Orellana L., ArielMoreno Vilches, Adrián AndrésFacultad de Ciencias de la SaludDepartamento de Ciencias Biológicas2019-03-292019-03-292008http://repositorio.unab.cl/xmlui/handle/ria/8349Tesis (Bioquímico, Magíster en Bioquímica)Esta Tesis se realizó en el Centro de Biotecnología Vegetal de la Facultad de Ecología y Recursos Naturales de la Universidad Andrés Bello y fue financiada por los proyectos FONDECYT N°:, 1070379, INICIATIVA CIENTIFICA MILENIO PCBP06-065-F y PROYECTO BASAL PFB-16/2007.El mecanismo de control de calidad del plegamiento de proteínas, en el turnen del retículo endoplasmatico, es un proceso fundamental durante la síntesis de proteínas glicosiladas. En la actualidad se han de.scrito varias proteínas que participan en este proceso, pero aún no se dilucida por completo cuáles son todos los productos génicos involucrados en él. Específicamente en plantas, se han identificado varios componentes que participarían en este proceso, que muestran una alta homología con los componentes ya descritos en levaduras y mamíferos. Actualmente se propone que los transportadores de nucleótidos azúcar AtUTr 1 y AtUTr3 de Arabidopsis thaliana, participarían en el mecanismo de control de calidad del plegamiento suministrando UDP-glucosa al lumen del retículo endoplasmático, sustrato esencial para el proceso de reglucosilación de proteínas mal plegadas. En esta tesis, quisimos profundizar en la regulación transcripcional de estos transportadores de UDPglucosa que sólo se han identificado en plantas. Para esto, se analizó la acumulación de los transcritos de los genes AtUTr 1 y AtUTr3, mediante RT -PCR en tiempo real, en condiciones normales y de estrés inducido por tunicamicina y ditiotre itol. Los resultados obtenidos indican que los transcritos de los transpotiadores AtUTr1 y AtUTr3 presentan un comportamiento similar a los transcritos de genes que codifican para proteínas chaperonas de retículo endoplasmático. Junto a esto, el análisis de los promotores de estos genes mostró la existencia de elementos regulatorios en cis comunes a todos e llos, los que estarían asociados a la respuesta por estrés en el retículo endoplasmático.The quality control mechanism that takes place in the lumen of the endoplasmic reticulum (ER) is a fundamental process that ensures the proper folding of N-glycoproteins synthesized at that organelle. To date, severa! proteins that play a role in this process have been described, however, it is still not clear what are al! the gene products involved in this process. Recently it has been proposed that the nucleotide-sugar transporters AtUTr 1 and AtUTr3 from Arabidopsis thaliana would play a role in the quality control mechanism by providing UDP-glucose into the lumen of the endoplasmic reticulum, an essential substrate for the reglucosylation of unfolded proteins mediated by the enzyme UDP-glucose glycoprotein glucosyltransferase (UGGT). In this thesis, we analyzed the transcriptional regulation of these UDP-glucose transporters that have been identified only in plants. Towards this end, we analyzed, by using real time RT-PCR, the transcript accumulation ofthe genes AtUTr1 and AtUTr3 in normal plants and in those where ER stress was induced by tunicamycin or dithiothreitol (DTT). The results showed that the transcripts of the transpotiers AtUTr 1 and AtUTr3 exhibit a similar behavior to the transcripts of genes encoding for ER chaperones. This analysis also showed that the transcript for the UGGT gene is up-regulated under conditions that promote ER stress. The analysis of the promoter regions of the genes up-regulated by stress shows cis regulatory elements that are common among them, which would be associated to the ER stress response. The exposure of Arabidopsis thaliana plants to other stress conditions, such as incubation with glucose, showed that the AtUTr 1 and AtUTr3 transcripts are accumulated in a different manner compared to the genes for chaperones. Furthermore, the analysis of the promoter regions does not show the presence of common cis elements that are associated with this response, which agrees with the results obtained by real time RT-PCR. To determine whether the accumulation of the AtUTr 1 and AtUTr3 transcripts is only the result of a transcriptional regulation event, the promoter regions of both genes were cloned and fused to the uidA (GUS) gene and then tested in a heterologous system. Moreover, they were used to generate Arabidopsis thaliana transgenic plants in order to monitor the expression of the genes AtUTr 1 and AtUTr 3 throughout plant development and in different organs. On the other hand, these plants are a powe¡Jul too! to analyze the role of the promoter in the response to different stress conditions. In the laboratory we al so have insertional mutants in the genes AtUTr 1 and AtUTr3 that are "genetrap enhancer Iines" . On these plants, the uidA (GUS) reporter gene is under the control ofthe promoter that belongs to the gene that is interrupted by the insertion. Only the mutant line for the AtUTr3 gene showed ~-glucuronid ase activity, in eariy stages of development and during the reproductive development, with a high expression in pollen. The present work provide evidence that strongly support the hypothesis that the transporters AtUTr 1 and AtUTr3 play a role in the qua! ity control process required for the folding ofN-glycoproteins in the lumen ofthe ER. In addition, we showed for the first time that the transcript for the UGGT gene is differentially accumulated under conditions that promote ER stress.esTransporte BiológicoTesis