Structural and Functional Characterization of a Phosphatase Domain within Yeast General Transcription Factor IIIC

Saccharomyces cerevisiae tau 55, a subunit of the RNA polymerase III-specific general transcription factor TFIIIC, comprises an N-terminal histidine phosphatase domain (tau 55-HPD) whose catalytic activity and cellular function is poorly understood. We solved the crystal structures of tau 55-HPD and its closely related paralogue Huf and used in silico docking methods to identify phosphoserine- and phosphotyrosine-containing peptides as possible substrates that were subsequently validated using in vitro phosphatase assays. A comparative phosphoproteomic study identified additional phosphopeptides as possible targets that show the involvement of these two phosphatases in the regulation of a variety of cellular functions. Our results identify tau 55-HPD and Huf as bona fide protein phosphatases, characterize their substrate specificities, and provide a small set of regulated phosphosite targets in vivo.