A single heterologously expressed plant cellulose synthase isoform is sufficient for cellulose microfibril formation in vitro
- 19 September 2016
- journal article
- Published by Proceedings of the National Academy of Sciences in Proceedings of the National Academy of Sciences of the United States of America
- Vol. 113 (40), 11360-11365
- https://doi.org/10.1073/pnas.1606210113
Abstract
Plant cell walls are a composite material of polysaccharides, proteins, and other noncarbohydrate polymers. In the majority of plant tissues, the most abundant polysaccharide is cellulose, a linear polymer of glucose molecules. As the load-bearing component of the cell wall, individual cellulose chains are frequently bundled into micro and macrofibrils and are wrapped around the cell. Cellulose is synthesized by membrane-integrated and processive glycosyltransferases that polymerize UDP-activated glucose and secrete the nascent polymer through a channel formed by their own transmembrane regions. Plants express several different cellulose synthase isoforms during primary and secondary cell wall formation; however, so far, none has been functionally reconstituted in vitro for detailed biochemical analyses. Here we report the heterologous expression, purification, and functional reconstitution of Populus tremula x tremuloides CesA8 (PttCesA8), implicated in secondary cell wall formation. The recombinant enzyme polymerizes UDP-activated glucose to cellulose, as determined by enzyme degradation, permethylation glycosyl linkage analysis, electron microscopy, and mutagenesis studies. Catalytic activity is dependent on the presence of a lipid bilayer environment and divalent manganese cations. Further, electron microscopy analyses reveal that PttCesA8 produces cellulose fibers several micrometers long that occasionally are capped by globular particles, likely representing PttCesA8 complexes. Deletion of the enzyme’s N-terminal RING-finger domain almost completely abolishes fiber formation but not cellulose biosynthetic activity. Our results demonstrate that reconstituted PttCesA8 is not only sufficient for cellulose biosynthesis in vitro but also suffices to bundle individual glucan chains into cellulose microfibrils.Keywords
Funding Information
- U.S. Department of Energy (DE-SC0001090)
This publication has 44 references indexed in Scilit:
- Tertiary model of a plant cellulose synthaseProceedings of the National Academy of Sciences of the United States of America, 2013
- Structure of Cellulose Microfibrils in Primary Cell Walls from CollenchymaPlant Physiology, 2012
- Radiometric and spectrophotometric in vitro assays of glycosyltransferases involved in plant cell wall carbohydrate biosynthesisNature Protocols, 2012
- Moss cell walls: structure and biosynthesisFrontiers in Plant Science, 2012
- Nanostructure of cellulose microfibrils in spruce woodProceedings of the National Academy of Sciences of the United States of America, 2011
- RING domain dimerization is essential for RNF4 functionBiochemical Journal, 2010
- Biosynthesis of Callose and Cellulose by Detergent Extracts of Tobacco Cell Membranes and Quantification of the Polymers Synthesized in vitroJournal of Integrative Plant Biology, 2010
- TOPCONS: consensus prediction of membrane protein topologyNucleic Acids Research, 2009
- Characterization of the Purified Hyaluronan Synthase from Streptococcus equisimilisBiochemistry, 2004
- Cellulose structure and biosynthesis: What is in store for the 21st century?Journal of Polymer Science Part A: Polymer Chemistry, 2003