期刊
JOURNAL OF CELLULAR PHYSIOLOGY
卷 212, 期 2, 页码 551-561出版社
WILEY-BLACKWELL
DOI: 10.1002/jcp.21048
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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 (VDAC 1). In this study, further localization and possible roles of the proposed VDAC I-NBS were investigated using site-directed mutagenesis. The predicated NBS of murine VDAC I (mVDAC 1) was mutated by replacing two glycine residues with alanines or a conserved lysine residue with a serine. Expression of the G2IA,G23A- and K20S-mVIDACI s in human T-REx-293 cells in which endogenous VDAC I expression had been silenced affected cell growth and cytosolic ATP levels. While G2IA,G23A-mVDACI expressing cells displayed growth rates similar to native-mVDACI-expressing cells, the K20S-mVDACI-expressing cells displayed significantly retarded growth and increased resistance to cell death. Cells expressing either mVIDAC I mutant also displayed significantly reduced cellular ATP levels. When K20S-mutant mVDAC I was expressed in porin I -less yeast, the transformed cells grew slower on non-fermentable carbon sources, while isolated mitochondria expressing either mVDAC I mutant showed significant reduction in ATP synthesis. Purified K20S-mVDAC I displayed a significant decrease in [alpha-P-32]BzATP-binding and altered channel properties, that is, reduced ion selectivity, while the G2IA,G23A-mutant protein displayed only a mild reduction in channel selectivity. These results suggest that mutations in the proposed VDACI-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 VDACI-NBS in the control of mitochondrial ATP synthesis, cell growth and viability is discussed.
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