Figure 6.

Interaction of ascorbate biosynthesis enzymes assessed by Y2H analysis and Co-IP. A, The Y2HY2H assay detected no direct interaction between the enzymes, but GMP, GME, and GPP, which contain predicted dimerization/trimerization domains (B), form dimers/trimers. B, Protein scheme for each of the enzymes assayed in the Y2H assay showing length (in amino acids), annotated domains and predicted dimer/trimerization interfaces. C–G, The Co-IP assay reported association in vivo in N. benthamiana between consecutive steps as well as between the first (GMP) and the last (L-GalDH) steps occurring in the cytosol. GFP-tagged enzyme from N. benthamiana crude protein extracts containing two overexpressed consecutive enzymes, GFP and HA-tagged, was pulled-down using α-GFP agarose beads and coIP protein was detected using α-HA antibody. CBB: Coomassie Brilliant Blue.