Study of interaction the Arabidopsis PP2A regulatory B’ subunits with mitochondrial and cytoplasmic proteins
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Protein phosphatase 2A (PP2A) is a ubiquitous serine/threonine-specific phosphatase comprising a catalytic subunit C, a scaffolding subunit A and a highly variable regulatory subunit B. The regulatory subunits are essential for substrate specificity and subcellular localization of the PP2A holoenzyme and are classified into B/B55, B’, and B” non-related families in higher plants. Previous studies had shown that PP2A B’ regulatory subunits have a role in regulation of energy metabolism. An Arabidopsis B’ θ mutant had shown growth retardation in sucrose free medium, and cytosolic aconitase 3 (ACO3) was shown to interact with B´ζ and B’γ; furthermore, B´ζ had shown interaction with Arabidopsis mitochondrial succinate fumarate translocator (AtmSFC). In this thesis, wanted to investigate possibility of interactions between AtmSFC and other PP2A B’ regulatory subunits (B’α, B’β and B’θ). Furthermore, wanted to study probability of interaction between B´ζ with ACO1 and truncated ACO1 in which last 192 bp had been removed. Protein-protein interaction were analyzed by bimolecular fluorescence complementation (BiFC) assay. The regulatory subunits were tagged with C-terminal fragment of fluorescent protein Venus while ACO1, truncated ACO1 and AtmSFC were tagged with N-terminal fragment of fluorescent protein Venus. Tagged proteins were transiently expressed in onion epidermal cells. Fluorescence in the cytoplasm was detected in combinations of both ACO1 and B´ζ, whereas no interaction was observed with truncated ACO1. This result indicates that C -terminal end of ACO1 appeared to be necessary for interaction with B´ζ, because the truncated ACO1 that lacked the 64 C-terminal amino acids did not interact with B’´ζ. The Fluorescence was also detected between AtmSFC and PP2A B’β.
Master's thesis in Biological chemistry