Identification of novel oxidized protein substrates and physiological partners of the mitochondrial ATP-dependent Lon-like protease Pim1

Abstract

ATP-dependent proteases are currently emerging as key regulators of mitochondrial functions. Among these proteolytic systems, Pim1, a Lon-like serine protease in Saccharomyces cerevisiae, is involved in the control of selective protein turnover in the mitochondrial matrix. In the absence of Pim1, yeast cells have been shown to accumulate electron-dense inclusion bodies in the matrix space, to lose integrity of mitochondrial genome, and to be respiration-deficient. Because of the severity of phenotypes associated with the depletion of Pim1, this protease appears to be an essential component of the protein quality control machinery in mitochondria and to exert crucial functions during the biogenesis of this organelle. Nevertheless, its physiological substrates and partners are not fully characterized. Therefore, we used the combination of different proteomic techniques to assess the nature of oxidized protein substrates and physiological partners of Pim1 protease under non-repressing growth conditions. The results presented here supply evidence that Pim1-mediated proteolysis is required for elimination of oxidatively damaged proteins in mitochondria.

FIGURE 1.

Processing of Pim1 preprotein, ATP-stimulated proteolytic activity, and oxidized protein content in WT and Δpim1 mitochondria. WT and Δpim1 cells were grown at 30 °C in Raffinose medium. A, immunoblot analysis (50 μg of protein/lane) of mitochondrial proteins (M), mitochondrial membrane fractions (Mb), and total fractions (T) isolated from WT and Δpim1 cells, using antibodies directed against Pim1 (p, pro-form; m, mature form), Por1, or Om45. B, mitochondrial ATP-stimulated proteolytic activity was assessed in WT and Δpim1 mitochondria by analysis of the proteolytic breakdown of casein-fluorescein isothiocyanate (for details, see “Experimental Procedures”). Fluorescence released into supernatant fractions was quantified, and Vi values, corresponding to casein degradation-hydrolyzing activity, were calculated for WT (6 ± 0.24 pmol/min/mg) and Δpim1 (3.9 ± 0.17 pmol/min/mg). Vi was taken as 100% for the wild type (n = 3). The data in the bar graph represent means ± S.E. The asterisk indicates the level of statistical significance: *, p < 0.03 according to Student's t test. C, representative OxyBlot showing oxidized protein content in WT and Δpim1 mitochondria. 10 μg of mitochondrial proteins were loaded/lane (see details under “Experimental Procedures”).

FIGURE 2.

Comparative analysis of mitochondrial oxidized proteomes between WT and Δpim1 cells. Mitochondrial fractions were isolated from WT and Δpim1 cells grown at 30 °C in raffinose medium and were subjected to two-dimensional GE analysis. Following two-dimensional GE, gels were either stained with colloidal Coomassie Brilliant Blue G to detect proteins (A and B) or were electrotransferred onto nitrocellulose membranes to subsequently detect carbonylated proteins using the OxyBlot technique (C and D), as described under “Experimental Procedures.” A and C, WT mitochondrial extracts; B and D, Δpim1 mitochondrial extracts. The arrows and numbers refer to spots accumulating in Δpim1 mitochondria, which are identified in Fig. 3 and listed in supplemental Table S1 and Table 1. Presented results are from one representative experiment of four independent experiments.

FIGURE 3.

Identification of spots of interest. Spots of interest were identified using mass spectrometry as described under “Experimental Procedures” (see supplemental Table S1 and “Supplemental Proteomic Data” for details). Enlarged areas of two-dimensional gels (colloidal Coomassie Brilliant Blue G) and two-dimensional OxyBlot regions containing spots of interest are presented for WT and Δpim1 mitochondria. Spots of interest are indicated with an arrow. The corresponding identified proteins are shown on two-dimensional gels, whereas spots are numbered on two-dimensional OxyBlots (as in Fig. 2). Presented results are from one representative experiment of four independent experiments.

FIGURE 4.

Influence of dextrose versus raffinose medium on the steady state level of Pim1 degradation substrates. A, mitochondria were isolated from WT and Δpim1 cells grown either in dextrose (D) or in raffinose (R) medium at 30 °C. Immunodetection of Pim1 substrates was performed using specific antibodies (25 μg of mitochondrial protein/lane). Immunodetection of Por1 (mitochondrial porin) and Phb1 (prohibitin 1) was used as loading control. B, immunoblot analysis of total cell extracts (T; 50 μg of protein/lane) or mitochondrial extracts (M; 20 μg of protein/lane) from WT cells grown either in dextrose or in raffinose medium was performed using specific antibodies directed against various mitochondrial proteins, especially Pim1 substrates (Hsp60, Lat1, Kgd2, Mrp20, Cox4, Ilv5, Atp7, and Rip1). Immunoblot of Hsp104 (cytosolic heat shock protein 104) was used as a control.

FIGURE 5.

Identification of Pim1 interacting partners using TAP. Cells from SC0000 and SC0437 strains were grown in YPGR at 30 °C. A, mitochondrial protein extracts (50 μg of protein/lane) from SC0000 and SC0437 strains were electrotransferred onto nitrocellulose membrane and incubated with anti-Pim1 or anti-TAP tag antibodies. Immunodetection of Por1 (porin) was used as loading control. B, TAP purification was performed using SC0000 and SC0437 mitochondrial fractions as described under “Experimental Procedures.” Elution fractions were analyzed on one-dimensional gel using silver staining. Protein bands, obtained from the SC0437 strain, were identified by mass spectrometry (for more details, see “Experimental Procedures” and see supplemental Table S2 and “Supplemental Proteomic Data”). Immunoblot analysis of mitochondrial fractions from the SC0437 strain (M; 25 μg of protein) and elution fractions from SC0000 or SC0437 mitochondria (same volume of elution fraction for each) was performed using an antibody directed against the TAP tag. The presented results are from one representative experiment of three independent experiments. C, Pim1 and its interacting partners (Phb1, Phb2, and Atp7) were confirmed by immunoblotting of the elution fraction from the TAP purification performed with SC0437 mitochondria using specific antibodies. WB, Western blot.

FIGURE 6.

Pim1 protease is involved in mitochondrial biogenesis and integrity. Pim1 degrades various mitochondrial protein substrates. The turnover of unassembled respiratory chain subunits by Pim1 protease could be mediated by the prohibitin complex (Phb1/2) in the inner membrane. Many substrates of Pim1 protease are part of mtDNA nucleoid proteins, underscoring the role of this protease in mtDNA metabolism. In addition, Pim1-mediated proteolysis is responsible for elimination of oxidatively damaged proteins. Accordingly, depletion of Pim1 protease leads to a mitochondrial stress response (overexpression of Hsp78, Hsp60, and Sod2). PDH, pyruvate dehydrogenase complex (E1, E2, and E3 subunits); M, matrix; IM, inner mitochondrial membrane; IMS, intermembrane space; OM, outer mitochondrial membrane. Proteins of interest identified in this study are shown in red.