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

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.

Keywords: myoglobin; proteinase K; skeletal muscle; submitochondrial localization.

FIGURE 1

Schematic representation of biochemical approaches in the present study. (a) Scheme for isolating mitochondria from rat gastrocnemius. (b) Scheme for performing the treatment with different PK concentrations. (c–d) Scheme for performing the mitochondrial protease protection assay. IMM, inner mitochondrial membrane; OMM, outer mitochondrial membrane; OS, osmotic shock; PK, proteinase K; TCA, trichloroacetic acid; and Tx‐100, Triton X‐100.

FIGURE 2

Mb is localized inside the mitochondria of skeletal muscle. (a) Western blotting was performed with antibodies for Mb, Tom20, Mic60/Mitofilin and PDH on isolated mitochondria treated with different PK concentrations. (b–e) Quantification of the immunoreactivities of all analyzed proteins in PK treated mitochondria (n = 3 from separate rats). The immunoreactivities of proteins of untreated mitochondria were set as 100%. The values are means ± SD. Significant differences were assessed using one‐way ANOVA and Bonferroni's post hoc test. *, †, §, and ¶ indicate significantly different from untreated, 5 μg/ml PK, 10 μg/ml PK, and 20 μg/ml PK treated mitochondria, respectively (p < 0.05). IMM, inner mitochondrial membrane; Mb, myoglobin; Mic60/Mitofilin, MICOS complex subunit Mic60; OMM, outer mitochondrial membrane; PDH, pyruvate dehydrogenase; PK, proteinase K; and Tom, translocase of outer mitochondrial membrane.

FIGURE 3

Mitochondrial protease protection assay. (a) Western blotting was performed with antibodies for Mb, Tom20, Mic60/Mitofilin and PDH on isolated mitochondria treated with OS, Tx‐100 and/or PK. (b–e) Quantification of the immunoreactivities of all analyzed proteins in PK treated mitochondria (n = 3 from separate rats). The immunoreactivities of proteins of untreated mitochondria were set as 100%. The values are means ± SD. Significant differences were assessed using one‐way ANOVA and Bonferroni's post hoc test. *, †, §, and ¶ indicate significantly different from untreated, PK treated, OS treated, and OS + PK treated mitochondria, respectively (p < 0.05). IMM, inner mitochondrial membrane; Mb, myoglobin; Mic60/Mitofilin, MICOS complex subunit Mic60; OMM, outer mitochondrial membrane; OS, osmotic shock; PDH, pyruvate dehydrogenase; PK, proteinase K; Tom, translocase of outer mitochondrial membrane; and Tx‐100, Triton X‐100.

FIGURE 4

Mb is partially unanchored in the IMS. (a) Western blotting was performed with antibodies for Mb, Cyt c, and Mic60/Mitofilin on untreated and OS treated mitochondria. (b–d) Quantification of the immunoreactivities of all analyzed proteins in PK treated mitochondria (n = 3 from separate rats). The immunoreactivities of proteins of untreated mitochondria were set as 100%. The values are means ± SD. Significant differences were assesses using an unpaired t test. * indicates significantly different from untreated mitochondria (p < 0.05). (e) Presence of Mb in the supernatant and pellet fractions following OS treatment. Cyt c, cytochrome c; IMM, inner mitochondrial membrane; IMS, intermembrane space; M, mitochondria; Mb, myoglobin; Mic60/Mitofilin, MICOS complex subunit Mic60; OS, osmotic shock; P, pellet; and S, supernatant.