TOM complex‐independent transport pathway of myoglobin into mitochondria in C2C12 myotubes

Abstract

Recently, we found that myoglobin (Mb) localizes in both the cytosol and mitochondrial intermembrane space in rodent skeletal muscle. Most proteins of the intermembrane space pass through the outer mitochondrial membrane via the translocase of the outer membrane (TOM) complex. However, whether the TOM complex imports Mb remains unknown. The purpose of this study was to investigate the involvement of the TOM complex in Mb import into the mitochondria. A proteinase K protection assay of mitochondria from C2C12 myotubes confirmed that Mb integrated into the mitochondria. An immunoprecipitation assay verified the interaction of Mb and TOM complex receptors (Tom20, Tom70) in isolated mitochondria. The assay showed a clear interaction of Mb with Tom20 and Tom70. A knockdown experiment using siRNA for TOM complex receptors (Tom20, Tom70) and TOM complex channel (Tom40) did not alter the amount of Mb expression in the mitochondrial fraction. These results suggested that Mb does not necessarily require the TOM complex for mitochondrial import of Mb. Although the physiological role of Mb interactions with TOM complex receptors remains unclear, further studies are needed to clarify how Mb enters the mitochondria independently of the TOM complex.

Endurance training increases mitochondrial myoglobin and enhances its interaction with complex IV in rat plantaris muscle

“…45 If Mb also localizes in the endoplasmic reticulum, it is possible that Mb is also imported into the mitochondria through the same pathway as AIF. 14 Further studies are needed to elucidate the mechanisms of mitochondrial transport of Mb and to examine whether endurance training activates these mitochondrial transport pathways.…”

“…A major unresolved question in the present study is how mitochondrial import of Mb was promoted by endurance training. Continuous exercise is suggested to promote mitochondrial protein import by the TOM complex, which is the main entry gate across the outer mitochondrial membrane, in skeletal muscle; 39 however, mitochondrial import of Mb is independent of the TOM complex 14 . Therefore, we have considered two pathways of Mb transport to mitochondria independent of TOM complex.…”

“…A previous study has reported that apoptosis inducing factor (AIF) is imported from the endoplasmic reticulum into the mitochondria via transport vesicles during the mitochondrial fusion/fission process independent of TOM complex 45 . If Mb also localizes in the endoplasmic reticulum, it is possible that Mb is also imported into the mitochondria through the same pathway as AIF 14 . Further studies are needed to elucidate the mechanisms of mitochondrial transport of Mb and to examine whether endurance training activates these mitochondrial transport pathways.…”

“…Mb is responsible for oxygen (O 2 ) storage, buffering intracellular O 2 concentrations and facilitating O 2 diffusion 9,10 . Although Mb has been treated as a cytoplasmic protein, 9,11,12 our laboratory and a previous study found that some Mb localizes within mitochondria in myocytes and brown adipocytes 8,13,14 . Since noncanonical mitochondrial proteins, such as Stat3 and neuroglobin, which are mainly expressed in the nucleus and/or cytosol, regulate mitochondrial functions, 15,16 it is possible that mitochondrial Mb also regulates mitochondrial functions.…”

“…9,10 Although Mb has been treated as a cytoplasmic protein, 9,11,12 our laboratory and a previous study found that some Mb localizes within mitochondria in myocytes and brown adipocytes. 8,13,14 Since noncanonical mitochondrial proteins, such as Stat3 and neuroglobin, which are mainly expressed in the nucleus and/or cytosol, regulate mitochondrial functions, 15,16 it is possible that mitochondrial Mb also regulates mitochondrial functions. Indeed, Mb overexpression results in increased mitochondrial respiratory function in myocytes and brown adipocytes.…”