Molecular and functional characterization of VDAC2 purified from mammal spermatozoa
Résumé
VDAC (Voltage Dependent Anion selective Channel) is the pore-forming protein located in the outer mitochondrial membrane. In higher eukaryotes three genes encode for VDAC. Nevertheless the knowledge of VDAC isoforms is mainly restricted to VDAC1, the only isoform characterized from living tissues until now. We have highly enriched the isoform VDAC2 using as starting material bovine spermatozoa. VDAC2 was obtained in the hydroxyapatite/celite pass-through of sperm proteins solubilized with Triton X-100. This fraction showed in SDS-PAGE two major and one-faint bands in the Mr range of 30-35 kDa. Two-dimensional electrophoresis resolved these bands in ten spots with various Coomassie staining intensities. Western blot analysis with antibodies monospecific for each isoform and MS peptide sequencing showed that the main protein resolved in electrophoresis was VDAC2 with minor contaminations of the other isoforms. Proteomic analysis of the higher Mw VDAC2 protein allowed the coverage of the whole protein with the exception of the tri-peptide A24AR26. In the same material it was revealed the presence of two possible amino acid substitutions (T88 to L88 and A97 to Q97). Reconstitution of VDAC2 pores in planar lipid bilayers showed typical features of mitochondrial porins. Step-wise increases in membrane conductance were observed with a predominant conductance of about 3.5 nS in 1 M KCl. Very often small short-lived fluctuations were observed with single-channel conductance of about 1.5 nS. Bovine spermatozoa VDAC2 was anion-selective and showed voltage dependence. This is the first work reporting the purification and characterization of VDAC2 from a mammalian tissue.
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