Investigations on N-Terminal Chimeras of VDAC Isoforms

Abstract

VDAC (Voltage Dependent Anion-selective Channels, [1]) are pore-forming proteins mainly found in the outer mitochondrial membrane. In mammals three isoforms have been described [2]. The proposed VDAC structure has been found to form a β-barrel with the addition of 20 amino acids at the N-terminal that fold as an amphipathic α-helix [3]. We have produced swapping mutants (or chimeras) of human VDAC isoforms carrying exchanged N-termini. In particular the addition of VDAC1 or VDAC2 N-termini to the barrel-forming sequence of VDAC3 transforms this latter protein from a poorly active behaviour into a normally active pore-forming protein [4]. The functionality of chimeras has been assayed in an S. cerevisiae strain lacking the endogenous porin. Then the activity of the chimeras has been proved in terms of its ability to complement the defective growth phenotype of yeast, due to a deficiency in the aerobic metabolism of the host cell [4].In this work we will present our investigations on the importance of the amino acid replacements expressed in the chimeras. In addition, the characterization of expressed and purified chimeras in reconstituted systems in planar lipid bilayers will be presented. The overall picture emerging from our experiments is that the VDAC N-terminal peptide plays a role in the proper function of this protein during cell life events.[1] Shoshan-Barmatz et al (2010) Mol. Aspects Med. 31, 227-85; [2] De Pinto et al (2010) Biochim. Biophys. Acta. 1797, 1268-75; Hiller et al (2010) Trends Biochem Sci. 35, 514-21; [4] Reina et al FEBS Lett. (2010) 584, 2837-44Acknowledgements: The authors acknowledge the financial support of PRIN 2008SW44CS_004. S.R. was recipient of a fellowship from INBB, Rome.