Membrane Protein Degradation by AAA Proteases in Mitochondria Klaus Leonhard, Bernard Guiard, Giovanna Pellecchia, Alexander Tzagoloff, Walter Neupert, Thomas Langer  Molecular Cell  Volume 5, Issue 4, Pages 629-638 (April 2000) DOI: 10.1016/S1097-2765(00)80242-7

Figure 1 Degradation of Yme2p Imported into the Mitochondrial Inner Membrane (A) Submitochondrial localization of newly imported Yme2p. Import of radiolabeled Yme2p and mitochondrial subfractionation was performed as described in Experimental Procedures. Immunoblots using antisera directed against cytochrome b2 (Cyt b2), the ADP/ATP carrier (AAC), and Mge1p are shown in the lower panel. An N-terminal fragment of AAC is generated by proteinase K (PK) in mitoplasts (Endres et al. 1999). The cartoon shows the topology of Yme2p in the mitochondrial inner membrane (Hanekamp and Thorsness 1996). Abbreviations: p, precursor form of Yme2p; m, mature form of Yme2p; Pe, pellet; Sup, supernatant; IMS, intermembrane space; IM, inner membrane. (B) Stability of newly imported Yme2p at 25°C and at 37°C. After completion of import, samples were split into halves and incubated at 25°C (closed squares) or 37°C (closed circles). Aliquots were removed at the time points indicated, chilled on ice, and analyzed by SDS-PAGE. Yme2p was quantified by laser densitometry and is given as percentage of imported Yme2p. Molecular Cell 2000 5, 629-638DOI: (10.1016/S1097-2765(00)80242-7)

Figure 2 Unfolding of Solvent-Exposed Domains at Both Membrane Surfaces Triggers Proteolysis (A) Topology of Yme2ΔN33, DHFRWT-Yme2ΔN, and DHFRmut-Yme2ΔN in the mitochondrial inner membrane. DHFR moieties of the hybrid proteins are shown in grey. (B) Stability of Yme2ΔN33, DHFRWT-Yme2ΔN, and DHFRmut-Yme2ΔN in mitochondria. After import of the precursor proteins and digestion of nonimported preproteins with trypsin (Tryp), samples were divided into halves and further incubated at 25°C or 37°C as indicated. (C) Topology of DHFRmut-TM-DHFRWT and DHFRmut-TM-DHFRmut in the mitochondrial inner membrane. (D) Stability of DHFRmut-TM-DHFRWT and DHFRmut-TM-DHFRmut in mitochondria. The stability of the hybrid proteins after completion of import was analyzed at 25°C and 37°C. Abbreviations: p, precursor form; m, mature form; IMS, intermembrane space; IM, inner membrane. Molecular Cell 2000 5, 629-638DOI: (10.1016/S1097-2765(00)80242-7)

Figure 3 Degradation of Yme2p and Derivatives with Domains on Either Membrane Side by m- and i-AAA Protease (A and B) Yme2p, (C) DHFRmut-Yme2ΔN, (D) DHFRmut-TM-DHFRWT, and (E) DHFRmut-TM-DHFRmut were imported into mitochondria isolated from wild-type cells (WT), Δyme1, and Δyta10 mutant cells. After trypsin treatment of the mitochondria (+Tryp), degradation of imported proteins at 37°C was assessed. The time course of the proteolytic breakdown of Yme2p in WT, Δyme1, and Δyta10 mitochondria is shown in (A). Proteolytic fragments generated by trypsin treatment are marked with an asterisk (*). Degradation of Yme2p, DHFRmut-Yme2ΔN, DHFRmut-TM-DHFRWT, and DHFRmut-TM-DHFRmut was determined after an incubation of the mitochondria for 45 min at 37°C (B–E). Imported preprotein prior to proteolysis was set to 100%. The average of three independent experiments is shown. Molecular Cell 2000 5, 629-638DOI: (10.1016/S1097-2765(00)80242-7)

Figure 4 Overlapping Substrate Specificity of the m- and i-AAA Protease Wild-type (WT), Δyme1, Δyta12 Gal10-YTA12, and Δyme1/Δyta12 Gal10-YTA12 cells were grown at 30°C on galactose-containing YP medium, reisolated, and further cultivated for 48 hr in YP medium containing 2% glucose for depletion of Yta12p. (A) Steady-state levels of Yta12p, Yme1p, and, for control, Mge1p were determined by immunoblot analysis of cell extracts generated by alkaline lysis (Yaffe and Schatz 1984). (B) Synthetic lethality of mutations in YME1 and YTA12. We spotted 10-fold serial dilutions of cells onto YP plates containing 2% glucose and incubated for 24 hr at 30°C. (C) Stability of various Yme2p derivatives in wild-type (WT) and Δyme1 mitochondria after Yta12p depletion (Δyme1/Δyta12 Gal10-YTA12). Mitochondria were incubated for 30 min at 37°C after import to allow proteolysis to occur. Molecular Cell 2000 5, 629-638DOI: (10.1016/S1097-2765(00)80242-7)

Figure 5 Binding of the m-AAA Protease Requires Unfolding of a Protein Domain Protruding into the Matrix DHFRWT-Yme2ΔN and DHFRmut-Yme2ΔN were imported into mitochondria harboring proteolytically inactive Yta10E559Qp (yta10E559Q) or Yme1E541Qp (yme1E541Q). After lysis, coimmunoprecipitations with Yta10p- or Yme1p-specific antisera or preimmune serum (pre) were performed. Molecular Cell 2000 5, 629-638DOI: (10.1016/S1097-2765(00)80242-7)

Figure 6 Degradation of the Matrix Domain of Yme2p (A) Topology of Yme2ΔC23 and Yme2ΔC28 in the mitochondrial inner membrane. Yme2ΔC28 exposes 28 amino acid residues (including five C-terminal methionine residues) to the intermembrane space. Yme2ΔC23 has only 23 amino acid residues located in the intermembrane space. (B) Acetylation of Yme2ΔC23 accelerates proteolysis. Newly synthesized Yme2ΔC23 was either directly imported into wild-type mitochondria (closed circle) or acetylated prior to import to impair folding in mitochondria (closed square). Then, the stability of acetylated and nonacetylated Yme2ΔC23 at 37°C was examined. Yme2ΔC23 present in mitochondria is given as percentage of the imported protein. (C) Acetylation does not affect degradation of Yme2ΔC28. Acetylated (closed square) and nonacetylated (closed circle) Yme2ΔC28 was imported into wild-type mitochondria and the stability at 37°C was determined. (D) The m-AAA protease mediates the proteolytic breakdown of Yme2ΔC23. Newly synthesized Yme2ΔC23 was acetylated prior to import. The stability of Yme2ΔC23 at 37°C was analyzed after import in wild-type (closed circle), Δyme1 (open triangle), and Δyta10 (open square) mitochondria as in (B). (E) Both m- and i-AAA protease can degrade Yme2ΔC28. The stability of acetylated Yme2ΔC28 was examined in wild-type (closed circle), Δyme1 (open triangle), and Δyta10 (open square) mitochondria and in Δyme1/Δyta12 Gal10-YTA12 mitochondria depleted of Yta12p (inverted closed triangle) as in (B). Molecular Cell 2000 5, 629-638DOI: (10.1016/S1097-2765(00)80242-7)

Figure 7 Degradation of the Intermembrane Space Domain of Yme2p (A) Topology of Yme2ΔN17, Yme2ΔN28, and Yme2ΔN33 in the mitochondrial inner membrane. The Yme2p variants expose 17, 28, or 33 amino acid residues to the matrix but are otherwise identical. (B) Stability of Yme2ΔN17, Yme2ΔN28, and Yme2ΔN33 in wild-type mitochondria at 37°C. The preproteins Yme2ΔN17 (closed circle), Yme2ΔN28 (closed square), and Yme2ΔN33 (closed triangle) were imported into isolated mitochondria, and proteolysis at 37°C was analyzed. (C) Proteolysis of Yme2ΔN17 is mediated by the i-AAA protease. The stability of Yme2ΔN17 at 37°C was determined after import into wild-type (closed circle), Δyme1 (open triangle), and Δyta10 (open square) mitochondria. (D) Both m- and i-AAA protease can degrade Yme2ΔN33. Radiolabeled Yme2ΔN33 was imported into wild-type (closed circle), Δyta10 (open square), and Δyme1 (open triangle) mitochondria and in Δyme1/Δyta12 Gal10-YTA12 mitochondria depleted of Yta12p (inverted closed triangle), and the stability at 37°C was analyzed. Molecular Cell 2000 5, 629-638DOI: (10.1016/S1097-2765(00)80242-7)