IQUIBICEN   23947
INSTITUTO DE QUIMICA BIOLOGICA DE LA FACULTAD DE CIENCIAS EXACTAS Y NATURALES
Unidad Ejecutora - UE
congresos y reuniones científicas
Título:
In vivo oligomeric structure and partners of yeast PKA-R subunit through pull-down proteomics
Autor/es:
VALACCO, P; FERNANDEZ NUÑEZ,L., OCAMPO, J., GIOINO, P., BARDECI, N., ROSSI, S. AND MORENO, S; FERNANDEZ, G; TOFOLON, E; ROSSI, S
Lugar:
Cordoba
Reunión:
Congreso; LII Reunión Anual de la Sociedad Argentina de Investigación en Bioquímica y Biología Molecular; 2016
Institución organizadora:
SAIB
Resumen:
IN VIVO OLIGOMERIC STRUCTURE AND PARTNERS OF YEAST PKA-R SUBUNIT THROUGH PULL-DOWN PROTEOMICSTofolón|||E; Valacco|||MP; Fernández|||JG; Rossi|||S; Moreno|||SDepto Quimica Biológica, FCEN, UBA and IQUIBICEN/CONICETE-mail: enzotofolon@qb.fcen.uba.arPKA is a tetramer formed by a dimer of regulatory subunit (R2), which binds cAMP, and two catalytic subunits. In mammals, the N-terminus of 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 crystals and solution. To investigate the oligomerization state of Bcy1in vivo, we overexpressed the Tag-DD in yeast, with the rationale that it would interact with endogenous Bcy1 if a tetramer existed within the cell. A pull-down protocol was established comprising biological replicates of a WT strain with or without overexpression of the Tag-DD. Crude extracts were loaded onto Ni-agarose columns, the Tag-DD and its binding partners were eluted with 200 mM imidazol, and analyzed by nanoHPLC-ESI-Orbitrap. Bcy1 and the catalytic subunits (Tpk1,Tpk2,Tpk3) were detected in the replicates, indicating that in vivo, the overexpressed DD could interact with the Bcy1 subunit and with the PKA holoenzyme. We predict that this interaction is via the formation of a tetramer through the DD domain. The semi-quantitative comparison of the results yielded a set of specific proteins differentially observed in cells with the overexpressed DD, possible binders of Tag-DD, BCY1 or a new surface in the tetramer DD2/BCY12