Functional characterization of Glycine max primary cell wall CesAs.

From (a-c) Analytical size exclusion chromatography (Superose 6 Increase) of GmCesA1 (a), GmCesA3 (b), and GmCesA6 (c). Void volume (Vo) and trimer and monomer peaks are marked. A rerun of the trimer fraction for each species is shown as a dashed profile. Inset: Coomassie-stained SDS-polyacrylamide gel electrophoresis of the indicated elution volumes. (d) Catalytic activity of the purified CesAs. 3H-labeled cellulose synthesized by trimeric and monomeric species was degraded with cellulase, followed by quantification by scintillation counting. (DS) and (PS) indicate cellulase treatments during and after the synthesis reaction, respectively. DPM: disintegrations per minute. (e) pH optima for catalytic activity of CesA1, 3 and 6. Activities are normalized to the highest activity for each isoform. (f) Kinetic analyses of CesAs by titrating UDP-Glc and quantification of the released UDP using an UDP-Glo assay kit. (g) UDP inhibits CesAs. Cellulose biosynthesis in the presence of 1.4, 0.5, and 2.3 mM UDP-Glc for GmCesA1, 3, and 6, respectively, and the indicated increasing concentrations of UDP. Product yields in the absence of UDP were set as 100 %. Error bars in panels d-g represent deviations from the means of at least three replicas.

Soybean primary cell wall CesAs assemble into homotrimers.

(a) CryoEM maps of the CesA homotrimers contoured at 4.5-5.6 α. One subunit is shown in color, the others are shown in light and dark gray. The gray background indicates the estimated membrane boundaries. (b) Cartoon representation of a CesA6 protomer. The transmembrane region is shown in light and dark pink, interface helices (IF) are shown in orange, and the catalytic domain is colored gray. The PCR and CSR regions are shown in blue and yellow, respectively. (c) Cytosolic view of the CesA trimer highlighting the interactions of the PCR domains. (d) Comparison of CesA6 and CesA8. Soybean CesA6 is shown as a cartoon and overlaid with hybrid aspen CesA8 (surface, PDB: 6WLB). Transmembrane helix 7 is colored light and dark pink for CesA8 and CesA6, respectively. The black triangle indicates the 3-fold symmetry axis of the homotrimer. Zoom views: Surface representations of CesA6 (left) and CesA8 (right) highlighting the lateral window. TM7b refers to TM helix 7 of another protomer. (e) CryoEM map of the CesA1 trimer shown at a low contour level (1.4 ο). The CesA1 structure is shown as a cartoon with one protomer colored yellow. The resolved CSR N– and C-terminal regions are colored blue and red, respectively. Bottom panel: Sequence alignment of the CSR regions of CesA1, 3 and 6 generated in Clustal Omega (Larkin et al., 2007).

In vitro interactions between different CesA isoforms.

Tandem pull-down experiments using Ni-NTA and Strep-Tactin resin. Experiments were performed with homotrimers of the indicated CesA isoforms tagged N-terminally either with His– or TwinStrep-tags. Material eluted from the Ni-NTA resin was loaded onto the Strep-Tactin beads. Top panels: Coomassie stained SDS-PAGE, bottom panels: Western blots using anti penta-His or anti-Strep primary antibodies. (a-c) Trimer-trimer interactions between CesA1 and CesA3, CesA6 and CesA3, and CesA6 and CesA1, respectively. (d) Differently tagged homotrimers of the same isoform do not interact. Tandem purification of a mixture of His– and TwinStrep-tagged CesA1. (e-h) Control binding of His-CesAs to Strep-Tactin beads and Strep-CesAs to Ni-NTA resin. F, W, E: Flow through, wash, and eluted fractions.

Clustering of CesA homotrimers.

(a-c) Uranylformate stained EM images of homotrimers of purified GmCesA1 (A), GmCesA3 (b) and GmCesA6 (c). The proteins were incubated overnight on ice prior to grid preparation. (d) The same for an equimolar mixture of all three CesA isoforms, incubated overnight, separated from individual trimers by size exclusion chromatography, and imaged by negative stain EM. Selected clusters are encircled. Scale bar: 100 nm. (e-g) Isoform interactions do not depend on the NTD. (e) Activity comparison of full-length and N-terminally truncated CesA isoforms. DPM: disintegrations per minute. (f) Tandem pull-down experiments as in Figure 3 but with N-terminally truncated homotrimers of CesA1 and 3. Top panel: SDS-PAGE, bottom panel: Western blots using anti-His and anti-Strep primary antibodies. (g) Control binding of His-ΔNCesA1 to Strep-Tactin beads and Strep-ΔNCesA3 to Ni-NTA resin. F, W, E: Flow through, wash, and eluted fractions.

Synergistic catalytic activity. Cellulose biosynthesis from mixtures of CesA isoforms.

The formation of 3H-labeled cellulose was quantified by scintillation counting for reaction mixtures containing one CesA isoform at 20 µM concentration and the other isoform at the indicated increasing concentrations. Blue, magenta and green columns represent activities measured for the individual single isoforms alone. Gray columns represent the calculated theoretical activities for the isoform mixtures by adding the individually determined activities. Red columns represent the experimentally determined activities for the isoform mixtures. (a and b) CesA120 μM + CesA31-20 μM and CesA320 μM + CesA11-20 μM; (c and d) CesA320 μM + CesA61-20 μM and CesA620 μM + CesA31-20 μM; and (e and f) CesA120 μM + CesA61-20 μM and CesA620 μM + CesA11-20 μM, respectively. (g) The same as for panel (a) but titrating the same CesA isoform (CesA120 μM + CesA11-20 μM). (h) The same as for panels a-f but for a combination of all three CesA isoforms. Individual and combined activities were determined at a concentration of 6.6 µM for each CesA isoform. DPM, disintegrations per minute.

CSC models consisting of different CesA isoforms.

(a) Association of homotrimers of three different CesA isoforms. Isoforms are indicated by different colors. The shapes represent the cytosolic CesA domains. (b and c) CSC assembly from heterotrimeric CesA complexes. Neither heterotrimer formation nor interactions between the same isoforms have been observed in vitro, rendering the models less likely. PCR: Plant conserved region, GT: Glycosyltransferase, CSR: Class specific region.