Hexokinase-binding properties of the mitochondrial VDAC protein: inhibition by DCCD and location of putative DCCD-binding sites
Related Experiment Videos
1Department of Chemistry and Biochemistry, Texas Tech University, Lubbock 79409.
Abstract:
The outer mitochondrial membrane receptor for hexokinase binding has been identified as the VDAC protein, also known as mitochondrial porin. The ability of the receptor to bind hexokinase is inhibited by pretreatment with dicyclohexylcarbodiimide (DCCD). At low concentrations, DCCD inhibits hexokinase binding by covalently labeling the VDAC protein, with no apparent effect on VDAC channel-forming activity. The stoichiometry of [14C]-DCCD labeling is consistent with one to two high-affinity DCCD-binding sites per VDAC monomer. A comparison between the sequence of yeast VDAC and a conserved sequence found at DCCD-binding sites of several membrane proteins showed two sites where the yeast VDAC amino acid sequence appears to be very similar to the conserved DCCD-binding sequence. Both of these sites are located near the C-terminal end of yeast VDAC (residues 257-265 and 275-283). These results are consistent with a model in which the C-terminal end of VDAC is involved in binding to the N-terminal end of hexokinase.
Insights
Voltage-dependent anion channel (VDAC) protein binds hexokinase to the outer mitochondrial membrane. Dicyclohexylcarbodiimide (DCCD) selectively inhibits this binding by labeling VDAC, identifying key binding sites near the C-terminus.
Area of Science:
- Mitochondrial biology
- Protein-protein interactions
- Biochemistry
Background:
- Hexokinase binding to the outer mitochondrial membrane is crucial for cellular energy regulation.
- The voltage-dependent anion channel (VDAC) protein has been implicated as the receptor for hexokinase.
- Understanding the precise interaction sites is key to elucidating mitochondrial function.
Purpose of the Study:
- To identify the specific VDAC protein regions involved in hexokinase binding.
- To investigate the mechanism by which dicyclohexylcarbodiimide (DCCD) inhibits VDAC-hexokinase interaction.
- To characterize the binding stoichiometry and location of DCCD modification sites on VDAC.
Main Methods:
- Pretreatment of VDAC with dicyclohexylcarbodiimide (DCCD) to assess inhibition of hexokinase binding.
- Radiolabeling ([14C]-DCCD) to quantify DCCD binding stoichiometry to VDAC monomers.
- Sequence analysis comparing yeast VDAC with conserved DCCD-binding motifs in other membrane proteins.
Main Results:
- Dicyclohexylcarbodiimide (DCCD) inhibits hexokinase binding to VDAC by covalently labeling the VDAC protein.
- DCCD labeling occurs at one to two high-affinity sites per VDAC monomer without affecting channel activity.
- Sequence analysis revealed two potential DCCD-binding sites in yeast VDAC (residues 257-265 and 275-283) near the C-terminus.
Conclusions:
- The C-terminal end of the VDAC protein is critically involved in the binding of hexokinase.
- Specific residues within the C-terminal region of VDAC serve as high-affinity binding sites for DCCD.
- These findings support a model where VDAC's C-terminus interacts with hexokinase's N-terminus.