<i>N</i>-glycosylation of RD21 demonstrates a Golgi maturation route.

<p><b>A</b>, Explanation of the generated <i>N</i>-glycosylation mutants. RD21 carries a putative <i>N</i>-glycosylation site (PGS) in the prodomain and the granulin domain (black stripes). The second PGS is removed in the –PGS mutant by introducing an N414A substitution. A PGS was introduced in the protease domain of the +PGS mutant by the substitutions D180N and I182T. This PGS is present at the same site in several RD21 orthologs. <b>B</b>, The size-shift of the +PGS mutant is not reverted by PNGaseF treatment after expression in wild-type <i>N. benthamiana</i> plants. –PGS (pJW03), WT (pRH628) and +PGS (pMS48) were expressed by agroinfiltration and protein extracts were treated with and without PNGaseF. Proteins were separated by SDS-PAGE and analyzed by immunoblotting using RD21 antiserum. <b>C</b>, PNGaseF sensitivity is gained by expression of RD21 in glyco-engineered <i>N. benthamiana</i> plants. Wild-type RD21 (WT) and the +PGS mutant RD21 (+PGS) were expressed by agroinfiltration into wild-type <i>N. benthamiana</i> (WT) and transgenic <i>N. benthamiana</i> plants silenced for β-1,2-xylosyltransferase and α-1,3-fucosyltransferase (ΔXF, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032422#pone.0032422-Strasser1" target="_blank">[27]</a>). Protein extracts were treated with and without EndoH or PNGaseF, separated by SDS-PAGE and analyzed by immunoblotting using RD21 antiserum.