An Early mtUPR: Redistribution of the Nuclear Transcription Factor Rox1 to Mitochondria Protects against Intramitochondrial Proteotoxic Aggregates

Abstract

The mitochondrial proteome is built mainly by import of nuclear-encoded precursors, which are targeted mostly by cleavable presequences. Presequence processing upon import is essential for proteostasis and survival, but the consequences of dysfunctional protein maturation are unknown. We find that impaired presequence processing causes accumulation of precursors inside mitochondria that form aggregates, which escape degradation and unexpectedly do not cause cell death. Instead, cells survive via activation of a mitochondrial unfolded protein response (mtUPR)-like pathway that is triggered very early after precursor accumulation. In contrast to classical stress pathways, this immediate response maintains mitochondrial protein import, membrane potential, and translation through translocation of the nuclear HMG-box transcription factor Rox1 to mitochondria. Rox1 binds mtDNA and performs a TFAM-like function pivotal for transcription and translation. Induction of early mtUPR provides a reversible stress model to mechanistically dissect the initial steps in mtUPR pathways with the stressTFAM Rox1 as the first line of defense.

Keywords: mitochondria-nuclear communication; mitochondrial protein import; presequence processing; proteostasis; proteotoxic; stress response; unfolded protein response.

Graphical abstract

Figure 1

Non-processed Precursor Proteins Form Aggregates inside Mitochondria and Escape Organellar Degradation (A) Coomassie-stained gels from SDS-PAGE of wild-type (WT) and mas1^ts mitochondria isolated from cells grown under respiratory conditions and separated into soluble (SN [supernatant]) and aggregated (P [pellet]) protein fractions. (B) Immunoblots of samples from (A) analyzed with antisera against indicated MPP substrate proteins. i, processing intermediate; m, mature; p, precursor. (C) Immunoblots of samples from (A) analyzed with antisera against non-processed proteins. (D) In organello degradation of indicated precursor (p) and mature (m) forms of Cox4, Rip1, and Isu1 in mas1^ts mitochondria. i, processing intermediate; Om45 and Tom70, non-processed control proteins. (E) Import of radiolabeled precursor proteins into isolated WT or mas1^ts mitochondria followed by separation into soluble (SN) and aggregated protein (P) fraction. T, total, non-lysed mitochondria. Where indicated the membrane potential (Δψ) was depleted prior to the import reaction. See also Figure S1.

Figure 2

Accumulation of Aggregation-Prone Precursor Proteins inside Mitochondria Causes a Transcriptional Stress Response (A) Cell death determined by flow cytometric quantification of propidium iodide (PI) staining indicative of loss of plasma membrane integrity of wild-type (WT) and mas1^ts cells after shift to 37°C for indicated time. n = 12; data represent mean ± SEM; n.s., not significant. (B) Determination of clonogenicity via survival plating of WT and mas1^ts cells after shift to 37°C for the indicated time. n = 6; data represent mean ± SEM. (C) Immunoblot analysis of WT and mas1^ts mitochondria isolated from strains shifted for indicated times to non-permissive temperature. Sod2^preseq., antibody generated against presequence peptide of Sod2. (D) Distribution of transcripts quantified by RNA-seq in WT and mas1^ts cells. Displayed are Benjamini-Hochberg adjusted p values. FDR, false discovery rate. (E) Highlighted transcripts analyzed in (D) for indicated Gene Ontology (GO) terms provided by Saccharomyces Genome Database. See also Figure S2 and Tables S1 and S2.

Figure 3

Identification of Rox1 as a Mediator of the Early Mitochondrial Unfolded Protein Response (mtUPR) Pathway (A) Synthetic growth defect in mas1^tsrox1Δ mutant at indicated temperatures on respiratory (YPglycerol) conditions. (B) Determination of cell death via PI staining and of clonogenic survival via survival plating in mas1^ts and mas1^tsrox1Δ cells (12 h induction). n = 4 for PI staining and n = 6 for clonogenic survival; data represent mean ± SEM. ^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.001. (C) Measurements of Δψ, ATP levels and ROS in mas1^ts and mas1^tsrox1Δ mitochondria (Δψ, ATP level 10 h, ROS 4 h induction). n = 3; data represent mean ± SEM. (D) Blue native PAGE autoradiography of assembled AAC2 complex after import into isolated mas1^ts and mas1^tsrox1Δ mitochondria. Quantification for n = 3; data represent mean ± SEM. (E) SDS-PAGE autoradiography analysis of cytosolic translation activity in mas1^ts and mas1^tsrox1Δ cells (4 h induction). Pgk1, cytosolic marker as loading control. (F) Growth of rho⁺ (ρ⁺) and rho⁰ (ρ⁰) WT and mas1^ts cells on YPglucose plates at indicated temperature. (G) Determination of clonogenicity via survival plating of WT rho⁰ (ρ⁰) and mas1^ts rho⁰ (ρ⁰) cells. n = 8; data represent mean ± SEM. See also Figures S3 and S4.

Figure 4

Rox1 Translocates to Mitochondria upon mtUPR (A) Immunoblot analysis of wild-type (WT) and mas1^ts mitochondria. m, mature; p, precursor. (B) Immunoblot analysis of mitochondria incubated in the absence or presence of proteinase K in iso-osmotic (SEM) or hypo-osmotic (EM) buffer or after lysis with non-ionic detergent (Dig.). Hsp10, matrix marker; Mpm1, intermembrane space; Tom22, outer membrane. (C) SDS-PAGE autoradiography of radiolabeled Rox1 precursor imported in isolated WT mitochondria for 30 min in the presence or absence of Δψ. Prec., precursor; Prot. K, proteinase K. (D) Import reaction as in (C), into WT and pam16-3 mitochondria for indicated time. Samples were treated with Prot. K. Por1, loading control. (E) Import of Rox1 and Abf2 precursor (as in C) into isolated WT and mas1^ts mitochondria. See also Figure S5.

Figure 5

Mitochondrial Rox1 Performs a TFAM-Like Function upon mtUPR (A) Autoradiography of gel mobility shift assay using recombinant Rox1 protein and ³³P-labeled HMG consensus sequences. (B) De novo DNA synthesis (labeled by ³³P-dCTP incorporation) in isolated mas1^tsrox1Δ mitochondria with and without prior import of Rox1 precursor protein. n = 3; data represent mean ± SEM. ^∗p < 0.05; ^∗∗p < 0.01. (C) De novo transcription (labeled by ³³P-UTP incorporation) in isolated mas1^tsrox1Δ mitochondria with and without prior import of Rox1 precursor protein. n = 3; data represent mean ± SEM. Mitochondrial rRNAs were stained with methylene blue as loading control. (D) Analysis of representative transcripts encoded by mitochondrial or nuclear DNA by qRT-PCR after cell growth at permissive (Ctrl.) or non-permissive (mtUPR) temperature. n = 6; data represent mean ± SEM (Table S2). (E) Mitochondrial translation in mas1^tsrox1Δ cells in the presence or absence of a Rox1-expressing plasmid. Labeled mtDNA-encoded proteins visualized by incorporation of ³⁵S-methionine. (F) Model of the early mtUPR pathway showing translocation of the stressTFAM Rox1 into mitochondria upon aggregation of accumulating precursor proteins in the matrix. Rox1 ensures maintenance and expression of mtDNA, thus protecting cells against the decline of transmembrane potential, respiratory activity, and cytosolic and mitochondrial translation and against accumulation of ROS. See also Figure S5 and Table S2.