Díaz-Moreno, I.

Díaz-Quintana, A.

Martínez-Fábregas, J.

Rosa, M.A. de la

Rubio Novella, S.

Repositorio Institucional de la Universidad de Córdoba

S2. Microbial Proteomics  Poster P22 132  HIGH-THROUGHPUT SCREENING OF CYTOCHROME C NOVEL PARTNERS IN CHLAMYDOMONAS REINDHARTII S. Rubio Novella(1), J. Martínez-Fábregas(1), A. Díaz-Quintana(1), I. Díaz-Moreno(1), M.Á. De La Rosa(1). (1) IBVF-CSIC-US Cytochrome c (Cc) has been described in mammals as a bifunctional protein, thus playing a double role in cell respiration as an electron carrier and in apoptosis as a triggering agent of the caspase-dependent apoptotic route [1]. In plants and green algae, the role of Cc in response to oxidative stress, or programmed cell death (PCD), is still unknown [2,3]. To identify novel targets of Cc in photosynthetic organisms, we have chosen the unicellular and flagellated alga Chlamydomonas reindhartii as a simple model system. We have designed a proteomic approach starting with an affinity chromatography step, in which Chlamydomonas Cc is covalently bound to thiol-Sepharose 4B [4]. First, the protein extracts from the algal cell cultures grown under normal or oxidative stress conditions were loaded into the affinity column. Further, the proteins purified by such affinity chromatography were analyzed by the Linear Trap Quadrupole (LTQ) technique so as to identify novel proteins interacting with Cc. Currently, the physiological relevance of such potential Cc targets are being confirmed in vivo by using the bimolecular fluorescence complementation assay (BiFC) [5,6].   [1] Rodríguez-Roldán et al. A comparative kinetic analysis of the reactivity of plant, horse and human respiratory cytochrome c towards cytochrome c oxidase (2006) Biochem. Biophys. Res. Commun.   [2] Sanmartin M et al. Caspases. Regulating death since the origin of life (2005) Plant Physiol.  [3] Zuppini A et al. Programmed cell death and adaptation: two different types of abiotic stress response in a unicellular chlorophyte (2010) Plant Cell. Physiol. [4] Azzi A et al. Affinity chromatography purification of cytochrome c binding enzymes(1982) Proc. Natl. Acad. Sci. USA [5] Fujii et al. In vitro reconstitution of an abscisic acid signalling pathway (2009) Nature  [6] Gandia et al. Detection of higher-order G protein-coupled receptor oligomers by a combined BRET–BiFC technique (2008) FEBS Lett.   

High-trhoughput screening of cytochrome C novel partners chlamydomonas ReindhartII

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