Multicopy Suppressors of Phenotypes Resulting from the Absence of Yeast VDAC Encode a VDAC-like Protein

Abstract

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 (⌬por1) 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 genomic library for genes which, when overexpressed, can correct the growth defect of ⌬por1 yeast grown on glycerol at 37°C. This screen identified a second yeast VDAC gene, POR2, encoding a protein (YVDAC2) with 49% amino acid sequence identity to the previously identified yeast VDAC protein (YVDAC1). YVDAC2 can functionally complement defects present in ⌬por1 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 ⌬por1 single mutants. Like ⌬por1 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, ⌬por1 cells, ⌬por1 ⌬por2 cells, and ⌬por1 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 ⌬por1 yeast cells, VDAC-like channels could be 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.

The voltage-dependent anion channel-1 modulates apoptotic cell death

“…26,27 To identify possible Ca 2 þ binding sites in VDAC and their involvement in VDAC regulation of cell life and death, recombinant murine VDAC (mVDAC)1 or rat VDAC (rVDAC)1, modified by site-directed mutagenesis, were constructed and expressed in Saccharomyces cerevisiae (S. cerevisiae) and U-937 cells. Accordingly, recombinant native and mutated VDAC1 were expressed in the S. cerevisiae strain lacking VDAC1 (M22-2) 28 from which mitochondria were isolated and tested for HK-I binding, and VDACs purified from these mitochondria were tested for their bilayer-reconstituted channel activity. Finally, we examined the effect of native and mutated VDAC expression in U-937 cells on cell survival and RuR and HK-I protection against apoptotic cell death.…”

“…The expression of native and mutated mVDAC genes was conducted in the S. cerevisiae por1À mutant strain M22-2 (MATa, lys2 his4 trp1 ade2 ura3) 28 under the control of the yeast porin1 (YVDAC1) promoter in a lowcopy number plasmid (pSEYC58). S. cerevisiae M22-2 and its evolved mutant were cultured at 301C in selective minimal medium containing ammonium sulfate, yeast nitrogen base, the required amino acids and 2% glucose or in rich medium containing 1% yeast extract, 2% peptone (YP) with 2% lactate (YPL) or glucose (YPD) at pH 5.5.…”

Bax and Bcl-xL independently regulate apoptotic changes of yeast mitochondria that require VDAC but not adenine nucleotide translocator

“…Since mammalian cells lacking VDAC and ANT are not available, analysis of these proteins using yeast mutants seems to be useful for obtaining genetic evidence on the involvement of such proteins in apoptotic mitochondrial changes. Yeast cells carry two vdac genes and three ant genes (Blanchly-Dyson et al, 1997;Nelson et al, 1993).…”

Cytoprotective activity of minocycline includes improvement of mitochondrial coupling: the importance of minocycline concentration and the presence of VDAC

“…For example, it is known that H 2 O 2 induces apoptosis in S. cerevisiae as well as in mammalian cells (Magherini et al 2007). In addition, S. cerevisiae mitochondria express two VDAC isoforms (VDAC1 and VDAC2) of which only VDAC1, encoded by the POR1 gene, has been proven to form a channel with properties highly conserved in other species (Blachly-Dyson et al 1997;Lee et al 1998). The presence of only one channel-forming VDAC isoform in S. cerevisiae mitochondria simplifies studies of the channel.…”

“…The following Saccharomyces cerevisiae strains were used: the isogenic wild type M3 (MATa, lys2 his4 trp1 ade2 leu2 ura3) and VDAC1 (porin1)-depleted mutant M22-2 (Δpor1) (Blachly-Dyson et al 1997;Lee et al 1998). Yeast cells were grown at 28°C in YPG medium (1 % yeast extract, 2 % peptone, 3 % glycerol) at pH 5.5.…”