Localization Of Myoglobin In Mitochondria

Abstract

PURPOSE: Mitochondria play a principal role for metabolism and have a primary role in regulating respiration and energy expenditure. Recently, we showed that the muscle- specific protein myoglobin (Mb) interacted with complex IV to augment mitochondrial respiratory capacity in skeletal muscles. However, the precise mechanism for the Mb- mediated upregulation remains under debate. The present study has focused on localizing Mb within the mitochondria. METHODS: Muscle specimen from deep portion of m. Gastrocnemius in Wistar rat was homogenized. Crude mitochondria were isolated by differential centrifugations and washed with the mitochondrial isolation buffer. The isolated mitochondria were treated with proteinase K (PK), osmotic shock (OS), and SDS (or TriX) in order to digest proteins on the outer membrane and in the intramembrane. The final samples were subjected to SDS-PAGE and immunoblotting using antibodies to localize the proteins in the mitochondria. RESULTS: Western blotting analysis revealed that the PK digests Tom20, which localized on the outer membrane of mitochondria. The Tom20 band intensity decreased with the amount of PK used. Other mitochondrial proteins such as cytochrome c (intermembrane space), COX-IV (inner membrane), and PDH (matrix), were not affected by PK treatment. PK treatment did not affect Mb. The results suggested that Mb did not localize on the outer membrane of mitochondria. The combined treatment of PK, OS and SDS (or TrX) allowed immunoblotting detection of the mitochondrial proteins in specific regions of the mitochondria. For example, cytochrome c disappeared with OS treatment. Timm22 disappeared with PK+OS treatment. However, Mb was detected with either PK or OS treatment. But it cannot be detected with a combined PK+OS treatment. The results suggest that Mb associated with the inner membrane (intramembrane side, not matrix side) of the mitochondria. CONCLUSIONS: We conclude that Mb in muscle cells localizes in the cytosol and in the mitochondrial intermembrane space. Since exercise training increases Mb expressions of skeletal muscle, the increased Mb concentration may play a direct role in modulation skeletal muscle respiration and oxidative phosphorylation capacity.