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Molecular dissection of Phaseolus vulgaris polygalacturonase-inhibiting protein 2 reveals the presence of hold/release domains affecting protein trafficking toward the cell wall.

ABSTRACT: 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.

SUBMITTER: De Caroli M

PROVIDER: S-EPMC4550104 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Molecular dissection of Phaseolus vulgaris polygalacturonase-inhibiting protein 2 reveals the presence of hold/release domains affecting protein trafficking toward the cell wall.

De Caroli Monica M Lenucci Marcello S MS Manualdi Francesca F Dalessandro Giuseppe G De Lorenzo Giulia G Piro Gabriella G

Frontiers in plant science 20150826

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 ...[more]

PMID: 26379688

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Project description:BACKGROUND: The PolyGalacturonase-Inhibiting Proteins (PGIP) of plant cell wall limit the invasion of phytopathogenic organisms by interacting with the enzyme PolyGalacturonase (PG) they secrete to degrade pectin present in the cell walls. PGIPs from different or same plant differ in their inhibitory activity towards the same PG. PGIP2 from Phaseolus vulgaris (Pv) inhibits the PG from Fusarium moniliforme (Fm) although PGIP1, another member of the multigene family from the same plant sharing 99% sequence similarity, cannot. Interestingly, PGIP3 from Glycine max (Gm) which is a homologue of PGIP2 is capable of inhibiting the same PG although the extent of similarity is lower and is 88%. It therefore appears that subtle changes in the sequence of plant PGIPs give rise to different specificity for inhibiting pathogenic PGs and there exists no direct dependence of function on the extent of sequence similarity. RESULTS: Structural information for any PGIP-PG complex being absent, we resorted to molecular modelling to gain insight into the mechanism of recognition and discrimination of PGs by PGIPs. We have built homology models of PvPGIP1 and GmPGIP3 using the crystal structure of PvPGIP2 (1OGQ) as template. These PGIPs were then docked individually to FmPG to elucidate the characteristics of their interactions. The mode of binding for PvPGIP1 to FmPG considerably differs from the mode observed for PvPGIP2-FmPG complex, regardless of the high sequence similarity the two PGIPs share. Both PvPGIP2 and GmPGIP3 despite being relatively less similar, interact with residues of FmPG that are known from mutational studies to constitute the active site of the enzyme. PvPGIP1 tends to interact with residues not located at the active site of FmPG. Looking into the electrostatic potential surface for individual PGIPs, it was evident that a portion of the interacting surface for PvPGIP1 differs from the corresponding region of PvPGIP2 or GmPGIP3. CONCLUSION: van der Waals and electrostatic interactions play an active role in PGIPs for proper recognition and discrimination of PGs. Docking studies reveal that PvPGIP2 and GmPGIP3 interact with the residues constituting the active site of FmPG with implications that the proteins bind/block FmPG at its active site and thereby inhibit the enzyme.

Dataset Information

Molecular dissection of Phaseolus vulgaris polygalacturonase-inhibiting protein 2 reveals the presence of hold/release domains affecting protein trafficking toward the cell wall.

Publications

Molecular dissection of Phaseolus vulgaris polygalacturonase-inhibiting protein 2 reveals the presence of hold/release domains affecting protein trafficking toward the cell wall.

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