Bacterial Expression, Purification and Characterization of a Rice Voltage-Dependent, Anion-Selective Channel Isoform, OsVDAC4

Abstract

The voltage-dependent anion-selective channel (VDAC) is the most abundant protein in the mitochondrial outer membrane and forms the major conduit for metabolite transport across this membrane. VDACs from different sources show varied primary sequence but conserved functional properties. Here, we report on the characterization of a rice channel, OsVDAC4, which complements a VDAC1 deficiency in yeast. We present a consensus secondary structure prediction of an N-terminal α-helix and 19 β-strands. Bacterially expressed OsVDAC4 was purified from inclusion bodies into detergent-containing solution, where it is largely helical. Detergent-solubilized OsVDAC4 inserts spontaneously into artificial membranes of two topologies-spherical liposomes and planar bilayers. Insertion into liposomes results in an increase in β-structure. Transport of polyethylene glycols was used to estimate a pore diameter of ~2.6 nm in liposomes. Channels formed in planar bilayers exhibit large conductance (4.6 ± 0.3 nS in 1 M KCl), strong voltage dependence and weak anion selectivity. The open state of the channel is shown to be permeable to ATP. These data are consistent with a large β-barrel pore formed by OsVDAC4 on inserting into membranes. This study forms a platform to carry out studies of the interaction of OsVDAC4 with putative modulators.

Development of the Structural Core and of Conformational Heterogeneity during the Conversion of Oligomers of the Mouse Prion Protein to Worm-like Amyloid Fibrils

“…56 Dynamic light scattering measurements on a DynaPro-99 instrument (Protein Solutions) indicated that the average diameter of the vesicles was 460 ± 65 nm. Liposomes were subsequently incubated with moPrP aggregates for 1 h before performing the liposome swelling assay.…”

Genomic survey and gene expression analysis of the VDAC gene family in rice

“…Four VDAC genes have been reported previously in O. sativa (Roosens et al, 2000;Al Bitar et al, 2002Godbole et al, 2011). This study has shown that there are 8 rice VDAC genes, which is the complete rice VDAC gene family.…”

“…At least three isoforms of VDAC are present in Nicotiana tabacum ) and five different VDACs are present in Arabidopsis thaliana, Lotus japonicus, and Medicago truncatula (Clausen et al, 2004;Wandrey et al, 2004;Lee et al, 2009). There are 4 VDACs reported in Oryza sativa (Roosens et al, 2000;Al Bitar et al, 2002Godbole et al, 2011). Interestingly, cultivated rice has more VDAC isoforms than the wild Oryza officinalis group (Ma et al, 2008).…”

Molecular identification and interaction assay of the gene (OsUbc13) encoding a ubiquitin-conjugating enzyme in rice

“…VDAC is a mitochondrial outer membrane ion channel and a critical participant in the apoptosis of animal and plant cells (Al Bitar et al, 2003;Godbole et al, 2003). In plants, over-expression of rice VDAC (OsVDAC4) induces apoptosis (Godbole et al, 2011). In animal cells, knocking down VDAC1 by small interfering RNA attenuates endostatin (ES)-induced apoptosis, while over-expression of VDAC1 enhances the sensitivity of endothelial cells to ES (Yuan S. et al, 2008).…”