a b s t r a c tVoltage dependent anion channel (VDAC) is a vital ion channel in mitochondrial outer membranes and its structure was recently shown to be a 19 stranded b-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 (scV-DAC) has its C-terminus exposed to the cytoplasm whilst two others are more equivocal and, when combined with published data, suggest a dynamic behavior.

BackgroundSignal peptide peptidase (SPP), a member of the presenilin-like intra-membrane cleaving aspartyl protease family, migrates on Blue Native (BN) gels as 100 kDa, 200 kDa and 450 kDa species. SPP has recently been implicated in other non-proteolytic functions such as retro-translocation of MHC Class I molecules and binding of misfolded proteins in the endoplasmic reticulum (ER). These high molecular weight SPP complexes might contain additional proteins that regulate the proteolytic activity of SPP or support its non-catalytic functions.ResultsIn this study, an unbiased iTRAQ-labeling mass spectrometry approach was used to identify SPP-interacting proteins. We found that vigilin, a ubiquitous multi-KH domain containing cytoplasmic protein involved in RNA binding and protein translation control, selectively enriched with SPP. Vigilin interacted with SPP and both proteins co-localized in restricted intracellular domains near the ER, biochemically co-fractionated and were part of the same 450 kDa complex on BN gels. However, vigilin does not alter the protease activity of SPP, suggesting that the SPP-vigilin interaction might be involved in the non-proteolytic functions of SPP.ConclusionsWe have identified and validated vigilin as a novel interacting partner of SPP that could play an important role in the non-proteolytic functions of SPP. This data adds further weight to the idea that intramembrane-cleaving aspartyl proteases, such as presenilin and SPPs, could have other functions besides the proteolysis of short membrane stubs.

negatively charged CTT of tubulin penetrates into VDAC pore, reaching through the channel at application from both sides of the membrane and interacting with high affinity with the positively charged channel lumen. We used the VDAC-tubulin specific interaction feature to probe orientation of VDAC in a planar membrane and extrapolate the results to the mitochondria outer membrane. We found that after in vitro phosphorylation by PKA or GSK3b cytosolic kinases the tubulin binding to VDAC (from rat liver mitochondria) becomes highly asymmetrical. When tubulin was added to the cis side of the membrane (side of VDAC addition) k on was more than 10 times higher than with tubulin added to the trans side. Untreated VDAC interacts symmetrically with tubulin. Considering putative PKA and GSK3b phosphorylation sites on the cytosolic loops 3, 5 and 7, we conclude that these loops face cis side in the VDAC reconstitution system. Our preliminary data show that some of the specific antibodies raised against different VDAC peptides, compete with tubulin-VDAC binding when added to the membrane bathing solution and therefore could be employed to probe VDAC orientation and positioning of the loops. Recent VDAC three-dimensional structures are compared with the functional data of VDAC-tubulin binding.