A more detailed view on the interaction between KOR1 and the CESA proteins using truncated versions of KOR1 revealed that all portions which contain the TMD were able to interact and this led to the conclusion that KOR1 transmembrane domain is required for the interaction with CESAs. These data suggest that KOR1 and CESA associate in the CSC in the plasma membrane, between the TMD of KORRIGAN and the TMDs of the CESA proteins. Surprisingly, despite showing interaction in vitro, CESA6 and CESA4 show a very weak to no interaction with KOR1TMD in planta suggesting that at least for the two mentioned CESAs the presence of TMD is essential but not sufficient for interaction and would require more than just the TMD. It is interesting to note that some proteins required for cellulose synthesis are found in sterol-rich lipid rafts . It is possible that KOR1 and CESAs traffic to the plasma membrane separately, but are then concentrated in specific lipid domains, favoring interactions. This domain is also important in the homodimerisation of the KOR1 protein as all partial proteins containing this domain are able to interact, however other domains of the KOR1 protein might also play a role in the dimerization as the combination KOR1C and KOR1N only showed a weak interaction in both assays. The function of this dimerization is thus far unknown, however one could speculate that the dimer enables a more efficient hydrolysis of the glucan chains or binding to the cellulose, or the interlinking of KORRIGAN might result in a more stable rosette complex.
The interactions between KOR1 and three members of the primary cell wall cellulose-synthesizing rosette complex (CESA1, CESA3 and CESA6) have been demonstrated in vitro using the membrane based yeast two hybrid system . The results indicated that KOR1 is able to interact with all three of the primary CESA proteins as yeast colonies were able to grow on selective medium with KOR1 as bait and the primary CESAs as prey. Here the converse experiments, in which the different CESA proteins were the bait and the KOR1 the prey were carried out to confirm the findings, and showed that all combinations were able to induce the reporter genes, allowing the yeast to grow on selective medium (). The lack of growth in the negative control indicated that the interaction with KOR1 was specific as an unrelated protein expressed as prey is not able to activate the system. We conclude that KOR1 can interact with CESA1, CESA3, and CESA6 in vitro.
Cellulose is synthesized by large cellulose synthase complexes ..
N2 - A brief history of the literature dealing with cellulose microfibril assembly is presented, and a current summary of cellulose microfibril synthesizing complexes among eukaryotic cells is given. Terminal complexes not described before include the following: linear terminal complexes (TCs) with three rows in Eremosphaera, Microdictyon and Chaetomorpha; globular terminal complexes in Ophioglossum, Psilotum, Equisetum and Gingko. Cellulose microfibril assembly in Acetobacter xylinum is described very briefly and compared with the process among eukaryotic cells. Particular emphasis on structures that may be involved in the spatial control of cellulose synthesis is given. Among these are cytoplasmic structures such as microtubules and microfilaments. Microfilament structures are shown to clearly surround individual microtubules that lie adjacent to the plasma membrane. Using freeze-fracture techniques, these labile associations have been shown for the first time. Microfibril orientation may be mediated through an interaction of cortical microtubules in association with microfilaments. A review of Mueller and Brown's membrane flow model for microfibril orientation is presented. Cellulose terminal complex clustering and its role in gravitropic response is covered. Definitive membrane changes with TC clustering/disaggregation and intramembranous particle frequencies, occur within 12 min following gravistimulation. These differences are pronounced in the cells from upper and lower hemicylinders of rapidly frozen tissue, which was studied by the freeze-fracture method. A hypothesis for cellulose microfibril interaction in controlling the constraint of the growth axis is presented, and the supporting data for terminal complex clustering/disaggregation as well as fluorescent brightener inhibition of the gravitropic response support this hypothesis. The onset and regulation of cellulose microfibril assembly is presented for synchronized protoplasts generated by Boergesenia, using inhibitors of transcription and translation. These results suggest dynamic turnover of terminal complex subunits during the assembly of the cellulose microfibril. This study is concluded with a brief discussion of possible phylogenetic trends in the evolution of cellulose synthesis. A principal underlying theme is that the specific arrangement and consolidation of the terminal complex subunits determine to a large degree the size and shape of the microfibril, its crystallinity, as well as intramicrofibrillar associations. Three basic types of TCs appear among all eukaryotic cells studied so far: namely, the rosette, the globular and the linear complex.