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.
Department of Biochemistry & Molecular Biology, The Pennsylvania State University, University Park, PA 16802, U.S.A.
Sung Hyun
SH
Cho
Department of Biochemistry & Molecular Biology, The Pennsylvania State University, University Park, PA 16802, U.S.A.
Juan
J
Du
Department of Chemical Engineering, The Pennsylvania State University, University Park, PA 16802, U.S.A.
Ian
I
Sines
Department of Biochemistry & Molecular Biology, The Pennsylvania State University, University Park, PA 16802, U.S.A.
Venkata Giridhar
VG
Poosarla
Department of Biochemistry & Molecular Biology, The Pennsylvania State University, University Park, PA 16802, U.S.A.
Venkata
V
Vepachedu
Department of Chemical Engineering, The Pennsylvania State University, University Park, PA 16802, U.S.A.
Kabindra
K
Kafle
Department of Biology, The Pennsylvania State University, University Park, PA 16802, U.S.A.
Yong Bum
YB
Park
Department of Chemical Engineering, The Pennsylvania State University, University Park, PA 16802, U.S.A.
Seong H
SH
Kim
Department of Chemical Engineering, The Pennsylvania State University, University Park, PA 16802, U.S.A. manish.kumar@psu.edu btn1@psu.edu.
Manish
M
Kumar
Department of Biochemistry & Molecular Biology, The Pennsylvania State University, University Park, PA 16802, U.S.A. manish.kumar@psu.edu btn1@psu.edu.
B Tracy
BT
Nixon
D019068:Bryopsida / Q000737:chemistry*; D002482:Cellulose / Q000737:chemistry*; D005964:Glucosyltransferases / Q000737:chemistry*; D008565:Membrane Proteins / Q000737:chemistry*; D020894:Microfibrils / Q000737:chemistry*; D037121:Plant Lectins / Q000737:chemistry; D010940:Plant Proteins / Q000737:chemistry*; D011523:Protoplasts / Q000737:chemistry*
D008565:Membrane Proteins; D037121:Plant Lectins; D010940:Plant Proteins; D002482:Cellulose; D005964:Glucosyltransferases; C478648:cellulose synthase
Physcomitrella patens; cellulose synthase 5 (CesA5); cellulose synthase complex; protoplasts; transmission electron microscopy (TEM)