Complementation of lethal conditional mutant yeast by RPL10 from Arabidopsis thaliana

FALCONE FERREYRA, LORENA; LUCIANI D´OTTAVIO, MARIANELA DANA; PEZZA, ALEJANDRO; CASATI, PAULA

Congreso; XLVI Reunión anual de la Sociedad Argentina de Investigaciones en Bioquímica y Biología Molecular (SAIB); 2010

Ribosomal protein L10 (RPL10) is a key factor in joining the 40S and 60S ribosomal subunits into a functional 80S ribosome. In yeast, a mutation in Rpl10 demonstrated that it is essential for viability. The Arabidopsis genome contains three rpl10 genes encoding RPL10A-C. Previously, we showed that A. thaliana RPL10s are not functionally equivalent, being involved in plant development, translation and UV-B response. On the basis of the high degree of amino acid sequence identity between Arabidopsis and yeast RPL10s (67%); the objective of this work was to analyze the ability of AtRPL10s to complement lethal conditional mutant yeast. We used the AJY2104 strain where RPL10 is expressed from its genomic locus but under control of GAL1 promoter, consequently, this strain is inviable under repressing condition (glucose) unless functional RPL10 is episomally provided. First, cDNAs encoding to AtRPL10A-C were amplified by RT-PCR from A. thaliana leaves and cloned under control of constitutive GDP promoter. After transformation with these constructions, complementation was evaluated. Serial dilutions of the AtRPL10s expressed in AJY2104 (GAL1::RPL10) showed growth onto selective plates under repressing condition for genomic RPL10 indicating complementation by AtRPL10s. This result was also supported by growth assay in liquid medium. In addition, western blot with antibodies raised against human RPL10 showed an immunoreactived band indicating the presence of RPL10. Further studies of polysome profile will allow analyzing the 60S biogenesis and translation in this complemented strain.