Molecular characterization of the B' regulatory subunit gene family of Arabidopsis protein phosphatase 2A

Abstract

Type 2A serine/threonine protein phosphatases (PP2A) have been implicated as important mediators of a diverse array of reversible protein phosphorylation events in plants. We have identified a novel Arabidopsis gene (AtB'δ) which encodes a 55-kDa B' type regulatory subunit of PP2A. The protein encoded by this gene is 57-63% identical and 69-74% similar to the previously identified AtB' genes. The AtB'δ gene appears to be expressed in all Arabidopsis organs indicating its protein product has a basic housekeeping function in plant cells. Unlike certain mRNAs derived from the AtB'γ gene, AtB'δ mRNAs do not fluctuate significantly in response to heat stress. Further analysis of eDNA sequences derived from the AtB' genes identified an alternatively spliced eDNA derived from AtB'γ. This cDNA differs from the previously identified AtB'γ cDNA by the absence of a 133- bp region in its 5' untranslated region. The missing 133-bp region appears to constitute an unspliced intron and its presence in the AtB'γ gene was confirmed by PCR using Arabidopsis genomic DNA as a template. AtB'γ mRNA containing the 133-bp intron accumulate in all Arabidopsis organs and their levels fluctuate differentially in response to heat stress. The 133-bp insert contains two short open reading frames arid hence might serve as a translational control mechanism affecting AtB'γ protein synthesis. Finally we show, using both the yeast two hybrid system and in vitro binding assays, that the B' subunit of Arabidopsis PP2A is able to associate with other PP2A subunits, supporting the notion that the B' protein serves as a regulator of PP2A activity in plants.