Multicopy suppressors of phenotypes resulting from the absence of yeast VDAC encode a VDAC-like protein

The permeability of the outer mitochondrial membrane to most metabolites is believed to be based in an outer membrane, channel-forming protein known as VDAC (voltage-dependent anion channel). Although multiple isoforms of VDAC have been identified in multicellular organisms, the yeast Saccharomyces cerevisiae has been thought to contain a single VDAC gene, designated POR1. However, cells missing the POR1 gene (Δpor1) were able to grow on yeast media containing a nonfermentable carbon source (glycerol) but not on such media at elevated temperature (37°C). If VDAC normally provides the pathway for metabolites to pass through the outer membrane, some other protein(s) must be able to partially substitute for that function. To identify proteins that could functionally substitute for POR1, we have screened a yeast generate library for genes which, when overexpressed, can correct the growth defect of Δpor1 yeast grown on glycerol at 37°C. This screen identified a second yeast VDAC gene, POR2, encoding a protein (YVDAC2) with 49% amine acid sequence identity to the previously identified yeast VDAC protein (YVDAC1). YVDAC2 can functionally complement defects present in Δpor1 strains only when it is overexpressed. Deletion of the POR2 gene alone had no detectable phenotype, while yeasts with deletions of both the POR1 and POR2 genes were viable and able to grow on glycerol at 30°C, albeit more slowly than Δpor1 single mutants. Like Δpor1 single mutants, they could not grow on glycerol at 37°C. Subcellular fractionation studies with antibodies which distinguish YVDAC1 and YVDAC2 indicate that YVDAC2 is normally present in the outer mitochondrial membrane. However, no YVDAC2 channels were detected electrophysiologically in reconstituted systems. Therefore, mitochondrial membranes made from wild-type cells, Δpor1 cells, Δpor1 Δpor2 cells, and Δpor1 cells overexpressing YVDAC2 were incorporated into liposomes and the permeability of resulting liposomes to nonelectrolytes of different sizes was determined. The results indicate that YVDAC2 does not confer any additional permeability to these liposomes, suggesting that it may not normally form a channel. In contrast, when the VDAC gene from Drosophila melanogaster was expressed in Δpor1 yeast cells, VDAC-like channels could he detected in the mitochondria by both bilayer and liposome techniques, yet the cells failed to grow on glycerol at 37°C. Thus, channel-forming activity does not seem to be either necessary or sufficient to restore growth on nonfermentable carbon sources, indicating that VDAC mediates cellular functions that do not depend on the ability to form channels.