Summary
The outer mitochondrial membranes of all organisms so far examined contain a protein which forms voltage-dependent anion selective channels (VDAC) when incorporated into planar phospholipid membranes. Previous reports have suggested that the yeast (Saccharomyces cerevisiae) outer mitochondrial membrane component responsible for channel formation is a protein of 29,000 daltons which is also the major component of this membrane. In this report, we describe the purification of this 29,000-dalton protein to virtual homogeneity from yeast outer mitochondrial membranes. The purified protein readily incorporates into planar phospholipid membranes to produce ionic channels. Electrophysiological characterization of these channels has demonstrated they have a size, selectivity and voltage dependence similar to VDAC from other organisms. Biochemically, the purified protein has been characterized by determining its amino acid composition and isoelectric point (pI). In addition, we have shown that the purified protein, when reconstituted into liposomes, can bind hexokinase in a glucose-6-phosphate dependent manner, as has been shown for VDAC purified from other sources. Since physiological characterization suggests that the functional parameters of this protein have been conserved, antibodies specific to yeast VDAC have been used to assess antigenic conservation among mitochondrial proteins from a wide number of species. These experiments have shown that yeast VDAC antibodies will recognize single mitochondrial proteins fromDrosophila, Dictyostelium andNeurospora of the appropriate molecular weight to be VDAC from these organisms. No reaction was seen to any mitochondrial protein from rat liver, rainbow trout,Paramecium, or mung bean. In addition, yeast VDAC antibodies will recognize a 50-kDa mol wt protein present in tobacco chloroplasts. These results suggest that there is some antigenic as well as functional conservation among different VDACs.
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References
Benz, R. 1985. Porin from bacterial and mitochondrial outer membranes.CRC Crit. Rev. Biochem. 19:145–190
Capaldi, R., Vanderkooi, G. 1972. The low polarity of many membrane proteins.Proc. Natl. Acad. Sci. USA 69:930–932
Colombini, M. 1979. A candidate for the permeability pathway of the outer mitochondrial membrane.Nature (London) 279:643–645
Colombini, M. 1980. Structure and mode of action of a voltage dependent anion-selective channel (VDAC) located in the outer mitochondrial membrane.Ann. N.Y. Acad. Sci. 341:552–563
Colombini, M. 1983. Purification of VDAC (voltage-dependent anion-selective channel) from rat liver mitochondria.J. Membrane Biol. 74:115–121
Felgner, P.L., Messer, J., Wilson, J.E. 1979. Purification of a hexokinase binding protein from the outer mitochondrial membrane.J. Biol. Chem. 254:4946–4949
Fiek, C., Benz, R., Roos, N., Brdiczka, D. 1982. Evidence for identity between the hexokinase-binding protein and the mitochondrial porin in the outer membrane of rat liver mitochondria.Biochim. Biophys. Acta 688:429–440
Flugge, U.I., Benz, R. 1984. Pore-forming activity in the outer membrane of the chloroplast envelope.FEBS Lett. 169:85–90
Forte, M., Guy, H.R., Mannella, C. 1987. Molecular genetics of the VDAC ion channel: Structural model and sequence analysis.J. Biochembr. Bioenerg., (in press)
Freitag, H., Benz, R., Neupert, W. 1983. Isolation and properties of the porin of the outer mitochondrial membrane fromNeurospora crassa Methods Enzymol. 97:286–294
Freitag, H., Neupert, W., Benz, R. 1982. Purification and characterization of a pore protein of the outer mitochondrial membrane fromNeurospora crassa.Eur. J. Biochem. 123:629–636
Gasser, S.M., Schatz, G. 1983. Import of proteins into mitochondria.J. Biol. Chem. 258:3427–3430
Glass, W., Briggs, R., Hnillica, L. 1981. Identification of tissue-specific nuclear antigens transferred to nitrocellulose from polyacrylamide gels.Science 211:70–72
Laemmli, U.K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4.Nature (London) 227:680–685
Linden, M., Gellerfors, P., Nelson, B.D. 1982a. Purification of a protein having pore forming activity from the rat liver mitochondrial outer membrane.Biochem. J. 208:77–82
Linden, M., Gellerfors, P., Nelson, B.D. 1982b. Pore protein and the hexokinase-binding protein from the outer membrane of rat liver mitochondria are identical.FEBS Lett. 141:189–192
Mannella, C.A., Colombini, M. 1984. Evidence that the crystalline arrays in the outer membrane ofNeurospora mitochondria are composed of the voltage-dependent channel protein.Biochim. Biophys. Acta 774:206–214
Markwell, M.K., Hass, S.M., Bieber, L.L., Tolbert, N.E. 1978. A Modification of the Lowry procedure to simplify protein determination in membrane and lipoprotein samples.Anal. Biochem. 87:206–210
Mihara, K., Blobel, G., Sato, R. 1982.In vitro synthesis and integration into mitochondria of porin, a major protein of the outer mitochondrial membrane ofSaccharomyces cerevisiae.Proc. Natl. Acad. Sci. USA 79:7102–7106
Mihara, K., Sato, R. 1985. Molecular cloning and sequencing ofcDNA for yeast porin, an outer mitochondrial membrane protein: A search for targeting signal in the primary structure.EMBO J. 4:769–774
Montal, M., Mueller, P. 1972. Formation of biomolecular membranes from lipid monolayers and a study of their electrical properties.Proc. Natl. Acad. Sci. USA 69:3561–3566
Nakashima, R.A., Mangan, P.S., Colombini, M., Pedersen, P.L. 1986. Hexokinase receptor complex in hepatoma mitochondria: Evidence from N,N′-1 dicyclohexylcarbodiimide-labeling studies for the involvement of the pore-forming protein VDAC.Biochemistry 25:1015–1021
O'Farrell, P.H. 1975. High resolution two-dimensional electrophoresis of proteins.J. Biol. Chem. 250:4007–4021
Roos, N., Benz, R. Brdiczka, D. 1982. Identification and characterization of the pore-forming protein in the outer membrane of rat liver mitochondria.Biochim. Biophys. Acta 686:204–214
Schein, S.J., Colombini, M., Finkelstein, A. 1976. Reconstitution in planar lipid bilayers of a voltage-dependent anion-selective channel obtained from paramecium mitochondria.J. Membrane Biol. 30:99–120
Tremblath M., Tzagoloff, A. 1979. Large- and small-scale preparations of yeast mitochondria.Methods Enzymol. 50:160–163
Zalman, L.S., Nikaido, H., Kagawa, Y. 1980. Mitochondrial outer membrane contains a protein producing nonspecific diffusion channels.J. Biol. Chem. 255:1771–1774
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Forte, M., Adelsberger-Mangan, D. & Colombini, M. Purification and characterization of the voltage-dependent anion channel from the outer mitochondrial membrane of yeast. J. Membrain Biol. 99, 65–72 (1987). https://doi.org/10.1007/BF01870622
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DOI: https://doi.org/10.1007/BF01870622