Involvement of 14-3-3 protein post-translational modifications in Giardia duodenalis encystation.
14-3-3s are a family of phosphoserine/phosphothreonine binding proteins directly affecting many protein functions by regulating enzyme activity, intracellular localisation or mediating protein-protein interaction. The single 14-3-3 (g14-3-3) of the flagellated parasite Giardia duodenalis is phosphorylated at residue threonine 214 (T214) and polyglycylated at the extreme C-terminus in a stage-specific manner. To define the role of each post-translational modification, Giardia transgenic lines expressing a N-terminally FLAG-tagged g14-3-3, or the single point mutant T214A, or the E246A and the E247A mutants of the putative polyglycylation sites, were generated in this study. By affinity chromatography and MALDI-MS analysis, Glu246 was identified as the only site of polyglycylation. The absence of a polyglycine chain results in the nuclear localisation of the protein at any parasite life-stage, suggesting a role for polyglycylation in 14-3-3 nucleo/cytoplasm shuttling. Moreover, cyst formation was strongly induced in parasites expressing the E246A mutant and delayed in those harbouring the T214A mutant. Finally, in vitro overlay assays with a GST_T214E mutant indicated that phosphorylation can alter in vitro the binding properties of 14-3-3. The present data suggest that g14-3-3 post-translational modifications act in combination to affect encystation efficiency in Giardia.
Lalle
Marco
M
Department of Infectious, Parasitic and Immunomediated Diseases, Istituto Superiore di Sanità, 00161 Rome, Italy. marco.lalle@iss.it
Bavassano
Carlo
C
Fratini
Federica
F
Cecchetti
Serena
S
Boisguerin
Prisca
P
Crescenzi
Marco
M
Pozio
Edoardo
E
D048948:14-3-3 Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005872:Giardia / Q000235:genetics / Q000254:growth & development* / Q000378:metabolism*; D010455:Peptides / Q000378:metabolism; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D021381:Protein Transport; D015800:Protozoan Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D048948:14-3-3 Proteins; D010455:Peptides; D015800:Protozoan Proteins; C011080:polyglycine