Characterization of calcium-dependent signaling pathway in the secretion and cellu...
| Grant number: | 05/54618-9 |
| Support type: | Research Projects - Thematic Grants |
| Duration: | March 01, 2006 - February 28, 2009 |
| Field of knowledge: | Biological Sciences - Genetics - Plant Genetics |
| Principal Investigator: | Márcio de Castro Silva Filho |
| Grantee: | Márcio de Castro Silva Filho |
| Home Institution: | Escola Superior de Agricultura Luiz de Queiroz (ESALQ). Universidade de São Paulo (USP). Piracicaba , SP, Brazil |
Abstract
This laboratory has as one of its main Lines of Research studies aiming at understanding the molecular mechanisms responsible for the protein localization inside of the plant cells, especially the proteins directed to mitochondria and chloroplasts. In this proposal, it is intended to advance in the characterization of the regulatory mechanisms involved in the intracellular localization of different proteins, also integrating an evolutionary approach. Four sub-projects are presented. The first one aims to identify proteins interacting with THIl protein of Arabidopsis thaliana on thiamin biosynthesis, by using the yeast double-hybrid technique. Moreover, it is intended to identify proteins responsible for the control of the double targeting of this protein, since it was shown by this group that this protein is directed simultaneously to mitochondria and chloroplasts by a post-transcriptional mechanism. The second sub-project aims to study in details the thiamin biosynthesis in plants. Preliminary studies carried through by this group involving techniques of in-silico analysis and molecular phylogenetics suggest that biosynthesis of thiamin in plants can involve more than one subcellular compartment, opposing previous works that suggest the occurrence of the pathway only in the plastids. In addition to a deep in-silico analysis there experiments of functional complementation in yeast cells of plant genes involved on thiamin biosynthesis will be carried out. Furthermore, the determination of the subcellular localization of all proteins involved in the process will be provided by gene fusions with the green fluorescent protein (GFP). The third project aims at to understand one of the most fascinating mechanisms of gene regulation recently described: riboswitches. The presence of structures in mRNA capable to interact directly with molecules intervening with its own translation has driven the attention of several groups worldwide. In the case of one of genes involved in thiamin biosynthesis, thic of Arabidopsis thaliana, it was shown that its mRNA presents an aptamer-like structure that allows its binding to either thiamin pirophosfate (TPP) or thiamin, regulating its expression. It is intended to verify experimentally if the occurrence of this structure intervenes with the expression of the thic gene as well as to the subcellular localization of the THIC protein. Moreover, a study will be carried through in silico in all genes whose products are involved in thiamin biosynthesis in order to identify the occurrence of aptamer-like structures and, in positive case, to verify its function experimentally. The fourth sub-project involves the characterization of the mechanism responsible for the localization of one metaloprotease of the thylakoid membrane, the FtsH-p1. Members of this family had been described as belonging to the Tat system (twin-arginine translocation system) for insertion in the thylakoid membrane. In a previous work of this group, it was shown that a member of this family does not present a classical RR motif responsible for its insertion in the membrane. Therefore, it is intended to characterize the mechanism responsible for the localization of protein, which suggests a distinct mechanism of those described in literature. (AU)