Voltage-dependent anion-selective channel (VDAC) interacts with the dynein light chain Tctex1 and the heat-shock protein PBP74.
The voltage-dependent anion-selective channel 1 (VDAC1), i.e. eukaryotic porin, functions as a channel in membranous structures as described for the outer mitochondrial membrane, the cell membrane, endosomes, caveolae, the sarcoplasmatic reticulum, synaptosomes, and post-synaptic density fraction. The identification of VDAC1 interacting proteins may be a promising approach for better understanding the biological context and function of the channel protein. In this study human VDAC1 was used as a bait protein in a two-hybrid screening, which is based on the Sos recruitment system (SRS). hVDAC1 interacts with the dynein light chain Tctex-1 and the heat-shock protein peptide-binding protein 74 (PBP74)/mitochondrial heat-shock protein 70 (mtHSP70)/glucose-regulated protein 75 (GRP75)/mortalin in vivo. Both interactions were confirmed by overlay-assays using recombinant partner proteins and purified hVDAC1. Indirect immunofluorescence on HeLa cells indicates a co-localisation of hVDAC1 with the dynein light chain and the PBP74. In addition, HeLa cells were transfected transiently with enhanced green fluorescent protein (EGFP)-hVDAC1 fusion proteins, which also clearly co-localise with both proteins. The functional relevance of the identified protein interactions was analysed in planar lipid bilayer (PLB) experiments. In these experiments both recombinant binding partners altered the electrophysiological properties of hVDAC1. While rTctex-1 increases the voltage-dependence of hVDAC1 slightly, the rPBP74 drastically minimises the voltage-dependence, indicating a modulation of channel properties in each case. Since the identified proteins are known to be involved in the transport or processing of proteins, the results of this study represent additional evidence of membrane-associated trafficking of the voltage-dependent anion-selective channel 1.
Schwarzer
Christian
C
Max-Planck-Institute for Experimental Medicine, Department of Immunochemistry, Hermann-Rein Street 3, 37075 Göttingen, Germany.
Barnikol-Watanabe
Shitsu
S
Thinnes
Friedrich P
FP
Hilschmann
Norbert
N
D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D004398:Dyneins; D004594:Electrophysiology; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D007150:Immunohistochemistry; D007473:Ion Channels / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D008051:Lipid Bilayers / Q000378:metabolism; D008868:Microtubule Proteins / Q000235:genetics / Q000378:metabolism*; D008869:Microtubule-Associated Proteins; D008928:Mitochondria / Q000378:metabolism*; D009687:Nuclear Proteins; D018272:Porins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D020798:Two-Hybrid System Techniques; D050995:Voltage-Dependent Anion Channel 1; D050994:Voltage-Dependent Anion Channels; D056427:t-Complex Genome Region
D002352:Carrier Proteins; C533386:Dynlt1b protein, mouse; D018840:HSP70 Heat-Shock Proteins; C083794:Hspa9 protein, mouse; D007473:Ion Channels; D008051:Lipid Bilayers; D008868:Microtubule Proteins; D008869:Microtubule-Associated Proteins; D009687:Nuclear Proteins; D018272:Porins; D011993:Recombinant Fusion Proteins; C494762:VDAC1 protein, human; D050994:Voltage-Dependent Anion Channels; D050995:Voltage-Dependent Anion Channel 1; D004398:Dyneins