Sampathkumar Padmapriya, Mak Michelle W, Fischer-Witholt Sarah J, Guigard Emmanuel, Kay Cyril M, Lemieux M Joanne
Department of Biochemistry, University of Alberta, Edmonton, Alberta, Canada T6G 2H7.
Biochim Biophys Acta. 2012 Dec;1818(12):3090-7. doi: 10.1016/j.bbamem.2012.08.004. Epub 2012 Aug 18.
Rhomboid peptidases (proteases) play key roles in signaling events at the membrane bilayer. Understanding the regulation of rhomboid function is crucial for insight into its mechanism of action. Here we examine the oligomeric state of three different rhomboid proteases. We subjected Haemophilus influenzae, (hiGlpG), Escherichia coli GlpG (ecGlpG) and Bacillus subtilis (YqgP) to sedimentation equilibrium analysis in detergent-solubilized dodecylmaltoside (DDM) solution. For hiGlpG and ecGlpG, rhomboids consisting of the core 6 transmembrane domains without and with soluble domains respectively, and YqgP, predicted to have 7 transmembrane domains with larger soluble domains at the termini, the predominant species was dimeric with low amounts of monomer and tetramers observed. To examine the effect of the membrane domain alone on oligomeric state of rhomboid, hiGlpG, the simplest form from the rhomboid class of intramembrane proteases representing the canonical rhomboid core of six transmembrane domains, was studied further. Using gel filtration and crosslinking we demonstrate that hiGlpG is dimeric and functional in DDM detergent solution. More importantly co-immunoprecipitation studies demonstrate that the dimer is present in the lipid bilayer suggesting a physiological dimer. Overall these results indicate that rhomboids form oligomers which are facilitated by the membrane domain. For hiGlpG we have shown that these oligomers exist in the lipid bilayer. This is the first detailed oligomeric state characterization of the rhomboid family of peptidases.
菱形蛋白酶在膜双层的信号传导事件中发挥关键作用。了解菱形蛋白酶功能的调节对于深入了解其作用机制至关重要。在此，我们研究了三种不同菱形蛋白酶的寡聚状态。我们将流感嗜血杆菌（hiGlpG）、大肠杆菌GlpG（ecGlpG）和枯草芽孢杆菌（YqgP）置于去污剂溶解的十二烷基麦芽糖苷（DDM）溶液中进行沉降平衡分析。对于hiGlpG和ecGlpG，菱形蛋白酶分别由不含和含有可溶性结构域的核心6个跨膜结构域组成，而YqgP预计有7个跨膜结构域，在末端有较大的可溶性结构域，主要物种为二聚体，观察到少量单体和四聚体。为了研究仅膜结构域对菱形蛋白酶寡聚状态的影响，我们进一步研究了hiGlpG，它是膜内蛋白酶菱形蛋白酶家族中最简单的形式，代表了六个跨膜结构域的典型菱形蛋白酶核心。使用凝胶过滤和交联技术，我们证明hiGlpG在DDM去污剂溶液中是二聚体且具有功能。更重要的是，免疫共沉淀研究表明二聚体存在于脂质双层中，提示其为生理性二聚体。总体而言，这些结果表明菱形蛋白酶形成寡聚体，膜结构域促进了这一过程。对于hiGlpG，我们已经表明这些寡聚体存在于脂质双层中。这是首次对菱形蛋白酶家族进行详细的寡聚状态表征。
