Synthesis and self-assembly of cellulose microfibrils from reconstituted cellulose synthase

Cellulose, the major component of plant cell walls, can be converted to bioethanol and is thus highly studied. In plants, cellulose is produced by cellulose synthase, a processive family-2 glycosyltransferase. In plant cell walls, individual β-1,4-glucan chains polymerized by CesA are assembled into microfibrils that are frequently bundled into macrofibrils. An in vitro system in which cellulose is synthesized and assembled into fibrils would facilitate detailed study of this process. Here, we report the heterologous expression and partial purification of His-tagged CesA5 from Physcomitrella patens. Immunoblot analysis and mass spectrometry confirmed enrichment of PpCesA5. The recombinant protein was functional when reconstituted into liposomes made from yeast total lipid extract. The functional studies included incorporation of radiolabeled Glc, linkage analysis, and imaging of cellulose microfibril formation using transmission electron microscopy. Several microfibrils were observed either inside or on the outer surface of proteoliposomes, and strikingly, several thinner fibrils formed ordered bundles that either covered the surfaces of proteoliposomes or were spawned from liposome surfaces. We also report this arrangement of fibrils made by proteoliposomes bearing CesA8 from hybrid aspen. These observations describe minimal systems of membrane-reconstituted CesAs that polymerize β-1,4-glucan chains that coalesce to form microfibrils and higher-ordered macrofibrils. How these micro- and macrofibrils relate to those found in primary and secondary plant cell walls is uncertain, but their presence enables further study of the mechanisms that govern the formation and assembly of fibrillar cellulosic structures and cell wall composites during or after the polymerization process controlled by CesA proteins.