Proteolytic regulation of mitochondrial magnesium channel by m-AAA protease and prohibitin complex

Abstract

Mitochondrial membrane phospholipid cardiolipin is essential for the stability of several inner mitochondrial membrane protein complexes. We recently showed that the abundance of mitochondrial magnesium channel MRS2 is reduced in models of Barth syndrome, an X-linked genetic disorder caused by a remodeling defect in cardiolipin. However, the mechanism underlying the reduced abundance of MRS2 in cardiolipin-depleted mitochondria remained unknown. In this study, we utilized yeast mutants of mitochondrial proteases to identify an evolutionarily conserved m-AAA protease, Yta10/Yta12, responsible for degrading Mrs2. The activity of m-AAA protease is regulated by the inner mitochondrial membrane scaffolding complex prohibitin, and consistent with this role, we find that Mrs2 turnover is increased in yeast prohibitin mutants. Importantly, we find that deleting Yta10 in cardiolipin-deficient yeast cells restores the steady-state levels of Mrs2 to the wild-type cells, and the knockdown of AFG3L2, a mammalian homolog of Yta12, increases the abundance of MRS2 in a murine muscle cell line. Thus, our work has identified the m-AAA protease/prohibitin complex as an evolutionarily conserved regulator of Mrs2 that can be targeted to restore Mrs2 abundance in cardiolipin-depleted cells.

Keywords: m-AAA protease; Mitochondria; Mrs2; cardiolipin; prohibitin.

Fig. 1.

Evolutionarily conserved m-AAA protease regulates Mrs2 abundance. a) A schematic representation of the mitochondrial proteases and their submitochondrial localization in yeast S. cerevisiae. b) List of yeast proteases and their corresponding human homologs. c) SDS-PAGE immunoblot analysis of Mrs2-V5 in mitochondria isolated from WT and the indicated yeast protease deletion strains that were transformed with plasmid expressing Mrs2-V5. The mitochondrial outer membrane protein Por1 is used as a loading control. d) Quantification of Mrs2-V5 from (c) by densitometry. e) SDS-PAGE immunoblot analysis of Mrs2-V5 exogenously expressed through a plasmid in WT and yta10Δ cells treated with protein translation inhibitor CHX for the indicated time, Por1 is used as a control. f) Densitometric quantification of relative levels of Mrs2-V5 [from panel (e)] normalized to 0 h of CHX treatment. Data in (d, f) are shown as mean ± SD, with n = 3 biological replicates. ***P < 0.001, *P < 0.05.

Fig. 2.

Deletion of Phb increases the rate of Mrs2 degradation. a) A schematic depiction of a ring-like Phb complex composed of alternating Phb1 and Phb2 subunits. b) Phb complex embedded in the IMM containing defined proteins and phospholipids. Left: Phb as protein scaffold in a stoichiometric complex with m-AAA protease in the IMM. Right: Phb as a lipid scaffold defining the local phospholipid composition and creating a protein-free phospholipid environment. PS, phosphatidylserine; CL, cardiolipin; PE, phosphatidylethanolamine; PC, phosphatidylcholine; PA, phosphatidic acid; PI, phosphatidylinositol. c) SDS-PAGE immunoblot-based detection of Mrs2-V5 in mitochondria isolated from indicated yeast mutants expressing either the empty vector or V5-tagged Mrs2. Por1 is used as a loading control. d) Densitometric quantification of relative levels of Mrs2-V5 from (c). e) qPCR-based measurements of MRS2 mRNA in the indicated yeast cells. MRS2 expression was normalized to ACT1. f) SDS-PAGE immunoblot analysis of cell lysate from the indicated yeast mutants treated with protein translation inhibitor CHX for the indicated time. Por1 is used as a loading control. g) Quantification of the relative level of Mrs2 from (f), normalized to 0 h of CHX treatment. h, i) SDS-PAGE immunoblot analysis of mitochondria isolated from WT and phb1/2Δ yeast cells for the indicated respiratory chain complex subunits (h), and the outer and IMM proteins (i). The data from panels (h) and (i) are from the same mitochondrial isolates, where Por1 is used as a loading control. Data shown in panels (c), (f), (h), and (i) are representative of 3 biological replicates. Data in (d), (e), and (g) are shown as mean ± SD. ***P < 0.001, **P < 0.01, *P < 0.05, ns, not significant.

Fig. 3.

Mrs2 abundance is restored in CL-deficient yeast cells upon deletion of Yta10. a) SDS-PAGE immunoblot analysis of Mrs2-V5 expressed in WT, crd1Δ, or crd1Δyta10Δ yeast cells. Por1 is used as a loading control. Western blot image is a representative blot from 3 independent experiments. b) Quantification of the relative level of Mrs2 [from panel (a)] normalized to WT levels, n = 3 biological replicates. c) SDS-PAGE immunoblot analysis of Mrs2 levels in mitochondria isolated from the indicated yeast mutants. Western blot is a representative blot from 3 independent experiments. d) Quantification of the relative level of Mrs2 [from panel (c)], n = 3 biological replicates. e) SDS-PAGE immunoblot analysis of Mrs2-V5 exogenously expressed through a plasmid in crd1Δ and yta10Δcrd1Δ cells treated with protein translation inhibitor CHX for the indicated time, Por1 is used as a control. f) Densitometric quantification of relative levels of Mrs2-V5 [from panel (e)] normalized to 0 h of CHX treatment. g) SDS-PAGE immunoblot analysis of Mrs2-V5 in mitochondria isolated from WT and the indicated yeast deletion strains transformed with Mrs2-V5 plasmid. The mitochondrial outer membrane protein Por1 is used as a loading control. h) Quantification of Mrs2-V5 from (g) by densitometry. Data in panels (b), (d), (f), and (h) are shown as mean ± SD. ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05, n = 3 biological replicates.

Fig. 4.

Knockdown of m-AAA protease AFG3L2 increases MRS2 abundance in mammalian cells. a) SDS-PAGE immunoblot analysis of AFG3L2 and PHB1 in control and BTHS patient B-lymphocytes. VDAC1 is used as a loading control. b) Quantification of the relative level of PHB1 and AFG3L2 [from panel (a)], n = 3 biological replicates. c, d) BN-PAGE immunoblot analysis of the digitonin-solubilized mitochondria isolated from control and BTHS patient B-lymphocytes probed with anti-PHB1 (c) or anti-AFG3L2 (d). e) qPCR-based measurements of AFG3L2 mRNA from control and BTHS patient B-lymphocytes. AFG3L2 expression was normalized to ACTB. f) qPCR-based measurements of Afg3l2 mRNA from murine C2C12 cells infected either with an empty vector, pLKO.1 (Control), or with one of the 2 independent shRNAs targeting AFG3L2 (KD1 and KD2). AFG3L2 expression was normalized to ACTB. g) SDS-PAGE immunoblot analysis of AFG3L2 and MRS2 in mitochondria isolated from C2C12 cells infected with the shRNA. VDAC1 is used as a loading control. h) Quantification of the relative level of MRS2 [from panel (g)], n = 3 biological replicates. Data in panels (b), (e), (f), and (h), are shown as mean ± SD. ***P < 0.001, **P < 0.01, *P < 0.05, ns, not significant.

Fig. 5.

A model showing CL and Phb-based regulation of Mrs2. Activation of m-AAA protease Yta10 either by deletion of prohibitins (Phb1/Phb2) or depletion in CL levels in crd1Δ/taz1Δ would lead to increased turnover of Mrs2 resulting in its reduced abundance. IMM, inner mitochondrial membrane.