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A high-throughput screening for mammalian cell death effectors identifies the mitochondrial phosphate carrier as a regulator of cytochrome c release

Oncogene volume 27, pages 44–54 (2008)Cite this article

Abstract

Functional annotation of complex genomes requires the development of novel experimental platforms with increased capacity. Here, we describe a high-throughput system designed to identify cDNAs whose overexpression induces morphologically distinct cell death modalities. The methodology incorporates two robotized steps, and relies on coexpression of library clones with GFP to reveal the morphological features presented by the dying cells. By using this system we screened 135 000 cDNA clones and obtained 90 independent molecules. Interestingly, three death categories were identified, namely; apoptotic, vacuolated and autophagic. Among the pro-apoptotic clones, we found four members of the mitochondrial carrier family: the phosphate and adenine nucleotide (type 3) transporters, and the mitochondrial carrier homologs (MTCHs) 1 and 2. Expression of these molecules induced cytochrome c release and caspase-9-dependent death. One of them, the phosphate carrier, was able to interact with members of the permeability transition pore complex ANT1 and VDAC1, and its binding to ANT1 was stabilized in the presence of apoptotic activators. Depletion of this carrier by siRNA delayed cytochrome c mobilization and apoptosis. These results attribute a previously undescribed apoptotic function to the phosphate carrier and, more generally, suggest that a common property of various mitochondrial transporters was exploited during evolution to regulate apoptosis.

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Abbreviations

PARP:

poly(ADP-ribose) polymerase

PI:

propidium iodide

STS:

staurosporine

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Acknowledgements

We thank Dr Brian Seed, in whose laboratory this work was initiated, Dr Atan Gross for the Ab3 anti-MTCH2 antibody, Dr Richard Mulligan for the pMMP, pMD-G and pMD-gag-pol vectors, Dr Felix Randow for P12-MMP, Dr Ute Moll for the LF-OTC-flag construct and CIC members for support. This work was funded by Grants SAF2002-00193, SAF2005-01208 and GEN2003-20239-C06-05 from the Spanish MEC. Additional funding came from the FEDER program of the European Union. SA, MK and JF hold predoctoral fellowships from the Junta de Castilla y León (SA) or the FPU program of the Spanish Government (MK and JF). AF holds a long-term EMBO postdoctoral fellowship. FXP is an investigator affiliated to the University of Salamanca and funded by the Ramón y Cajal program (Spanish Government).

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  1. Instituto de Biología Molecular y Celular del Cáncer, Centro de Investigación del Cáncer, CSIC-Universidad de Salamanca, Campus Miguel de Unamuno, Salamanca, Spain

    S Alcalá, M Klee, J Fernández, A Fleischer & F X Pimentel-Muiños

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  1. S Alcalá
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Correspondence to F X Pimentel-Muiños.

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Alcalá, S., Klee, M., Fernández, J. et al. A high-throughput screening for mammalian cell death effectors identifies the mitochondrial phosphate carrier as a regulator of cytochrome c release. Oncogene 27, 44–54 (2008). https://doi.org/10.1038/sj.onc.1210600

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