Membrane protein turnover by the m-AAA protease in mitochondria depends on the transmembrane domains of its subunits - PubMed (original) (raw)

Membrane protein turnover by the m-AAA protease in mitochondria depends on the transmembrane domains of its subunits

Daniel Korbel et al. EMBO Rep. 2004 Jul.

Abstract

AAA proteases are membrane-bound ATP-dependent proteases that are present in eubacteria, mitochondria and chloroplasts and that can degrade membrane proteins. Recent evidence suggests dislocation of membrane-embedded substrates for proteolysis to occur in a hydrophilic environment; however, next to nothing is known about the mechanism of this process. Here, we have analysed the role of the membrane-spanning domains of Yta10 and Yta12, which are conserved subunits of the hetero-oligomeric m-AAA protease in the mitochondria of Saccharomyces cerevisiae. We demonstrate that the m-AAA protease retains proteolytic activity after deletion of the transmembrane segments of either Yta10 or Yta12. Although the mutant m-AAA protease is still capable of processing cytochrome c peroxidase and degrading a peripheral membrane protein, proteolysis of integral membrane proteins is impaired. We therefore propose that transmembrane segments of m-AAA protease subunits have a direct role in the dislocation of membrane-embedded substrates.

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Figures

Figure 1

Figure 1

Restoration of respiratory growth of Δ_yta10_ and Δ_yta12_ cells on expression of Yta10ΔTM or Yta12ΔTM, respectively. (A–C) Cells were grown at 30°C on selective media containing the required auxotrophs and glucose (2%) as the sole carbon source. Fivefold serial dilutions of logarithmically growing cultures were spotted onto yeast peptone medium containing glycerol (3%) and incubated for 2 days at 30°C. (D) Co-immunoprecipitation of Yta12 with Yta10ΔTM. Mitochondria isolated from wild-type (WT), Δ_yta10_ and yta10_Δ_TM cells were solubilized in digitonin and incubated with Yta10specific antibodies. The immunoprecipitate was analysed by SDS–PAGE and immunoblotting using Yta12-specific antiserum.

Figure 2

Figure 2

Respiratory competence of yta10_Δ_TM cells depends on the proteolytic activity of Yta12 and the ATPase but not the proteolytic activity of Yta10ΔTM. Cells were grown at 30°C on YP medium containing glycerol. (A) Expression of a proteolytic site mutant variant of Yta10ΔTM in Δ_yta10_ cells (yta10 E559Q_Δ_TM) and in Δ_yta10_Δ_yta12_ cells expressing proteolytically inactive Yta12E614Q (yta10 E559Q_Δ_TM yta12 E614Q) (B) Expression of Yta10ΔTM variants harbouring mutations in conserved arginine residues of the SRH region in Δ_yta10_ cells.

Figure 3

Figure 3

Impaired proteolysis of misfolded integral inner membrane proteins in yta10_Δ_TM and yta12_Δ_TM mitochondria. The stability of newly imported, 35S-labelled Yme2ΔC (A) and Oxa1ts (B) was analysed at 37°C in mitochondria isolated from wild-type (WT), Δ_yta10_, yta10_Δ_TM, Δ_yta12_ and yta12_Δ_TM cells. The average of at least three independent experiments (±s.e.m.) is shown.

Figure 4

Figure 4

Processing of Ccp1 by a mutant m_-AAA protease lacking the transmembrane domain of one subunit. Mitochondria were isolated from wild-type (WT), Δ_yta10, yta10_Δ_TM, Δ_yta12_ and yta12_Δ_TM cells and fractionated by SDS–PAGE (30 μg). Samples were analysed by immunoblotting using polyclonal antisera directed against Ccp1 (anti-Ccp1) and, as gel loading controls, cytochrome _b_2 (anti-Cyt b2) and Mge1 (anti-Mge1). p, precursor form of Ccp1; m, mature form of Ccp1.

Figure 5

Figure 5

Degradation of Atp7 by a mutant m_-AAA protease harbouring truncated subunits. Radiolabelled Atp7 was imported into mitochondria isolated from wild-type (WT), Δ_yta10, yta10_Δ_TM, Δ_yta12_ and yta12_Δ_TM cells, and the stability of newly imported Atp7 at 37°C was determined as in Fig 3. The average of four independent experiments (±s.e.m.) is shown.

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