INBA   12521
INSTITUTO DE INVESTIGACIONES EN BIOCIENCIAS AGRICOLAS Y AMBIENTALES
Unidad Ejecutora - UE
congresos y reuniones científicas
Título:
MOLECULAR CHARACTERIZATION OF PKA FROM YARROWIA LIPOLITICA
Autor/es:
KRONBERG FLORENCIA; GIACOMETTI ROMINA; PASSERON SUSANA
Lugar:
San Miguel de Tucuman, Tucuman
Reunión:
Congreso; XLV Reunión Anual SAIB; 2009
Resumen:
The dimorphic yeast Yarrowia lipolytica has gained interest in recent years due to its biotechnological and basic application. We have previously shown that the cAMP/PKA signal transduction pathway regulates different physiological processes in this fungus, including dimorphism, being cAMP a strong inhibitor of yeast to mycelium transition. In this work, several biochemical and structural characteristics of the PKA holoenzyme were studied. In this organism, both PKA catalytic (C) and regulatory (R) subunits are codified by single genes, named TPK1 and RKA1 respectively. Since in silico analysis of the N-terminal of R sequence showed low homology with the dimerization domain present in almost all eukaryotic R, we assessed the polymeric nature of the holoenzyme and of the R subunit by sucrose gradient analysis. Partially purified holoenzyme and R subunit were obtained from wild-type and tpk1 strains, and were characterised by phosphortransferase activity, cAMP-binding activity and western-blot recognition. The holoenzyme sedimented as a dimmer with an apparent sedimentation coefficient (S) of 5.6, while dissociated R and C subunits appeared as monomeric proteins with S of 3.6 and 2.5 respectively. Furthermore, cloning, expression and characterization of R subunits in E. coli, indicated that recombinant R appeared only as a monomeric protein. Supported by ANCyP and CONICET.