Abstract
Mitochondrial porins or voltage-dependent anion-selective channels are channel-forming proteins mainly found in the mitochondrial outer membrane. Genome sequencing of the fruit fly Drosophila melanogaster revealed the presence of three additional porin-like genes. No functional information was available for the different gene products. In this work we have studied the function of the gene product closest to the known Porin gene (CG17137 coding for DmPorin2). Its coding sequence was expressed in Escherichia coli. The recombinant DmPorin2 protein is able to form channels similar to those formed by DmPorin1 reconstituted in artificial membranes. Furthermore, DmPorin2 is clearly voltage-independent and cation-selective, whereas its counterpart isoform 1 is voltage-dependent and anion-selective. Sequence comparison of the two porin isoforms indicates the exchange of four lysines in DmPorin1 for four glutamic acids in DmPorin2. We have mutated two of them (Glu-66 and Glu-163) to lysines to investigate their role in the functional features of the pore. The mutants E163K and E66K/E163K are endowed with an almost full inversion of the ion selectivity. Both single mutations partially restore the voltage dependence of the pore. We found that an additional effect with the double mutant E66K/E163K was the restoration of voltage dependence. Protein structure predictions highlight a 16 beta-strand pattern, typical for porins. In a three-dimensional model of DmPorin2, Glu-66 and Glu-163 are close to the rim of the channel, on two opposite sides. DmPorin2 is expressed in all the fly tissues and in all the developmental stages tested. Our main conclusions are as follows. 1) The CG17137 gene may express a porin with a functional role in D. melanogaster. 2) We have identified two amino acids of major relevance for the voltage dependence of the porin pore.
Highlights
Mitochondrial porins or voltage-dependent anion-selective channels are channel-forming proteins mainly found in the mitochondrial outer membrane
Mitochondrial porins or voltage-dependent anion-selective channels (VDACs)1 are pore-forming proteins mainly found in the mitochondrial outer membrane, where they are able to form large hydrophilic channels [1,2,3]
The role of mitochondrial porin isoform 1 or VDAC1 in apoptosis was thoroughly investigated in the last few years [12,13,14,15,16]
Summary
Mitochondrial porins or voltage-dependent anion-selective channels (VDACs) are pore-forming proteins mainly found in the mitochondrial outer membrane, where they are able to form large hydrophilic channels [1,2,3]. Porin or VDAC is the major pore-forming protein of the mitochondrial outer membrane and has a unique role in energetic metabolism since all metabolites, including ADP and ATP, have to pass through the porin channels [9, 10]. It is the receptor where kinases bind to the mitochondrial surface, which is important for the metabolic energy channeling in the cell [11]. It has an identity of about 60% compared with those of the porin isoforms in mammals [26, 27]
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