Volume 85, Issue 6, Pages 875-885 (June 1996) The YTA10–12 Complex, an AAA Protease with Chaperone-like Activity in the Inner Membrane of Mitochondria  Heike Arlt, Raimund Tauer, Horst Feldmann, Walter Neupert, Thomas Langer  Cell  Volume 85, Issue 6, Pages 875-885 (June 1996) DOI: 10.1016/S0092-8674(00)81271-4

Figure 1 Yta10p and Yta12p Form a High Molecular Mass Complex in the Mitochondrial Inner Membrane (A) Superose 6 gel chromatography of detergent extracts of wild-type (closed circle, closed box), Δyta12 (open circle), and Δyta10 (open box) mutant mitochondria in the presence of ATP. Yta10p (closed circle, open circle) and Yta12p (closed box, open box) were detected in eluate fractions by Western blot analysis, using the enhanced chemiluminescence system. Protein amounts, determined by laser densitometry, are given as percent of Yta10p and Yta12p in the eluate (“total”). Hsp60 (840 kDa) and cytochrome b2 (“cyt b2,” 250 kDa) were used as standards for calibration. (B) Coimmunoprecipitation of Yta10p and Yta12p after fractionation of 35S-labeled mitochondrial extracts by sizing chromatography. 35S-labeled mitochondria (800 μg protein) were solubilized in buffer A containing 1 mM ATP and fractionated by Superose 6 gel chromatography. A portion (4%) of each fraction was removed and, after precipitation with TCA (12.5%), analyzed by SDS–PAGE and autoradiography. Peak fractions of Yta10p and Yta12p were determined in the eluate by immunostaining (data not shown). The remaining part of eluate fractions was divided into halves and subjected to coimmunoprecipitation with preimmune (not shown) or affinity-purified Yta10p-specific antibodies. The analysis of the immunoprecipitates by SDS–PAGE and autoradiography is shown in the upper panel. 35S-labeled Yta10p (open circle) and Yta12p (closed box) in the immunoprecipitates were quantified by laser densitometry. (C) Yta10p and Yta12p were the sole constituents of the 850 kDa complex. The 850 kDa form (eluted at 12.5 ml) and the 250 kDa form (eluted at 15 ml) of Yta10p were precipitated with preimmune serum or with affinity-purified Yta10p-specific antibodies as in (B) and analyzed by SDS–PAGE and autoradiography. (D) Submitochondrial localization of Yta12p. Mitochondria and mitoplasts, generated by hypotonic swelling, were incubated for 30 min at 4°C in the presence or absence of 50 μg/ml proteinase K (PK). For lysis, Triton X-100 was added to a final concentration of 0.1%. Mitoplasts were subjected to alkaline extraction (Na2CO3) and split by centrifugation for 60 min at 226,000 × g into pellet (P) and supernatant (S) fraction. Mitochondrial fractions were analyzed by SDS–PAGE and Western blot analysis, using specific antisera directed against Yta12p, Yta10p, cytochrome c peroxidase (CCPO), a soluble constituent of the intermembrane space, translocase of mitochondrial inner membrane protein 23 (TIM23), and mitochondrial GrpE (Mge1p), localized in the matrix space. Cell 1996 85, 875-885DOI: (10.1016/S0092-8674(00)81271-4)

Figure 2 Formation of the 850 kDa Complex of Yta10p and Yta12p Is Nucleotide-Dependent (A) Superose 6 gel chromatography of mitochondrial extracts in the presence (closed circle, closed box) or absence (open circle, open box) of 1 mM ATP. Yta10p (closed circle, open circle) and Yta12p (closed box, open box) were determined in the eluate as described in Figure 1. (B) Coimmunoprecipitation of Yta10p and Yta12p in the presence of various nucleotides. Mitochondria (200 μg protein) were solubilized in buffer B that was supplemented with 5 mM of the respective nucleotide, as indicated. After a clarifying spin, extracts were subjected to coimmunoprecipitation with Yta10p- and Yta12p-specific antisera, as described in Experimental Procedures. Yta10p or Yta12p were not precipitated with preimmune serum (data not shown). (C) Association of Yta10p and Yta12p within intact mitochondria required high levels of ATP. To vary ATP levels, mitochondria (300 μg protein) were incubated for 15 min at 30°C in import buffer (0.5 M sorbitol, 80 mM KCl, 10 mM Mg-acetate, 2 mM K-phosphate, 2 mM MnCl2, 3% fatty acid free bovine serum albumin, 50 mM HEPES·KOH [pH 7.2]) supplemented with 1 mM ATP (lanes 1 and 3) or 20 μM oligomycin and 40 U/ml apyrase (lanes 2 and 4), at a concentration of 0.4 mg/ml. Mitochondria were reisolated by centrifugation for 10 min at 9000 × g, washed twice with 1 ml SHKCl buffer, and then resuspended in buffer B containing 1 mM ATP at a concentration of 0.2 mg/ml. Samples were incubated for 15 min at 4°C under vigorous mixing and centrifuged for 15 min at 90,000 × g. The supernatant was divided into three aliquots and subjected to coimmunoprecipitation with Yta10p- and Yta12p-specific antiserum or preimmune serum (not shown). Precipitated proteins were analyzed by SDS–PAGE and immunostaining, using antisera directed against Yta12p (upper panel) or Yta10p (lower panel). Cell 1996 85, 875-885DOI: (10.1016/S0092-8674(00)81271-4)

Figure 3 Yta10p and Yta12p Are ATP Dependent Metallopeptidases in the Mitochondrial Inner Membrane (A) Degradation of polypeptides in the mitochondrial inner membrane requires Yta12p. Mitochondrial translation products were labeled in wild type (open triangle), Δyta12 mutant mitochondria (open box) and in Δyta12 mutant mitochondria containing Yta12E614Qp (closed box). Mitochondria were resuspended in translation buffer and incubated at 37°C. At the timepoints indicated, aliquots were withdrawn and radioactivity released into the TCA soluble fraction was measured, as described in Experimental Procedures. Total radioactivity in the TCA soluble and insoluble fraction at various timepoints was set to 100%. Newly synthesized polypeptides were tightly associated with the inner membrane and resistant to alkaline or urea extraction prior to proteolysis (data not shown). (B) Yta10E559Qp did not mediate the degradation of polypeptides in the mitochondrial inner membrane. Proteolysis of 35S-labeled mitochondrial translation products was monitored in wild-type (open triangle), Δyta10 mutant mitochondria (open circle), and Δyta10 mutant mitochondria containing Yta10E559Qp (closed circle) as in (A). (C) Newly imported Yta10E559Qp formed a complex with preexisting Yta12p. Yta10E559Qp was synthesized in rabbit reticulocyte lysate in the presence of 35S-methionine. Δyta10 mutant mitochondria (250 μg) were resuspended in import buffer containing 5 mM NADH and 2.5 mM ATP at a concentration of 0.5 mg/ml. For a control, Δyta10 mutant mitochondria (100 μg) were resuspended in import buffer supplemented with 0.5 μM valinomycin to dissipate the membrane potential (lanes 1 and 2). Reticulocyte lysate was added to a final concentration of 10% (v/v), and import was performed for 15 min at 25°C. Import was stopped by the addition of 0.5 μM valinomycin. Aliquots corresponding to 50 μg mitochondria were withdrawn (lanes 1 and 3). The remaining sample was further incubated for 20 min at 4°C in the presence of 50 μg/ml trypsin to digest nonimported precursor protein. The protease was blocked by adding a 10-fold molar excess of soybean trypsin inhibitor. After removal of a further aliquot (lanes 2 and 4), mitochondria were reisolated by centrifugation for 10 min at 9000 × g, washed with 0.5 ml SHKCl buffer containing trypsin inhibitor (0.6 mg/ml), and resuspended in buffer B supplemented with 1 mM ATP. The supernatant of a clarifying spin was then divided into halves and subjected to coimmunoprecipitation using preimmune serum or Yta12p-specific antiserum (lanes 5 and 6). Cell 1996 85, 875-885DOI: (10.1016/S0092-8674(00)81271-4)

Figure 4 Substrate Binding to the YTA10–12 Complex (A) Cross-linking of substrate polypeptides to Yta10p and Yta12p. Mitochondria (100 μg) containing labeled mitochondrial translation products were incubated for 15 min at 4°C (minus Chase) or 37°C (plus Chase) and then subjected to chemical cross-linking when indicated. For a reference, aliquots were withdrawn (“control”). Polypeptides cross-linked to Yta10p or Yta12p were isolated by immunoprecipitation, as described in Experimental Procedures, and analyzed by SDS–PAGE. Noncross-linked Yta10p and Yta12p migrated during electrophoresis as indicated. (B) Substrate binding was impaired in Δyta10 and Δyta12 mutant mitochondria. Mitochondrial translation was performed in isolated wild-type (WT), Δyta10, and Δyta12 mutant mitochondria (100 μg) followed by extensive washing of mitochondria and chemical cross-linking. To correct for differences in translation efficiency, the amount of protein subjected to immunoprecipitation was adjusted according to the incorporated radioactivity. Yta10p- or Yta12p-specific cross-link products were detected by immunoprecipitation and SDS–PAGE and quantified employing a phosphorimaging system. Polypeptides cross-linked to Yta10p or Yta12p in wild-type mitochondria were set to 100%. (C) Substrate polypeptides interacted with the YTA10–12 complex. After synthesis of mitochondrially encoded proteins and cross-linking, mitochondria (800 μg) were reisolated and lysed in buffer A containing 1 mM ATP. After a clarifying spin, solubilized mitochondrial proteins were fractionated by Superose 6 gel chromatography. 10% of each fraction was analyzed by SDS–PAGE. Peak fractions of Yta10p eluted at 12–13 ml (850 kDa) or at 14.5–15.5 ml (250 kDa), as demonstrated by immunoblotting (not shown). Yta10p-containing fractions were precipitated with TCA (12.5%), resuspended in 60 μl of 1% SDS, 5 mM phenylmethylsulfonyl fluoride, 100 mM Tris–HCl (pH 7.4) under vigorous shaking for 10 min at room temperature, and subsequently diluted 20-fold with buffer C. After a clarifying spin for 10 min at 25,000 × g, 10% of each fraction was removed (“control”). The remaining samples were subjected to immunoprecipitation with preimmune serum (not shown) or Yta10p-specific antibodies that have been covalently linked to Protein A–sepharose. Noncross-linked Yta10p migrated during electrophoresis as indicated. (D) Degradation and release of polypeptides bound to the YTA10–12 complex requires ATP hydrolysis. After labeling of mitochondrial translation products, mitochondria (100 μg) were reisolated by centrifugation for 10 min at 9000 × g, washed three times in wash buffer (1ml) supplemented with 1 mM ATP or AMP–PNP, and resuspended in translation buffer containing 1 mM ATP or AMP–PNP at a concentration of 1.2 mg mitochondrial protein/ml. Samples were incubated for 15 min at 4°C (minus Chase) or at 37°C (plus Chase) and subsequently subjected to cross-linking. An aliquot corresponding to 10% of the samples was withdrawn for a reference (“control”). Yta10p-specific cross-link products were identified by immunoprecipitation. (E) Substrate polypeptides remained associated with proteolytically inactive YTA10–12 complexes. Mitochondrially encoded proteins were labeled in wild-type mitochondria (WT), Δyta10 mutant mitochondria containing Yta10E559Qp, and Δyta12 mutant mitochondria containing Yta12E614Qp. Samples were incubated for 15 min at 4°C (minus Chase) or at 37°C (plus Chase), followed by extensive washing of mitochondria and chemical cross-linking. Incorporated radioactivity was measured to determine translation efficiency. The amount of protein that was used in the subsequent immunoprecipitation with Yta10p- and Yta12p-specific antiserum was adjusted according to the incorporated radioactivity. Immunoprecipitates were analyzed by SDS–PAGE and autoradiography and quantified using a phosphorimaging system. The amount of polypeptides cross-linked to Yta10p or Yta12p prior to chase was set to 100%. Cell 1996 85, 875-885DOI: (10.1016/S0092-8674(00)81271-4)

Figure 5 The Integrity of the HEXXH Binding Motif in Yta10p and Yta12p Is Not Required for Respiratory Competence Cultures of wild-type, Δyta10 mutant cells, and Δyta10 mutant cells expressing Yta10p or Yta10E559Qp (upper panel) and of wild-type, Δyta12 mutant cells, and Δyta12 mutant cells expressing Yta12p or Yta12E614Qp (lower panel) were grown on synthetic medium supplemented with 2% glucose at 30°C. 10-fold serial dilutions of logorithmically growing cultures were spotted onto synthetic medium containing 2% glycerol and incubated at 37°C for 2 days. Cell 1996 85, 875-885DOI: (10.1016/S0092-8674(00)81271-4)

Figure 6 The YTA10–12 Complex Mediates Oligomerization of ATPase 9 During Assembly of the ATP Synthase and Degrades Nonassembled ATPase 9 (A) Formation of the 48 kDa complex containing ATPase 9 depends on the presence of Yta10p and Yta12p. Mitochondrial translation products were labeled for 10 min at 25°C, as described in Experimental Procedures, in mitochondria isolated from chloramphenicol-pretreated wild-type and mutant cells (open triangle, wild-type; open circle, Δyta10; closed circle, Δyta10/Yta10E559Qp; open box, Δyta12; closed box, Δyta12/Yta12E614Qp). After inhibition of protein synthesis with puromycin (100 μM) and after adding cold methionine (20 mM), samples were further incubated at 37°C to allow the assembly of ATPase 9. At the timepoints indicated, mitochondria were reisolated and, without prior TCA precipitation, analyzed by SDS–PAGE. Formation of the 48 kDa oligomer was quantified by phosphorimaging analysis. The 48 kDa assembly intermediate is given as percent of total newly synthesized ATPase 9. (B) Nonassembled ATPase 9 is degraded by the YTA10–12 complex. Mitochondrial translation was carried out for 10 min at 25°C in mitochondria isolated from yeast cells not treated with chloramphenicol. After inhibition of protein synthesis, samples were further incubated for 30 min at 37°C to allow proteolysis to occur. Degradation of ATPase 9 was determined by SDS–PAGE and phosphorimaging analysis and is given as percent of newly synthesized ATPase 9 prior to proteolysis. Cell 1996 85, 875-885DOI: (10.1016/S0092-8674(00)81271-4)