Probing the orientation of yeast VDAC1 in vivo.
Voltage dependent anion channel (VDAC) is a vital ion channel in mitochondrial outer membranes and its structure was recently shown to be a 19 stranded beta-barrel. However the orientation of VDAC in the membrane remains unclear. We probe here the topology and membrane orientation of yeast Saccharomyces cerevisiae in vivo. Five FLAG-epitopes were independently inserted into scVDAC1 and their surface exposure in intact and disrupted mitochondria detected by immunoprecipitation. Functionality was confirmed by measurements of respiration. Two epitopes suggest that VDAC (scVDAC) has its C-terminus exposed to the cytoplasm whilst two others are more equivocal and, when combined with published data, suggest a dynamic behavior.
McDonald
Beth M
BM
The Institute for Cell and Molecular Biosciences, Newcastle University, Framlington Place, Newcastle-upon-Tyne NE2 4HH, UK.
Wydro
Mateusz M
MM
Lightowlers
Robert N
RN
Lakey
Jeremy H
JH
D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D003593:Cytoplasm / Q000235:genetics / Q000378:metabolism; D007473:Ion Channels / Q000235:genetics*; D008565:Membrane Proteins / Q000235:genetics; D026901:Membrane Transport Proteins / Q000235:genetics*; D008928:Mitochondria / Q000235:genetics / Q000378:metabolism; D051336:Mitochondrial Membranes / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics* / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D050994:Voltage-Dependent Anion Channels / Q000235:genetics*
D007473:Ion Channels; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D029701:Saccharomyces cerevisiae Proteins; D050994:Voltage-Dependent Anion Channels