Selectivity Changes in Site-Directed Mutants of the VDAC Ion Channel: Structural Implications

Abstract

The gene encoding the yeast mitochondrial outer membrane channel VDAC was subjected to site-directed mutagenesis to change amino acids at 29 positions to residues differing in charge from the wild-type sequence. The mutant genes were then expressed in yeast, and the physiological consequences of single and multiple amino acid changes were assessed after isolation and insertion of mutant channels into phospholipid bilayers. Selectivity changes were observed at 14 sites distributed throughout the length of the molecule. These sites are likely to define the position of the protein walls lining the aqueous pore and hence, the transmembrane segments. These results have been used to develop a model of the open state of the channel in which each polypeptide contributes 12 beta strands and one alpha helix to form the aqueous transmembrane pathway.

Probing the orientation of yeast VDAC1 in vivo

“…The FLAG1, which is 11-20 residues from the N-terminus, will most likely have a trans exposure (see Fig. 1); especially if the Nterminus is as dynamic as proposed [8,13,[22][23][24][25]. Residues 1-10 continue through the lumen towards exposure of the extreme Nterminal residue to the cis side.…”

“…It has also been suggested to play a role in apoptosis [5] although this remains controversial [6]. Despite its importance, the structure of VDAC1 was unclear [7][8][9][10] until recently published high resolution structures of human and mouse VDAC1 revealed a 19 stranded b-barrel capable of dimerization and dynamic fluctuation [11][12][13] (Fig. 1).…”

Purification and Characterization of the Voltage-Dependent Anion-Selective Channel Protein from Wheat Mitochondrial Membranes

“…By transfecting the VDAC cDNA into a VDAC-deleted yeast strain and regaining complementation of the VDAC activity, BlachlyDyson et al (1990) verified that the previously purified 29-kD protein is indeed the cause of the VDAC activity. By imposing site-directed mutagenesis on the yeast VDAC cDNA and by transfecting the modified genes into a VDACdeleted yeast strain, they (Blachly-Dyson et al, 1990) determined the protein regions responsible for the unique VDAC properties. Moreover, a three-dimensional structure model of Blumenthal et ai.…”

“…Intensive research has been carried out during the last 15 years that has resulted in a comprehensive characterization of the fungal mitochondrial VDAC (Forte et al, 1987;Blachly-Dyson et al, 1990;Kiebler et al, 1990). In studies involving mainly S. cerevisiae and N. crassa, the VDAC proteins were purified (Freitag et al, 1982;Mihara and Sato, 1985;Forte et al, 1987), and their electrophysiological properties were revealed (Colombini, 1979;Freitag et al, 1982).…”

“…Vol. 101, 1993 the protein molecule was developed based on the results gained from these experiments (Blachly-Dyson et al, 1990). Finally, a major contribution has been made to the understanding of the VDAC targeting and import into mitochondria by the isolation of several important receptors that are related to these processes (Kiebler et al, 1990).…”