118) within the RsiW transmembrane segment based on mass spectrometry analysis, demonstrating that RasP is an intramembrane protease. Surprisingly, purified RasP failed to cleave His-MBP-FtsL(23-117). We propose that the lack of α-helix-breaking residues in the FtsL transmembrane segment creates a requirement for the membrane environment and/or an additional protein(s) in order for RasP to cleave FtsL. Intramembrane proteases govern important signaling pathways in nearly all organisms. In bacteria, they function in stress responses, cell division, pathogenesis, and other processes. Their membrane-associated substrates are typically inferred from genetic studies in the native bacterium. Evidence for direct effects has come sometimes from coexpression of the enzyme and potential substrate in a heterologous host and rarely from biochemical reconstitution of cleavage We applied these two approaches to the enzyme RasP and its proposed substrates RsiW and FtsL. We discovered potential pitfalls and solutions in heterologous coexpression experiments in , providing evidence that both substrates are cleaved by RasP but, surprisingly, that only RsiW was cleaved , suggesting that FtsL has an additional requirement.
RasP是一种预测的跨膜金属蛋白酶，有人提出它可切割应激反应抗σ因子RsiW和RsiV、细胞分裂蛋白FtsL以及它们跨膜段内的残余信号肽。为了提供RasP对假定底物直接作用的证据，我们开发了一种异源共表达系统。由于催化无活性的RasP E21A的表达抑制了[具体物种]中其他膜蛋白的表达，我们给RasP E21A添加了额外的跨膜段，这使得大多数其他膜蛋白得以积累。相应的活性形式的RasP似乎会随意切割共表达的膜蛋白，但那些具有大的周质结构域的蛋白除外。然而，即使在[具体物种]突变体中也未观察到稳定的切割产物。将FtsL和RsiW含跨膜段的部分与[具体物种]麦芽糖结合蛋白（MBP）融合，在[具体物种]中共表达时也产生了似乎是RasP底物的蛋白，包括具有全长C端结构域的FtsL（表明不需要位点1蛋白酶预先切割）和设计成模拟PrsW位点1切割产物的RsiW（表明不需要胞外蛋白酶进一步修剪）。基于质谱分析，纯化的RasP在RsiW跨膜段内切割His-MBP-RsiW(73 - 118)，证明RasP是一种跨膜蛋白酶。令人惊讶的是，纯化的RasP未能切割His-MBP-FtsL(23 - 117)。我们提出，FtsL跨膜段中缺乏破坏α螺旋的残基使得需要膜环境和/或其他额外的蛋白质才能使RasP切割FtsL。跨膜蛋白酶几乎在所有生物体中都控制着重要的信号通路。在细菌中，它们在应激反应、细胞分裂、致病机制和其他过程中发挥作用。它们与膜相关的底物通常是从天然细菌的遗传研究中推断出来的。直接作用的证据有时来自酶和潜在底物在异源宿主中的共表达，很少来自切割的生化重建。我们将这两种方法应用于[具体物种]的酶RasP及其假定底物RsiW和FtsL。我们在[具体物种]的异源共表达实验中发现了潜在的陷阱和解决方案，提供了两种底物都被RasP切割的证据，但令人惊讶的是，只有RsiW在[具体物种]中被切割，这表明FtsL还有额外的需求。
