An N-terminal nucleotide-binding site in VDAC1: involvement in regulating mitochondrial function.
In a previous study, we presented evidence for the existence of a nucleotide-binding site (NBS) in the N-terminal region of the voltage-dependent anion channel (VDAC1). In this study, further localization and possible roles of the proposed VDAC1-NBS were investigated using site-directed mutagenesis. The predicated NBS of murine VDAC1 (mVDAC1) was mutated by replacing two glycine residues with alanines or a conserved lysine residue with a serine. Expression of the G21A,G23A- and K20S-mVDAC1s in human T-REx-293 cells in which endogenous VDAC1 expression had been silenced affected cell growth and cytosolic ATP levels. While G21A,G23A-mVDAC1-expressing cells displayed growth rates similar to native-mVDAC1-expressing cells, the K20S-mVDAC1-expressing cells displayed significantly retarded growth and increased resistance to cell death. Cells expressing either mVDAC1 mutant also displayed significantly reduced cellular ATP levels. When K20S-mutant mVDAC1 was expressed in porin1-less yeast, the transformed cells grew slower on non-fermentable carbon sources, while isolated mitochondria expressing either mVDAC1 mutant showed significant reduction in ATP synthesis. Purified K20S-mVDAC1 displayed a significant decrease in [alpha-(32)P]BzATP-binding and altered channel properties, that is, reduced ion selectivity, while the G21A,G23A-mutant protein displayed only a mild reduction in channel selectivity. These results suggest that mutations in the proposed VDAC1-NBS, particularly the K20S, altered channel activity, thereby interfering with VDAC function as the major pathway for the transport of metabolites and adenine nucleotides across the outer mitochondrial membrane. Finally, involvement of the VDAC1-NBS in the control of mitochondrial ATP synthesis, cell growth and viability is discussed.
Yehezkel
Galit
G
Department of Life Sciences, Ben-Gurion University of the Negev, Beer-Sheva, Israel.
Abu-Hamad
Salah
S
Shoshan-Barmatz
Varda
V
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism*; D000409:Alanine / Q000737:chemistry; D017209:Apoptosis; D001665:Binding Sites; D002460:Cell Line; D049109:Cell Proliferation; D003600:Cytosol / Q000378:metabolism; D005998:Glycine / Q000737:chemistry; D006801:Humans; D008239:Lysine / Q000737:chemistry; D008928:Mitochondria / Q000378:metabolism* / Q000473:pathology; D051336:Mitochondrial Membranes / Q000378:metabolism*; D009154:Mutation; D010539:Permeability; D017434:Protein Structure, Tertiary; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism; D012694:Serine / Q000737:chemistry; D013997:Time Factors; D014162:Transfection; D050995:Voltage-Dependent Anion Channel 1 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D034741:RNA, Small Interfering; D029701:Saccharomyces cerevisiae Proteins; C494762:VDAC1 protein, human; C494763:Vdac1 protein, mouse; D012694:Serine; C033901:3'-O-(4-benzoyl)benzoyladenosine 5'-triphosphate; D000255:Adenosine Triphosphate; D050995:Voltage-Dependent Anion Channel 1; D008239:Lysine; D000409:Alanine; D005998:Glycine