Cho Sung Hyun, Du Juan, Sines Ian, Poosarla Venkata Giridhar, Vepachedu Venkata, Kafle Kabindra, Park Yong Bum, Kim Seong H, Kumar Manish, Nixon B Tracy
Department of Biochemistry & Molecular Biology, The Pennsylvania State University, University Park, PA 16802, U.S.A.
Department of Chemical Engineering, The Pennsylvania State University, University Park, PA 16802, U.S.A.
Biochem J. 2015 Sep 1;470(2):195-205. doi: 10.1042/BJ20141391. Epub 2015 Jun 30.
Plant cellulose synthases (CesAs) form a family of membrane proteins that are associated with hexagonal structures in the plasma membrane called CesA complexes (CSCs). It has been difficult to purify plant CesA proteins for biochemical and structural studies. We describe CesA activity in a membrane protein preparation isolated from protoplasts of Physcomitrella patens overexpressing haemagglutinin (HA)-tagged PpCesA5. Incubating the membrane preparation with UDP-glucose predominantly produced cellulose. Negative-stain EM revealed microfibrils. Cellulase bound to and degraded these microfibrils. Vibrational sum frequency generation (SFG) spectroscopic analysis detected the presence of crystalline cellulose in the microfibrils. Putative CesA proteins were frequently observed attached to the microfibril ends. Combined cross-linking and gradient centrifugation showed bundles of cellulose microfibrils with larger particle aggregates, possibly CSCs. These results suggest that P. patens is a useful model system for biochemical and structural characterization of plant CSCs and their components.
植物纤维素合酶（CesAs）构成了一类膜蛋白家族，这些膜蛋白与质膜中称为纤维素合酶复合体（CSCs）的六边形结构相关联。一直以来，纯化植物CesA蛋白用于生化和结构研究都很困难。我们描述了从过量表达血凝素（HA）标签的小立碗藓PpCesA5原生质体中分离出的膜蛋白制剂中的CesA活性。用UDP-葡萄糖孵育该膜制剂主要产生纤维素。负染电子显微镜显示出微纤丝。纤维素酶结合并降解了这些微纤丝。振动和频产生（SFG）光谱分析检测到微纤丝中存在结晶纤维素。经常观察到假定的CesA蛋白附着在微纤丝末端。交联和梯度离心相结合显示纤维素微纤丝束与更大的颗粒聚集体，可能是CSCs。这些结果表明，小立碗藓是用于植物CSCs及其组分的生化和结构表征的有用模型系统。
