IN VIVO RELEVANCE OF THE OLIGOMERIC STATE OF BCY1, THE REGULATORY SUBUNIT OF PKA IN Saccharomyces cerevisiae

TOFOLON, E.; ROSSI S; MORENO S

Parana

Congreso; LIV Reunion Anual de SAIB; 2018

SAIB (Sociedad Argentina de Investigacion en Bioquimica y Biologia Molecular) y PABMB (Pan American Association for Biochemistry and Molecular Biology)

PKA is a tetramer formed by a dimer of the regulatory subunit (R), and two catalytic subunits. R subunits have a dimerization and docking (DD)domain at its N-t. We have reported that Bcy1 (R subunit from S. cerevisiae) forms a tetramer in solution, unlike its dimeric mammaliancounterparts. A structural analysis by SAXS and crystallization of DD domain (1-50) showed that it forms a tetramer in vitro. A key residue fortetramerization is Arg45. Upon deletion of Gln44, the DDΔQ no longer forms tetramers but dimers. In order to study whether tetramerization isrelevant in vivo, a wt strain overexpressing either DDWT or DDΔQ was used to evaluate several phenotypes commonly used as read-outs ofPKA activity. The rationale for this approach was that a difference in phenotypic effects of the two constructs would reflect a differentialcapacity of competition with endogenous Bcy1, with its degree of oligomerization and therefore result in an alteration of PKA activity and/orlocalization. We analyzed growth, glycogen accumulation, heat stress and nuclear localization of the transcription factor Msn2. In most cases thedifference observed between the control strain and the one expressing DDWT was not significant; however, the expression of DDΔQ produced ahigher glucose accumulation, an increase in growth, and lower resistance to heat stress than the DDWT. The nuclear localization of Msn2-GFPwas completely impaired in the presence of either DDWT or DDΔQ. The phenotype analysis indicates that PKA function is differentially alteredby expression of DDΔQ or DDWT and suggests that the oligomeric state of Bcy1 in vivo is relevant for yeast physiology.