Assessment of Oxidative Modification of the Aac2p Carrier and Its Contribution to a PTP-Like

Assessment of oxidative modification of the Aac2p carrier and its contribution to a PTP-like function in yeast mitochondria

Dário Trindade1,2, Stephen Manon2, Manuela Côrte-Real1, Maria João Sousa1

1, Centre of Molecular and Environmental Biology (CBMA), Department of Biology, Universidado do Minho, Campus de Gualtar, 4710-057 Braga, Portugal; phone: +351253604313, Fax: +351253678980.

2, Institut de Biochimie et de Génétique Cellulaires du C.N.R.S. (IBGC), UMR5095 CNRS & Université de Bordeaux, 1 Rue de Camille Saint-Saëns, 33077 Bordeaux, France.

Mitochondria outer membrane permeabilization (MOMP) and cytochrome c (cyt c) release have been considered crucial steps of the intrinsic apoptotic pathway. Different mechanisms underlying such mitochondrial events have been suggested involving pro-apoptotic members of the Bcl-2 protein family, putative components of the permeability transition pore (PTP) or both. Recently, the ADP/ATP carrier (AAC), an yeast orthologue of mammalian adenine nucleotide translocator (ANT), has been identified as a necessary element for MOMP and cyt c release in yeast cells exposed to acetic acid. Such role of the AAC might be triggered by oxidative modifications mediated by ROS.

This work aims to understand whether oxidative modifications to the Aac2p may contribute to MOMP and consequent release of cyt c, during acetic acid induced programmed cell death (PCD) in Saccharomyces cerevisiae. Since ADP/ATP translocases are known to be sensitive to oxidative stress, especially in the thiol groups formed by cysteine residues, S. cerevisiae strain JL1-3, lacking the three forms of the AAC, was transformed with two different copies of the AAC2 gene, a wild-type (wt)and a cysteine-lessAAC2 (CL). Data collected in this work suggests that the cysteines in the Aac2p are not responsible, at least by themselves, for inducing a biochemical change capable of leading the Aac2p to a PTP-like function in yeast mitochondria. Survival assays indicate that there is no significant difference between the wt and CL strains tolerance to acetic acid. The membrane integrity and the mitochondrial membrane potential were also compared by PI exclusion and DiOC6 staining, respectively. Both experiments show no physiological differences between the death process of the wt and CL mutants. The levels of ROS, evaluated by DHE staining, are also identical in both cell types. Finally, Western-Blot detection of cytochrome c in mitochondria extracts, and the corresponding cytosolic fractions, demonstrated that the CL mutation does not prevent MOMP.

Our data indicates that the absence of the cysteines in the Aac2p, has no impact in the MOMP of S. cerevisiae cells during acetic acid-induced death. However, it is possible that a different set of residues, or a specific region of the ADP/ATP carrier, might be responsible for such process in yeast.

D.T. is supported by a PhD grant from F.C. Gulbenkian, n.º 104495. This work was financed by FEDER throughPOFC–COMPETE and national funds from FCT PEst-C/BIA/UI4050/2011.