Identification and Functional Characterization of Inhibitor-3, a Regulatory Subunit of Protein Phosphatase 1 in Plants

Protein phosphatase 1 (PP1) is a eukaryotic serine/threonine protein phosphatase, and mediates diverse cellular processes in animal systems via the association of a catalytic subunit (PP1c) with multiple regulatory subunits that determine the catalytic activity, the subcellular localization, and the substrate specificity. However, no regulatory subunit of PP1 has been identified in plants so far. In this study, we identified inhibitor-3 (Inh3) as a regulatory subunit of PP1 and characterized a functional role of Inh3 in Vicia faba and Arabidopsis (Arabidopsis thaliana). We found Inh3 as one of the proteins interacting with PP1c using a yeast two-hybrid system. Biochemical analyses demonstrated that Arabidopsis Inh3 (AtInh3) bound to PP1c via the RVxF motif of AtInh3, a consensus PP1c-binding sequence both in vitro and in vivo. AtInh3 inhibited the PP1c phosphatase activity in the nanomolar range in vitro. AtInh3 was localized in both the nucleus and cytoplasm, and it colocalized with Arabidopsis PP1c in these compartments. Disruption mutants of AtINH3 delayed the progression of early embryogenesis, arrested embryo development at the globular stage, and eventually caused embryo lethality. Furthermore, reduction of AtINH3 expression by RNA interference led to a decrease in fertility. Transformation of the lethal mutant of inh3 with wild-type AtINH3 restored the phenotype, whereas that with the AtINH3 gene having a mutation in the RVxF motif did not. These results define Inh3 as a regulatory subunit of PP1 in plants and suggest that Inh3 plays a crucial role in early embryogenesis in Arabidopsis.