IN VIVO OLIGOMERIC STRUCTURE AND PARTNERS OF YEAST PKA-R SUBUNIT THROUGH PULLDOWN PROTEOMICS

TOFOLÓN E; VALACCO MP; JORGE GERMÁN FERNÁNDEZ; ROSSI S; MORENO S

Córdoba

Congreso; 52 th Annual Meeting Argentine Society for Biochemistry and Molecular Biology; 2016

Sociedad Argentina de Investigación en Bioquímica y Biología Molecular

PKA is a tetramer formed by a dimer of regulatory subunit (R2), which binds cAMP, and two catalytic subunits. In mammals, the Nterminusof R2 (DD) is responsible for dimerization and binding to AKAPs. We have shown that Bcy1, the yeast PKA R subunit,binds specific proteins through its N-terminus and that a purified Tag-Bcy1(1-50) version of the DD forms tetramers both in crystalsand solution. To investigate the oligomerization state of Bcy1in vivo, we overexpressed the Tag-DD in yeast, with the rationale that itwould interact with endogenous Bcy1 if a tetramer existed within the cell. A pull-down protocol was established comprising biologicalreplicates of a WT strain with or without overexpression of the Tag-DD. Crude extracts were loaded onto Ni-agarose columns, theTag-DD and its binding partners were eluted with 200 mMimidazol, and analyzed by nanoHPLC-ESI-Orbitrap. Bcy1 and the catalyticsubunits (Tpk1,Tpk2,Tpk3) were detected in the replicates, indicating that in vivo, the overexpressed DD could interact with the Bcy1subunit and with the PKA holoenzyme. We predict that this interaction is via the formation of a tetramer through the DD domain. Thesemi-quantitative comparison of the results yielded a set of specific proteins differentially observed in cells with the overexpressedDD, possible binders of Tag-DD, BCY1 or a new surface in the tetramer DD2/BCY12