Molecular dissection of Phaseolus vulgaris polygalacturonase-inhibiting protein 2 reveals the presence of hold/release domains affecting protein trafficking toward the cell wall
The plant endomembrane system is massively involved in the synthesis, transport and
secretion of cell wall polysaccharides and proteins; however, the molecular mechanisms
underlying trafficking toward the apoplast are largely unknown. Besides constitutive, the
existence of a regulated secretory pathway has been proposed. A polygalacturonase
inhibitor protein (PGIP2), known to move as soluble cargo and reach the cell wall
through a mechanism distinguishable from default, was dissected in its main functional
domains (A, B, C, D), and C sub-fragments (C1–10), to identify signals essential for its
regulated targeting. The secretion patterns of the fluorescent chimeras obtained by fusing
different PGIP2 domains to the green fluorescent protein (GFP) were analyzed. PGIP2
N-terminal and leucine-rich repeat domains (B and C, respectively) seem to operate as
holding/releasing signals, respectively, during PGIP2 transit through the Golgi. The B
domain slows down PGIP2 secretion by transiently interacting with Golgi membranes.
Its depletion leads, in fact, to the secretion via default (Sp2-susceptible) of the ACD-GFP
chimera faster than PGIP2. Depending on its length (at least the first 5 leucine-rich repeats
are required), the C domain modulates B interaction with Golgi membranes allowing the
release of chimeras and their extracellular secretion through a Sp2 independent pathway.
The addition of the vacuolar sorting determinant Chi to PGIP2 diverts the path of the
protein from cell wall to vacuole, suggesting that C domain is a releasing rather than a
cell wall sorting signal