Interaction Of Myoglobin With Hsp70 Chaperone: Implication In Mitochondrial Translocation Via Tom Complex

Abstract

In contrast to conventional physiology, our studies have shown that muscle-specific protein myoglobin (Mb) is not only localized in the cytosol but also within the mitochondria and have suggested that Mb augments mitochondrial respiratory function by up-regulating complex IV activity. However, the uncertain mechanism of Mb import into mitochondria continues to challenge the hypothesis that Mb can directly affect respiration by modulating Complex IV activity. Proteins that lack the targeting sequence to transport from cytosol to the mitochondria through the TOM complex at the outer membrane must use the transport assistance of heat shock proteins (HSPs). Since Mb does not contain a mitochondrial targeting sequence, its import into the mitochondria may depend upon the HSPs-dependent TOM pathway. PURPOSE: The aim of this study was to investigate 1) the HSP dependent mechanisms of Mb translocation into mitochondria in skeletal muscle and 2) the conditions that promote Mb transport. METHODS: Muscle specimen from a deep portion of the gastrocnemius was taken from adult male Wistar rats. Crude mitochondria were isolated from the muscle sample by differential centrifugations. The isolated mitochondria were used for immunoprecipitation (IP) to ascertain whether the Mb is incorporated into HSPs-dependent TOM pathway. In addition, C2C12 myotubes were treated with 25 μM H2O2 for 6-h, and then the cells were harvested to isolate mitochondria. The final samples were subjected to immunoblotting. RESULTS: IP experiments showed the co-immunoprecipitation of Tom20 and Tom70 (TOM complex receptors) with Mb, suggesting that Tom20 and/or Tom70 recognized Mb. HSP70, but not HSP90, was also co-immunoprecipitated with Mb, implying that only HSP70 chaperoned Mb to the TOM complex receptors. The cell culture experiment showed that Mb expression in the mitochondrial fraction tended to increase by H2O2 treatment compared with non-treated group. CONCLUSION: The present results suggest that HSP70 chaperones the Mb translocation via the TOM complex. Oxidative stress could be a factor stimulating Mb transport into the mitochondria. Supported by Sasakawa Scientific Research Grant from The Japan Science Society.