| Popis: |
Formins are ubiquitous multidomain proteins involved in the nucleation of actin polymerization and cell cytoskeleton formation and regulation. These proteins invariably contain a formin homology 2 (FH2) domain that binds actin and a proline-rich formin homology 1 (FH1) domain that interacts with profilin, thereby enabling FH2 to nucleate actin assembly.Arabidopsis thaliana has twenty-one putative formins that are highly heterogeneous in terms of localization, domain structure, length and amino acid content. Some are unique in possessing a third domain that is extracellular (EC) and linked to the cytoplasmic FH1 and FH2 domains by a transmembrane (TM) module. The EC domains contain proline-rich motifs resembling those characteristic of extensins and arabinogalactan proteins (AGPs). Thus, it is predicted that the EC domains of plant formins are post-translationally modified to yield glycosylated hydroxyproline (Hyp).The present goal was to characterize the EC domain of Arabidopsis AtFH2 and determine if the EC domain are modified as predicted by Hyp O-contiguity hypothesis. The chimeric genes encoding the enhanced green fluorescent protein (EGFP) fused to the AFH2EC domain was expressed as secreted proteins in tobacco Bright Yellow 2 (BY-2) cells. The potential fusion glycoproteins of AFH2EC and afh2contigEC (a designed construct to verify the Hyp O-glycosylation pattern) were isolated by conventional chromatography and crosslink IP.Although we were unable to isolate and biochemically characterize the AFH2EC and afh2contigEC, I was able to uncover two interesting aspects:1)The MS analysis of the crosslink IP samples has shown that fragments from the cytoplasmic region of Arabidopsis AtFH2 were present in the media of transgenic tobacco cell cultures.2)The RNA sequences identified in the wild-type of tobacco cells and leaf tissue were identified to be identical to the EC of AtFH2EC antistrand. |
