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VDAC/porin is present in sarcoplasmic reticulum from skeletal muscle
V Shoshan-Barmatz1, N Hadad, W Feng
1Department of Life Sciences, Ben Gurion University of the Negev, Beer Sheva, Israel.
Abstract:
In this study we demonstrate the existence of a protein with properties of the voltage-dependent anion channel (VDAC) in the sarcoplasmic reticulum (SR) using multiple approaches as summarized in the following: (a) 35 and 30 kDa proteins in different SR preparations, purified from other membranal systems by Ca2+/oxalate loading and sedimentation through 55% sucrose, cross-react with four different VDAC monoclonal antibodies. (b) Amino acid sequences of three peptides derived from the SR 35 kDa protein are identical to the sequences present in VDAC1 isoform. (c) Similar to the mitochondrial VDAC, the SR protein is specifically labeled by [14C]DCCD. (d) Using a new method, a 35 kDa protein has been purified from SR and mitochondria with a higher yield for the SR. (e) Upon reconstitution into a planar lipid bilayer, the purified SR protein shows voltage-dependent channel activity with properties similar to those of the purified mitochondrial VDAC or VDAC1/porin 31HL from human B lymphocytes, and its channel activity is completely inhibited by the anion transport inhibitor DIDS and about 80% by DCCD. We also demonstrate the translocation of ATP into the SR lumen and the phosphorylation of the luminal protein sarcalumenin by this ATP. Both ATP translocation and sarcalumenin phosphorylation are inhibited by DIDS, but not by atractyloside, a blocker of the ATP/ADP exchanger. These results indicate the existence of VDAC, thought to be located exclusively in mitochondria, in the SR of skeletal muscle, and its possible involvement in ATP transport. Together with recent studies on VDAC multicompartment location and its dynamic association with enzymes and channels, our findings suggest that VDAC deserves attention and consideration as a protein contributing to various cellular functions.
Insights
Researchers found a voltage-dependent anion channel (VDAC) protein in the sarcoplasmic reticulum (SR), not just mitochondria. This VDAC protein facilitates ATP transport into the SR, suggesting a broader role for VDAC in cellular functions.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- The voltage-dependent anion channel (VDAC) is primarily known as a mitochondrial outer membrane protein.
- Its presence and function in other cellular compartments, like the sarcoplasmic reticulum (SR), remain largely unexplored.
Purpose of the Study:
- To investigate the presence and functional characteristics of VDAC in the sarcoplasmic reticulum (SR).
- To determine the role of SR VDAC in ATP transport and protein phosphorylation within the SR lumen.
Main Methods:
- Immunological cross-reactivity of SR proteins with VDAC antibodies.
- Peptide sequencing to compare SR protein with known VDAC isoforms.
- Chemical labeling with [14C]DCCD.
- Protein purification from SR and mitochondria.
- Reconstitution of purified SR protein into planar lipid bilayers for electrophysiological analysis.
- Inhibition studies using DIDS and atractyloside.
Main Results:
- A 35 kDa protein in SR preparations cross-reacted with VDAC antibodies and shared peptide sequences with VDAC1.
- The purified SR protein exhibited voltage-dependent channel activity similar to mitochondrial VDAC.
- This SR VDAC was inhibited by DIDS and DCCD.
- ATP translocation into the SR lumen and subsequent phosphorylation of sarcalumenin were demonstrated.
- These processes were inhibited by DIDS, indicating VDAC's involvement.
Conclusions:
- The study provides strong evidence for the existence of VDAC in the skeletal muscle SR.
- SR VDAC appears to mediate ATP transport into the SR lumen, influencing luminal protein phosphorylation.
- These findings expand the known cellular distribution of VDAC and suggest its involvement in diverse cellular functions beyond mitochondrial energy metabolism.