Lon Protease Mediated Degradation of Cytochrome c Oxidase Subunits and Altered Enzyme Activity During Hypoxia

Lon, a mitochondrial ATP dependent matrix protease, has been shown to be involved in the maintenance of mitochondrial DNA nucleoids and in the degradation of abnormal or misfolded proteins. We have previously shown that hypoxic stress induces the PKA dependent phosphorylation of cytochrome c oxidase (CcO) subunits I, IV and Vb and a time dependent reduction in subunit contents in raw 264.7 murine macrophages. Herein, we show that Lon is involved in the selective turnover of CcO subunits under hypoxic stress. We compared Lon protease expression levels and degradation of CcO subunits at various time points of hypoxic stress. Induction of Lon protease occurs as early as six hours of hypoxic stress and this increase coincides with lower CcO subunit contents. Overexpression of Lon by transient transfection accelerated the turnover of CcO subunits I, IV and Vb under hypoxic stress and also a marginal decrease in the CcO subunit levels under normoxic conditions. We next studied the role of Lon in degradation of wild type and phosphorylation site mutated CcO subunit Vb under normoxia and hypoxia. Overexpression of flag tagged wild type and mutant forms of Vb protein along with Lon caused selective degradation of wild type subunit but the mutant subunit was resistant to degradation. Our results suggest that Lon has a role in the regulation of CcO activity during hypoxia. Supported by NIH grant GM49683.