In vitro synthesis of cellulose microfibrils by a membrane protein from protoplasts of the non-vascular plant Physcomitrella patens

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. * CBM, : cellulose-binding module; CesA, : cellulose synthase; CSC, : cellulose synthase complex; EST, : expressed sequence tag; GraFix, : gradient fixation; HA, : haemagglutinin; KO, : knockout; Ni-NTA, : Ni2+-nitrilotriacetic acid; SFG, : sum frequency generation; TFA, : trifluoroacetic acid