A New Function in Translocation for the Mitochondrial i-AAA Protease Yme1: Import of Polynucleotide Phosphorylase into the Intermembrane Space

MPP mediates cleavage of PNPase. (A) Radiolabeled PNPase was synthesized in vitro and incubated with isolated WT mitochondria in the presence or absence of a Δψ at 25°C for 10 min. After import, samples were divided in equal aliquots for protease treatment with trypsin (Tryp) to remove nonimported precursor; protease activity was halted with soybean trypsin inhibitor. As a control, matrix-localized Su9-DHFR was also imported. Samples were analyzed by SDS-PAGE and fluorography. Standard (Std) refers to 10% of the radioactive precursor added to each assay. p, precursor; m, mature. (B) Import of PNPase and Su9-DHFR was performed as described for panel A into mas1 mitochondria. The inner membrane marker ADP/ATP carrier (AAC) was imported as a control; import reactions with AAC, which lacks a cleavable targeting sequence, were treated with protease followed by carbonate extraction to confirm insertion into the inner membrane. (C) Radiolabeled PNPase-DHFR (the N-terminal 157 amino acids of PNPase fused to DHFR) was imported into isolated WT mitochondria, and aliquots were removed at the designated time points. Nonimported precursor was removed with protease treatment. Standard (Std) represents 10% of the radioactive precursor in each time point. (D) PNPase-DHFR was imported into WT mitochondria (M) at 25°C for 10 min in the presence and absence of Δψ. Samples were incubated in hypotonic buffer to swell the outer membrane, generating mitoplasts (MP), in the presence and absence of trypsin (Tryp), followed by inactivation with trypsin inhibitor. Mitoplasts were recovered by centrifugation (P) and separated from the supernatant (S) containing the soluble intermembrane space contents. As a control, Su9-DHFR was imported and treated identically; relevant reactions representing import in the presence of Δψ and the recovered mitoplast fraction (P) treated with trypsin (+Tryp) are shown.

Recombinant MPP mediates cleavage of PNPase. (A) WT mitochondria were left untreated or were incubated in 10 mM EDTA, 2 mM o-phenanthroline (o-phe) for 15 min prior to the start of the import reaction to inhibit the endogenous MPP (52). Radiolabeled PNPase, Su9-DHFR, and AAC were imported as described for Fig. 2A. Nonimported AAC was removed by protease treatment, but protease addition was omitted for Su9-DHFR and PNPase. Standard (S) refers to 10% of the radioactive precursor added to each assay. p, precursor; m, mature. (B) Recombinant MPP (10) was purified over a Ni²⁺ column from a total lysate (T), followed by washing to remove unbound proteins. Purified MPP was eluted in fractions 2 and 3 in the presence of 300 mM imidazole. (C) In an in vitro cleavage assay (10), MPP was incubated with radiolabeled PNPase, Su9-DHFR, and cytochrome c₁. The standard (Std) is an equal volume of the radioactive precursor. i, intermediate. (D) Serial MPP dilutions were incubated with PNPase, PNPase-DHFR, and Su9-DHFR for 30 min at 30°C. The reactions were stopped with SDS sample buffer. Samples were separated by SDS-PAGE and detected by fluorography. (E) Recombinant PNPase-DHFR was purified using a C-terminal hexahistidine tag for an in vitro cleavage assay, and cleavage was confirmed by immunoblot analysis with a polyclonal antibody against DHFR (αDHFR). The cleaved DHFR was recovered for analysis by Edman degradation.

Yme1 is required for PNPase assembly in the intermembrane space. Radiolabeled PNPase, Su9-DHFR, and cytochrome c₁ were imported into Δyme1 (A), Δimp1 Δimp2 (B), yme1^E514Q (Su9-DHFR control omitted) (C), or Δtim54 (D) mitochondria in the presence and absence of Δψ as described for Fig. 2A. In half of the samples, nonimported precursor was removed by trypsin treatment, and analysis was done subsequently with SDS-PAGE and fluorography. p, precursor; i, intermediate; m, mature.

Yme1 is required for efficient translocation of PNPase and PNPase-DHFR. (A) PNPase-DHFR was imported into WT, mas1, Δyme1, and Δimp1 Δimp2 mitochondria (mitos) as described for Fig. 2A. (B) As shown in Fig. 2D in the same set of experiments, imported PNPase-DHFR was localized to the intermembrane space after osmotic shock. (C) Time course assays were performed for the import of PNPase, PNPase-DHFR, and Su9-DHFR (control) into WT, Δyme1, and Δtim54 mitochondria at the indicated times. Nonimported precursor was removed by trypsin treatment, and equal aliquots were separated by SDS-PAGE. The percent substrate imported was quantitated from fluorographs by use of a scanning densitometer. The amount of precursor that imported into WT mitochondria at the end time point was set at 100%.

Yme1 binds directly to PNPase and mediates translocation into the intermembrane space. (A) PNPase-DHFR was imported into WT mitochondria for 10 min at 25°C (−XL) and an aliquot was removed. Proteins were cross-linked (+XL) by the addition of 1.0 mM dithiobis(succinimidyl propionate) for 30 min followed by quenching with 0.1 M Tris-HCl. After an aliquot was removed, mitochondria were subsequently solubilized followed by immunoprecipitation (IP) with antibodies (α) against Yme1, Tim23, Tom40, and the control cytochrome b₂ (cyt b₂) Because cross-linked PNPase-DHFR migrates at a high molecular mass (indicated by the diamond), the immunoprecipitated cross-linking reactions were released with β-mercaptoethanol addition in the sample buffer, whereas reductant was omitted from the cross-linking reactions. The asterisks indicate cross-linked PNPase-DHFR (released by treatment with reductant) that copurified with the target protein of the antibody. (B) Cross-linking and immunoprecipitation assays were performed with full-length PNPase as described for panel A. (C) Cross-linking and immunoprecipitation assays were performed with PNPase-DHFR import into Δyme1 mitochondria. Note that PNPase-DHFR was not immunoprecipitated with antibodies against Yme1, confirming the specificity of the Yme1-PNPase interaction.

PNPase is not a proteolytic substrate of Yme1. (A) Radiolabeled Yta10-DHFR^m and PNPase-DHFR were imported into WT mitochondria for 30 min, and nonimported precursor was removed by protease treatment. The mitochondria were then incubated in the presence of an ATP-regenerating system at 37°C, and aliquots were removed at the indicated times to measure stability. The rate of proteolysis during the time course from three independent assays was quantitated using a Bio-Rad FX molecular imager and the affiliated Quantity 1 software. The amounts of Yta10-DHFR^m and PNPase-DHFR remaining at each chase time point are expressed as the percentages of the amounts detected for the respective strains at t = 0 (set at 100%; mean ± standard deviation; n = 3). As a control for Yme1-dependent degradation of Yta10-DHFR^m, a control reaction experiment was performed with Δyme1 mitochondria. (B) Mitochondria (Mito) and the postmitochondrial supernatant (PMS) were fractionated from Δyme1 and WT mitochondria expressing PNPase. Samples were separated by SDS-PAGE and blotted for PNPase and the mitochondrial marker AAC.