Elucidating the mechanisms of assembly and subunit interaction of the cellulose synthase complex of Arabidopsis secondary cell walls.
Cellulose is the most abundant biopolymer in nature; however, questions relating to the biochemistry of its synthesis including the structure of the cellulose synthase complex (CSC) can only be answered by the purification of a fully functional complex. Despite its importance, this goal remains elusive. The work described here utilizes epitope tagging of cellulose synthase A (CESA) proteins that are known components of the CSC. To avoid problems associated with preferential purification of CESA monomers, we developed a strategy based on dual epitope tagging of the CESA7 protein to select for CESA multimers. With this approach, we used a two-step purification that preferentially selected for larger CESA oligomers. These preparations consisted solely of the three known secondary cell wall CESA proteins CESA4, CESA7, and CESA8. No additional CESA isoforms or other proteins were identified. The data are consistent with a model in which CESA protein homodimerization occurs prior to formation of larger CESA oligomers. This suggests that the three different CESA proteins undergo dimerization independently, but the presence of all three subunits is required for higher order oligomerization. Analysis of purified CESA complex and crude extracts suggests that disulfide bonds and noncovalent interactions contribute to the stability of the CESA subunit interactions. These results demonstrate that this approach will provide an excellent framework for future detailed analysis of the CSC.
Atanassov
Ivan I
II
Faculty of Life Sciences, University of Manchester, Michael Smith Building, Oxford Road, Manchester M13 9PT, United Kingdom.
Pittman
Jon K
JK
Turner
Simon R
SR
D017360:Arabidopsis / Q000201:enzymology*; D029681:Arabidopsis Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D002473:Cell Wall / Q000201:enzymology*; D019281:Dimerization; D004220:Disulfides / Q000737:chemistry / Q000378:metabolism; D004795:Enzyme Stability / Q000502:physiology; D005964:Glucosyltransferases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D008958:Models, Molecular; D009097:Multienzyme Complexes / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*
D029681:Arabidopsis Proteins; D004220:Disulfides; D009097:Multienzyme Complexes; D005964:Glucosyltransferases; C478648:cellulose synthase