The prenylated Rab acceptor PRA1.F3 acts in vacuolar protein transport in Arabidopsis thaliana

Abstract

To date, many unknowns regarding the physiological roles and molecular machinery underlying vesicle trafficking in higher plants and the interactors of prenylated Ras related in brain (Rab) acceptor1 (PRA1) remain to be elucidated. The endomembrane system of eukaryotic cells is made up of a remarkably dynamic set of membrane-bounded organelles, which functions in cellular homeostasis and maintenance. Trafficking between organelles takes place in vesicles, accompanied by directional transferring of proteins and other cargo molecules. In yeast and mammal cells, evolutionarily conserved PRAs recruit the prenylated Rab proteins onto the membrane of a specific compartment and are proposed to interact with specific soluble N-ethylmaleimide sensitive factor attachment protein receptor (SNARE) proteins, which mediate the vesicle fusion, and, therefore, play a pivotal role in vesicle trafficking processes.

Here, the roles of PRA1.F3 in Arabidopsis (Arabidopsis thaliana) were investigated. To unravel the interactors of AtPRA1.F3, co-immunoprecipitation (Co-IP) assays and split ubiquitin yeast two-hybrid (SU-Y2H) assay were performed. RabG3e is the interactor of PRA1.F3. Further experiments show that members of the endosomal maturation- driving RabF/Rab5 and RabG/Rab7 protein families are the interactors of PRA1.F3. In addition, PRA1.F3 individually interacts with each of the SNAREs (SYP21/22-SYP51/52-VTI11/12-VAMP711/713) that participate in the formation of a tonoplast-localizing quaternary (Qa/Qb/Qc/R) trans-SNARE complex. However, PRA1.F3 does not interact with GDP dissociation inhibitor (GDI) proteins, suggesting that PRA1.F3-Rab interaction might not be restricted to only GDI-associated Rabs for only their recruitment.

PRA1.F3 gene is mainly expressed in cotyledon, guard cells, vasculatures of roots, flowers, and developing siliques. GFP-PRA1.F3 was shown to localize to the trans-Golgi, the trans-Golgi network (TGN), and the tonoplasts. Overexpression of PRA1.F3 influenced the trafficking of aleurain-GFP and sporamin-GFP to the vacuoles. However, overexpression of PRA1.F3 neither affected the trafficking of the secretory proteins invertase-GFP and pathogenesis-related 1 (PR1)-GFP nor the Golgi-localizing protein sialyltransferase (ST)-GFP. PRA1.F3 mediates the vacuolar protein transport on the post-Golgi. At the subcellular level, GFP-PRA1.F3 overexpression and pra1.f3 knockout plants displayed abnormally swollen vesicles associated with the TGN in contrast to wild-type plants. PRA1.F3 may play a role in the maintenance of salt homeostasis in Arabidopsis plants. PRA1.F3 may function as a regulator in vacuolar trafficking by interaction with the small GTPases Rab5/Rab7 and the SNARE complex members.