Localization of myoglobin in mitochondria: implication in regulation of mitochondrial respiration in rat skeletal muscle.

Rikuhide Koma,Tsubasa Shibaguchi,Thomas Jue,Toshihiko Oka,Claudia Pérez López,Kazumi Masuda,Hisashi Takakura

doi:10.14814/phy2.14769

Abstract

Mitochondria play a principal role in metabolism, and mitochondrial respiration is an important process for producing adenosine triphosphate. Recently, we showed the possibility that the muscle‐specific protein myoglobin (Mb) interacts with mitochondrial complex IV to augment the respiration capacity in skeletal muscles. However, the precise mechanism for the Mb‐mediated upregulation remains under debate. The aim of this study was to ascertain whether Mb is truly integrated into the mitochondria of skeletal muscle and to investigate the submitochondrial localization. Isolated mitochondria from rat gastrocnemius muscle were subjected to different proteinase K (PK) concentrations to digest proteins interacting with the outer membrane. Western blotting analysis revealed that the PK digested translocase of outer mitochondrial membrane 20 (Tom20), and the immunoreactivity of Tom20 decreased with the amount of PK used. However, the immunoreactivity of Mb with PK treatment was better preserved, indicating that Mb is integrated into the mitochondria of skeletal muscle. The mitochondrial protease protection assay experiments suggested that Mb localizes within the mitochondria in the inner membrane from the intermembrane space side. These results strongly suggest that Mb inside muscle mitochondria could be implicated in the regulation of mitochondrial respiration via complex IV.

Highlights

Mitochondria are essential organelles for cellular energy metabolism, and their respiration is an important process that requires oxygen (O2) to convert the energy stored in macronutrients into adenosine triphosphate (ATP)
The molecular machinery required for mitochondrial respiration is the electron transport chain (ETC), which is an Abbreviations: AP, alkaline phosphatase; ATP, adenosine triphosphate; Co-IP, co-immunoprecipitation; COX-IV, cytochrome c oxidase subunit IV; Cyt c, cytochrome c; ETC, electron transport chain; IMM, inner mitochondrial membrane; IMS, intermembrane space; Mb, myoglobin; Mic60/Mitofilin, MICOS complex subunit Mic60; O2, oxygen; OMM, outer mitochondrial membrane; OS, osmotic shock; PDH, pyruvate dehydrogenase; PK, proteinase K; SDS, sodium dodecyl sulfate; Tom, translocase of outer mitochondrial membrane; TOM, translocase of the outer membrane; Tx-1 00, Triton X-100
The PDH immunoreactivity showed no significant change at PK concentrations of 0–20 μg/ml, whereas the PDH immunoreactivity at PK concentrations of 40–8 0 μg/ml was significantly reduced compared to 10 μg/ml PK and 20 μg/ml PK treated mitochondria (p < 0.05); the mean value was higher than 76.5% (Figure 2a,e)

Summary

Introduction

Mitochondria are essential organelles for cellular energy metabolism, and their respiration is an important process that requires oxygen (O2) to convert the energy stored in macronutrients into adenosine triphosphate (ATP). A co-immunoprecipitation (Co-IP) analysis revealed that a portion of the Mb present in muscle binds cytochrome c oxidase subunit IV (COX-IV), a component of mitochondrial complex IV at the IMM. These findings suggest that Mb would be integrated into the mitochondria and would interact with complex IV in muscle cells. Questions remain about the precise mitochondrial localization of Mb. Differential centrifugation alone cannot remove proteins anchored in the OMM (Yamada et al, 2013), and previous studies cannot confidently exclude possible Mb interactions at the OMM (Postnikova et al, 2009; Taylor et al, 2003; Vernier et al, 2007), which would alter the interpretation of Co-IP results indicating Mb binding to COX-IV. This study aimed to investigate whether Mb is truly integrated into the mitochondria of skeletal muscle and its submitochondrial localization

Objectives

Findings

Discussion

Conclusion