The Italian Journal of Biochemistry Vol. 56 (4) 2007 279 Guarino et al. INTRODUCTION Mitochondrial PORINS or VDACs (voltage- dependent anion-selective channels) are transmem- brane pore-forming proteins in charge of the free dif- fusion of small solutes through the mitochondrial outer membrane (up to a well-defined exclusion limit of about 3000-5000 Da) [1-2]. Several important cel- lular pathways are interested by the VDAC activities: among them the most relevant are apoptosis [3-4] and the coordination of the cellular bioenergetics [5- 6]. In the evolution PORINS developed as slightly basic proteins with molecular masses around 30 kDa and a putative structural organization as a 16- strands beta barrel [7]. Gene duplication or other evolutionary events originated sets of porin genes in almost any organism studied so far. Three related but distinct isoforms of VDAC exist in vertebrates but several others possibilities have been found in other animals and in plants [8]. Interestingly in the course of biochemical purification experiments the so-called VDAC1 isoform was usually obtained. Its characteri- zation and functional reconstitution has given most of the information available for this protein [1-2]. Afterwards the isoforms VDAC2 and VDAC3 were found serendipitously during high-throughput searches of the genomes or the proteomes of sever- al organisms [9-10]. Genome projects have con- firmed that in vertebrates three paralog genes exist. The problem of the functional meaning of the addi- tional genes has been addressed only indirectly. The expression of the conceptual reading frames as recombinant proteins has been the preferred approach to investigate the function of these paralog genes [11-12]. This kind of experiments has anyway an obvious weakness: the native structure of the pro- teins with their potential post-translational modifica- tion is not ascertained at all, thus also the functional results obtained could be biased by the lack of the native conformation. In the fruit fly Drosophila melanogaster the porin gene (CG6647) was cloned, sequenced and mapped at 32B3-4 on the chromosome 2L. The completion of the fruit fly genome revealed the close proximity of three additional genes with significant homology to porin called CG17137, CG17139 and CG17140 [13]. The three additional genes are on the same strand as the porin gene, and they have a sim- ilar organization with two introns interrupting the cod- ing sequence. The phylogenetic analysis of this The Voltage Dependent Anion selective Channel family in Drosophila melanogaster Francesca Guarino 1,2 , Angela Messina 1,2 , Andrea Guarnera 2 , Giuseppe Puglia 2 , Francesco Bellia 2 , Simona Reina 2 , Vito De Pinto 1,2 , Valeria Specchia 3 and Maria Pia Bozzetti 3 1 Research Unit, National Institute for Biostructures and Biosystems, Roma, Italy 2 Department of Chemical Sciences, University of Catania, Catania, Italy 3 Department of Biological and Environmental Sciences, University of Salento, Ecotekne, Lecce, Italy Abstract. VDACs (voltage-dependent anion-selective channels) or mitochondrial PORINS are transmembrane proteins forming pores in the outer membrane. In eukaryotic genomes multiple genes coding for VDAC homologues have been discovered, but the functional meaning of this gene redundancy is unknown. In Drosophila melanogaster three additional genes homologous to the gene porin (CG6647) have been found. As in other occurences, the presence of a gene revealed by genome analysis raises the questions: are these genes really expressed? What are the molecular features of the putative proteins, if they are expressed? Where and when in the organism are they expressed? Consequently have they any specific activity justifying the presence of more isoforms in the organism? To answer to these questions we have produced antibodies against the recombinant proteins corresponding to the whole (VDAC1 and 2) or to substantial portions (VDAC3 and 4) of the known or predicted proteins. Immunohistological and transcriptional analysis has been performed, showing that VDAC2 and 3 are expressed, while VDAC4 was not detected. Structural predictions of VDAC3 are consistent with the presence of additional D-helices at the N-terminus of the protein. Key words: Voltage-dependent anion-selective channel isoforms, Drosophila melanogaster, Secondary structure prediction, RT-PCR, Immunolocalization