The ATP-dependent PIM1 protease is required for the expression of intron-containing genes in mitochondria

Abstract

The ATP-dependent PIM1 protease, a Lon-like protease localized in the mitochondrial matrix, is required for mitochondrial genome integrity in yeast. Cells lacking PIM1 accumulate lesions in the mitochondrial DNA (mtDNA) and therefore lose respiratory competence. The identification of a multicopy suppressor, which stabilizes mtDNA in the absence of PIM1, enabled us to characterize novel functions of PIM1 protease during mitochondrial biogenesis. The synthesis of mitochondrially encoded cytochrome c oxidase subunit I (CoxI) and cytochrome b (Cob) is impaired in pim1 mutants containing mtDNA. PIM1-mediated proteolysis is required for the translation of mature COXI mRNA. Moreover, deficiencies in the splicing of COXI and COB transcripts, which appear to be restricted to introns encoding mRNA maturases, were observed in cells lacking the PIM1 gene. Transcripts of COXI and COB genes harboring multiple introns are degraded in the absence of PIM1. These results establish multiple, essential functions of the ATP-dependent PIM1 protease during mitochondrial gene expression.

Figure 1

Respiratory deficiency of Δpim1 cells carrying intact mtDNA. Wild-type cells (WT), Δpim1 cells (Δpim1), and Δpim1 cells complemented with the E. coli Lon protease (Δpim1/LON), the suppressor gene (Δpim1/SUP), or both (Δpim1/LON/SUP), were grown in glucose-containing selective medium. Cells were harvested in exponential phase, spotted onto YEPG (rich medium containing 3% glycerol), and incubated at 30°C and 36°C for 7 and 10 days, respectively. MtDNA integrity (ρ⁺, ρ⁻) of various strains was examined by testing the respiratory competence of a diploid strain generated by mating with a ρ⁰ PIM1⁺ strain. (ρ⁺) Wild-type mtDNA; (ρ⁻) mutant mtDNA carrying deletions.

Figure 2

Requirement of PIM1 protease for the synthesis of CoxI and Cob. (A) Synthesis of mitochondrially encoded proteins in vivo. Mitochondrial translation products were labeled with [³⁵S]methionine in the presence of cycloheximide for 10 min (WT, Δpim1/LON, Δpim1/LON/SUP) or 30 min (Δpim1/SUP) at 30°C in vivo as described in Materials and Methods and analyzed by SDS-PAGE. The translation efficiency was reduced in Δpim1/SUP cells. A band marked with an asterisk (*) is not strain-specific and was also detected in ρ⁰ strains. (CoxI, CoxII, CoxIII) Subunits I, II, and III of cytochrome c oxidase, respectively; (Cob) cytochrome b; (Atp6, Atp8, Atp9) subunits 6, 8, and 9 of the F₀F₁–ATPase, respectively. (B) Degradation of newly synthesized CoxII and CoxIII in Δpim1/LON cells. Mitochondrially encoded polypeptides were synthesized in vivo for 30 min in the presence of [³⁵S]methionine. After addition of cold methionine (10 mm), cells were further incubated for the indicated time periods and then analyzed by SDS-PAGE. (C) Steady-state levels of CoxII and Cob in mitochondria lacking PIM1 protease. Mitochondria were isolated from wild-type, Δpim1/LON, and Δpim1/SUP cells, subjected to SDS-PAGE and analyzed by Western blotting with polyclonal antisera directed against CoxII (α-CoxII), Cob (α-Cob), and Yta10p (α-Yta10p) and the β-subunit of the F₁–ATPase (α-F1β) as gel loading controls. (D) Dependence of CoxI synthesis on PIM1-mediated proteolysis. Labeling of mitochondrial translation products was performed for 10 min at 30°C in Δpim1/LON cells expressing wild-type PIM1 or PIM1^S1015A from multicopy plasmids. Wild-type and mutant protease accumulated at similar levels in mitochondria.

Figure 2

Requirement of PIM1 protease for the synthesis of CoxI and Cob. (A) Synthesis of mitochondrially encoded proteins in vivo. Mitochondrial translation products were labeled with [³⁵S]methionine in the presence of cycloheximide for 10 min (WT, Δpim1/LON, Δpim1/LON/SUP) or 30 min (Δpim1/SUP) at 30°C in vivo as described in Materials and Methods and analyzed by SDS-PAGE. The translation efficiency was reduced in Δpim1/SUP cells. A band marked with an asterisk (*) is not strain-specific and was also detected in ρ⁰ strains. (CoxI, CoxII, CoxIII) Subunits I, II, and III of cytochrome c oxidase, respectively; (Cob) cytochrome b; (Atp6, Atp8, Atp9) subunits 6, 8, and 9 of the F₀F₁–ATPase, respectively. (B) Degradation of newly synthesized CoxII and CoxIII in Δpim1/LON cells. Mitochondrially encoded polypeptides were synthesized in vivo for 30 min in the presence of [³⁵S]methionine. After addition of cold methionine (10 mm), cells were further incubated for the indicated time periods and then analyzed by SDS-PAGE. (C) Steady-state levels of CoxII and Cob in mitochondria lacking PIM1 protease. Mitochondria were isolated from wild-type, Δpim1/LON, and Δpim1/SUP cells, subjected to SDS-PAGE and analyzed by Western blotting with polyclonal antisera directed against CoxII (α-CoxII), Cob (α-Cob), and Yta10p (α-Yta10p) and the β-subunit of the F₁–ATPase (α-F1β) as gel loading controls. (D) Dependence of CoxI synthesis on PIM1-mediated proteolysis. Labeling of mitochondrial translation products was performed for 10 min at 30°C in Δpim1/LON cells expressing wild-type PIM1 or PIM1^S1015A from multicopy plasmids. Wild-type and mutant protease accumulated at similar levels in mitochondria.

Figure 2

Requirement of PIM1 protease for the synthesis of CoxI and Cob. (A) Synthesis of mitochondrially encoded proteins in vivo. Mitochondrial translation products were labeled with [³⁵S]methionine in the presence of cycloheximide for 10 min (WT, Δpim1/LON, Δpim1/LON/SUP) or 30 min (Δpim1/SUP) at 30°C in vivo as described in Materials and Methods and analyzed by SDS-PAGE. The translation efficiency was reduced in Δpim1/SUP cells. A band marked with an asterisk (*) is not strain-specific and was also detected in ρ⁰ strains. (CoxI, CoxII, CoxIII) Subunits I, II, and III of cytochrome c oxidase, respectively; (Cob) cytochrome b; (Atp6, Atp8, Atp9) subunits 6, 8, and 9 of the F₀F₁–ATPase, respectively. (B) Degradation of newly synthesized CoxII and CoxIII in Δpim1/LON cells. Mitochondrially encoded polypeptides were synthesized in vivo for 30 min in the presence of [³⁵S]methionine. After addition of cold methionine (10 mm), cells were further incubated for the indicated time periods and then analyzed by SDS-PAGE. (C) Steady-state levels of CoxII and Cob in mitochondria lacking PIM1 protease. Mitochondria were isolated from wild-type, Δpim1/LON, and Δpim1/SUP cells, subjected to SDS-PAGE and analyzed by Western blotting with polyclonal antisera directed against CoxII (α-CoxII), Cob (α-Cob), and Yta10p (α-Yta10p) and the β-subunit of the F₁–ATPase (α-F1β) as gel loading controls. (D) Dependence of CoxI synthesis on PIM1-mediated proteolysis. Labeling of mitochondrial translation products was performed for 10 min at 30°C in Δpim1/LON cells expressing wild-type PIM1 or PIM1^S1015A from multicopy plasmids. Wild-type and mutant protease accumulated at similar levels in mitochondria.

Figure 2

Requirement of PIM1 protease for the synthesis of CoxI and Cob. (A) Synthesis of mitochondrially encoded proteins in vivo. Mitochondrial translation products were labeled with [³⁵S]methionine in the presence of cycloheximide for 10 min (WT, Δpim1/LON, Δpim1/LON/SUP) or 30 min (Δpim1/SUP) at 30°C in vivo as described in Materials and Methods and analyzed by SDS-PAGE. The translation efficiency was reduced in Δpim1/SUP cells. A band marked with an asterisk (*) is not strain-specific and was also detected in ρ⁰ strains. (CoxI, CoxII, CoxIII) Subunits I, II, and III of cytochrome c oxidase, respectively; (Cob) cytochrome b; (Atp6, Atp8, Atp9) subunits 6, 8, and 9 of the F₀F₁–ATPase, respectively. (B) Degradation of newly synthesized CoxII and CoxIII in Δpim1/LON cells. Mitochondrially encoded polypeptides were synthesized in vivo for 30 min in the presence of [³⁵S]methionine. After addition of cold methionine (10 mm), cells were further incubated for the indicated time periods and then analyzed by SDS-PAGE. (C) Steady-state levels of CoxII and Cob in mitochondria lacking PIM1 protease. Mitochondria were isolated from wild-type, Δpim1/LON, and Δpim1/SUP cells, subjected to SDS-PAGE and analyzed by Western blotting with polyclonal antisera directed against CoxII (α-CoxII), Cob (α-Cob), and Yta10p (α-Yta10p) and the β-subunit of the F₁–ATPase (α-F1β) as gel loading controls. (D) Dependence of CoxI synthesis on PIM1-mediated proteolysis. Labeling of mitochondrial translation products was performed for 10 min at 30°C in Δpim1/LON cells expressing wild-type PIM1 or PIM1^S1015A from multicopy plasmids. Wild-type and mutant protease accumulated at similar levels in mitochondria.

Figure 3

Mitochondrial protein synthesis in Δpim1 cells carrying intronless mtDNA. Δpim1/LON and Δpim1/SUP cells devoid of mitochondrial introns were generated by cytoduction. Mitochondrial translation products were labeled with [³⁵S]methionine for 10 min (WT; Δpim1/LON) or 30 min (Δpim1/SUP) at 30°C in vivo and analyzed by SDS-PAGE. The difference in the electrophoretic mobility of Var1 reflects gene polymorphism in the different mitochondrial genomes (Butow et al. 1985).

Figure 4

Northern blot analysis of COXI and COB transcripts in mitochondria lacking PIM1 protease. (A) mtRNA was isolated from wild-type (WT) and Δpim1/SUP cells, harboring an intron-containing or an intronless mitochondrial genome (±introns), and analyzed with COXI and COB-specific exon probes and a COXII probe for control as described in Materials and Methods. (B) mtRNA from wild-type (WT), Δpim1/LON, and Δpim1/SUP mitochondria with intron-containing mtDNA was analyzed as in A.

Figure 4

Northern blot analysis of COXI and COB transcripts in mitochondria lacking PIM1 protease. (A) mtRNA was isolated from wild-type (WT) and Δpim1/SUP cells, harboring an intron-containing or an intronless mitochondrial genome (±introns), and analyzed with COXI and COB-specific exon probes and a COXII probe for control as described in Materials and Methods. (B) mtRNA from wild-type (WT), Δpim1/LON, and Δpim1/SUP mitochondria with intron-containing mtDNA was analyzed as in A.

Figure 5

Characterization of COXI and COB pre-mRNA processing defects in pim1 mutants by Northern hybridization using intron-specific probes. mtRNA isolated from wild-type (WT) and Δpim1/SUP cells was analyzed with intron-specific COB and COXI probes. DNA probes specific for the group II introns bI1 of COB, aI1 and aI5γ of COXI and COXII, as a control, were employed.

Figure 6

Roles of PIM1 protease in mitochondrial biogenesis. PIM1-mediated proteolysis is required for mtDNA integrity and the expression of the intron-containing COXI and COB genes in mitochondria (see text for details). (*) The instability of pre-RNA in the absence of PIM1 may be a secondary effect of pre-mRNA processing deficiencies. (**) PIM1 protease is only required for translation of COXI mRNA. (E, E1–3) exons; (I, I1–3) introns; (M) mRNA maturases.