Unconventional Targeting of a Thiol Peroxidase to the Mitochondrial Intermembrane Space Facilitates Oxidative Protein Folding

Abstract

Thiol peroxidases are conserved hydrogen peroxide scavenging and signaling molecules that contain redox-active cysteine residues. We show here that Gpx3, the major H₂O₂ sensor in yeast, is present in the mitochondrial intermembrane space (IMS), where it serves a compartment-specific role in oxidative metabolism. The IMS-localized Gpx3 contains an 18-amino acid N-terminally extended form encoded from a non-AUG codon. This acts as a mitochondrial targeting signal in a pathway independent of the hitherto known IMS-import pathways. Mitochondrial Gpx3 interacts with the Mia40 oxidoreductase in a redox-dependent manner and promotes efficient Mia40-dependent oxidative protein folding. We show that cells lacking Gpx3 have aberrant mitochondrial morphology, defective protein import capacity, and lower inner membrane potential, all of which can be rescued by expression of a mitochondrial-only form of Gpx3. Together, our data reveal a novel role for Gpx3 in mitochondrial redox regulation and protein homeostasis.

Keywords: Gpx3; Mia40; alternative translation initiation; antioxidants; mitochondria; mitochondria biogenesis; oxidative protein folding; oxidative stress; protein targeting.

Graphical abstract

Figure 1

Gpx3 Is Localized in the Mitochondrial IMS (A) A Gpx3-GFP genomically tagged strain was transformed with a plasmid expressing mtRFP. Cells were grown either in SCD (fermentative) or SCGE (respiratory) media and exposed to 1 mM H₂O₂ for 1 hr. (B) A gpx3 mutant strain was transformed with empty vector (ev) or a plasmid expressing Gpx3-myc (WT). Cells were grown in SD media until mid-exponential phase and treated with 1 mM H₂O₂. Cells were then fractionated to separate the cytosolic (C) and mitochondrial (M) fractions and western blots probed using antibodies against Gpx3-myc, mitochondrial porin (αPor1), and cytosolic Pgk1 (αPgk1). (C) Fractionation of isolated WT yeast mitochondria (M). The outer mitochondrial membrane was removed with osmotic shock to create mitoplasts (MP) for protease (PK) access to the IMS. Soluble proteins (s) were obtained by carbonate extraction (CE), with the insoluble ones remaining in the pellet fraction (p). Western blots were probed with αErv1 and αTim10 for IMS proteins, αTom40 for the OM, and αHsp70 for the matrix. (D) Import of radiolabeled Gpx3 in WT yeast mitochondria for 20 min (M) (autoradiography). To verify the specificity of the import, samples were also treated with Triton X before exposure to protease (Tx). Further fractionation was additionally performed in a similar manner as in (C), using osmotic shock in the presence or absence of protease (MP samples). Finally, the mitochondrial soluble proteins were also obtained by carbonate extraction (CE). The 10% sample corresponds to the precursor that was used for the import reaction.

Figure 2

Gpx3 Is Localized to Mitochondria via an N-Terminal Extension Encoded from a Non-AUG Codon (A) Schematic representation of Gpx3 variants. WT Gpx3-myc has its 500 bp 5′ UTR. Δu18 is lacking 54 nt prior to the AUG codon. M1L has the AUG mutated to TTG. N18 (L(−18)M, M1L) and N16 (T(−16)M, M1L) have AUGs introduced at positions −54 and −48, respectively, and the normal AUG start codon is mutated to TTG. (B) Western blot analysis confirms protein expression from the Δu18 and M1L variants under fermentative growth and oxidative stress conditions. Cytosolic Pgk1 was used as a loading control. (C and D) gpx3 strains expressing ev, WT, Δu18, or M1L Gpx3 variants (C; depicted in A) were fractionated into cytosolic (C) and mitochondrial (M) fractions. gpx3 strains expressing ev, WT, N16, or N18 variant (D; depicted in A) were fractionated into cytosolic (C) and mitochondrial (M) fractions. Western blots were probed against cytosolic Pgk1 and mitochondrial Hsp70 as controls. (E) Import of radiolabeled precursors, Su9DHFR, DHFR, and N18DHFR in WT yeast mitochondria for 10 min. To verify the specificity of import, samples were treated with Triton X-100 before exposure to protease (Tx). Controls were similar to those used in Figure 1D. (F) A WT strain expressing both an N18-roGFP2 plasmid and an mtRFP plasmid was grown to mid-exponential phase, and the localization of both fluorescent probes was examined with light microscopy. (G) Schematic representation of the Gpx3 constructs used for the radioactive expression of the precursors in this study. Import of radiolabeled N18Gpx3 and Gpx3 in isolated WT yeast mitochondria for the indicated time points (autoradiography). Equal loading was verified using the known mitochondrial marker protein porin (αPor1). The 10% sample was used as a control, as in Figure 1D.

Figure 3

Strains Lacking Gpx3 Have Abnormal Mitochondrial Phenotypes (A) EM analysis of mitochondria of WT, gpx3, and yap1. Scale bar, 200 nm. Representative examples are shown. Quantitative analysis is shown Figure S3A. (B) WT and gpx3 yeast mitochondria were incubated with DISC₃(5) to assess the IM potential. Valinomycin was added to visualize the release of the dye from the mitochondria as a control. The signal of the dye in the reaction prior to the addition of mitochondria was set to 100%. Averages shown are from three repeats. (C) Import of radioactive Su9DHFR in WT and gpx3 yeast mitochondria for the indicated time points. Equal loading was verified using the mitochondrial marker protein αPor1. The 10% sample corresponds to the precursor that was used for the import. (D) Staining for mitochondrial ROS was performed using MitoSOX. Representative histograms from three experimental repeats are presented.

Figure 4

Mitochondrial Gpx3 Can Rescue Mitochondrial Defects of a gpx3 Mutant (A) Import of the radioactive precursor Su9DHFR in mitochondria isolated from gpx3 strains expressing different forms of Gpx3 for the indicated time points. Equal loading was verified using antibodies against mitochondrial Tom40. The 5% sample corresponds to the precursor that was used for the import reaction. (B) Same as (A), but the radioactive precursor of Tim10 was used instead of Su9DHFR. (C) EM analysis of the same cells grown to mid exponential phase. Quantification of occurrence of thinner mitochondria is presented. Mitochondria from 50 random cells were quantified. Statistical analysis was performed using Fisher’s exact test comparing the number of thinner mitochondria from the mutants to the WT form. ^∗∗∗p < 0.001.

Figure 5

IMS-Targeted Gpx3 Rescues gpx3 Phenotypes (A) Fractionation of cells expressing cyb2 Gpx3 into cytosolic (‘C)’ and mitochondrial (‘M)’ fractions. (B) Fractionation of isolated cyb2-Gpx3-Myc yeast mitochondria (M) as in Figure 1C. (C) Import of radioactive precursor Su9DHFR in WT, gpx3, and cyb2 Gpx3 yeast isolated mitochondria for 15 min. (D) As in Figure 3B, WT, gpx3, yap1, Δu18 Gpx3, and cyb2 Gpx3 yeast mitochondria were incubated with DISC₃(5) to assess membrane potential. Error bars show the average of three repeats. (E) Growth assays of WT and sod1 cells expressing the empty vector and a sod1 gpx3 strain expressing Gpx3, cyb2-Gpx3, or empty vector on fermentative or respiratory media. Cells were grown to stationary phase and dilutions of optical density (OD) 1, 0.1, and 0.01 were plated. Images were taken after 3 days of growth.

Figure 6

Gpx3 Interacts with Mia40 Both In Vitro and In Organello (A) Oxidized recombinant Gpx3His and reduced ΔN290Mia40His (Mia40) were incubated in vitro for the indicated times. Reactions were stopped with TCA and followed by AMS labeling. Samples were visualized by western blot analysis. (B) Same as in (A), except oxidized Gpx3His was radiolabeled and incubated with recombinant ΔN290Mia40His (Mia40) or the hydrophobic mutant (LMFFFM) of Mia40 (Mia40 mt). Samples were visualized by autoradiography. (C) Oxidized N18 Gpx3 was incubated with reduced ΔN290Mia40His (Mia40) in an assay similar to (B). (D) Import of radiolabeled Tim10, Gpx3, and N18Gpx3 in WT and Mia40-depleted yeast mitochondria for the indicated times. Samples were visualized with autoradiography prior to western blot analysis using mitochondrial Cpn10 for verification of equal loading. (E) Radioactive SPCMia40 was imported in gpx3-Gpx3 mitochondria for 20 min (M). Mitochondria were then solubilized with 0.16% n-Dodecyl β-D-maltoside (DDM), and the supernatant (s) was separated from the pellet (p) and was incubated with either αMyc or αGpx3 for 2 hr to immunoprecipitate Gpx3Myc (IP samples). Reactions with pre-immune (PI) serum as well as protein beads alone (pA and pG) were used as a control. The 10% and Tx control samples were also loaded, as in Figure 6D. The immunoprecipitation of Gpx3 was done using both αMyc and αGpx3 antibodies. (F) Western blot analysis of the redox state of endogenous Mia40 in isolated WT and gpx3 mitochondria that were blocked with TCA followed by AMS labeling.

Figure 7

Model Depicting the Cellular Localization of the H₂O₂ Sensor Gpx3 and Its Role and Interactions in Mitochondria Gpx3 is primarily found in the cytosol and in small amounts in the mitochondrial IMS. Both Gpx3 and N18 Gpx3 interact with Mia40, with a possible redox quality control role for the MIA pathway, and may serve an antioxidant function. Deletion of Gpx3 (shown in the right panel) results in elevated ROS levels, reduced mitochondrial IM potential, morphological anomalies, and defects in mitochondrial protein import pathways.