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Mistargeted mitochondrial proteins activate a proteostatic response in the cytosol

Abstract

Most of the mitochondrial proteome originates from nuclear genes and is transported into the mitochondria after synthesis in the cytosol. Complex machineries which maintain the specificity of protein import and sorting include the TIM23 translocase responsible for the transfer of precursor proteins into the matrix, and the mitochondrial intermembrane space import and assembly (MIA) machinery required for the biogenesis of intermembrane space proteins. Dysfunction of mitochondrial protein sorting pathways results in diminishing specific substrate proteins, followed by systemic pathology of the organelle and organismal death1,2,3,4. The cellular responses caused by accumulation of mitochondrial precursor proteins in the cytosol are mainly unknown. Here we present a comprehensive picture of the changes in the cellular transcriptome and proteome in response to a mitochondrial import defect and precursor over-accumulation stress. Pathways were identified that protect the cell against mitochondrial biogenesis defects by inhibiting protein synthesis and by activation of the proteasome, a major machine for cellular protein clearance. Proteasomal activity is modulated in proportion to the quantity of mislocalized mitochondrial precursor proteins in the cytosol. We propose that this type of unfolded protein response activated by mistargeting of proteins (UPRam) is beneficial for the cells. UPRam provides a means for buffering the consequences of physiological slowdown in mitochondrial protein import and for counteracting pathologies that are caused or contributed by mitochondrial dysfunction.

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Figure 1: The MIA pathway import efficiency regulates protein synthesis and proteasomal activity.
Figure 2: Defects in the presequence import pathway modulate protein synthesis and proteasomal activity.
Figure 3: Mitochondrial precursor proteins stimulate proteasomal activity.
Figure 4: Mistargeted proteins protect cells against stress.

Accession codes

Primary accessions

ArrayExpress

Data deposits

RNA-seq data have been submitted to the ArrayExpress database under accession number E-MTAB-3588. The mass spectrometry data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD001495.

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Acknowledgements

We thank A. Fergin, B. Knapp, B. Guiard, W. Voos, M. Glickman, A. Gornicka, A. Loniewska-Lwowska, and T. Wegierski for materials, experimental assistance and discussions. Deposition of the data to the ProteomeXchange Consortium is supported by PRIDE Team, EBI. Research in the B.W. laboratory is supported by the Deutsche Forschungsgemeinschaft and the Excellence Initiative of the German Federal & State Governments (EXC 294 BIOSS). Research in the A.C. laboratory was supported by Foundation for Polish Science – Welcome Programme co-financed by the EU within the European Regional Development Fund (L.W., M.E.S. and E.J.), National Science Centre grants 2011/02/B/NZ2/01402 (L.W., U.T. and A.V.) and 2013/11/B/NZ3/00974 (P.C.) and Ministerial Ideas Plus schema 000263 (E.J.). L.W. and U.T. were also supported by National Science Centre grant 2013/08/T/NZ1/00770 and Swiss National Science Foundation postdoctoral fellowship (PP300P3-147899), respectively. P.B. was supported by the National Science Centre grant 2013/11/D/NZ1/02294.

Author information

Authors and Affiliations

Authors

Contributions

P.B. and S.W. are joint second authors. L.W., U.T., P.B., M.E.S., A.V., P.C., S.M. and E.J. performed and analysed biochemical experiments. P.B. and M.L. performed RNA-seq and analyses. S.W. and S.O. performed the mass spectrometric measurements and analyses. A.C., B.W., M.K. and A.D. analysed and supervised the study. A.C and B.W. conceived the project. All authors interpreted the experiments. A.C. wrote the manuscript with the input of other authors.

Corresponding authors

Correspondence to Bettina Warscheid or Agnieszka Chacinska.

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Competing interests

The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Transcriptome and proteome analysis of WT and mia40-4int cells.

a, Distribution of transcripts quantified by RNA-seq in wild-type and mia40-4int cells based on the GO term for translation provided by the Saccharomyces genome database. b, c, Distribution of transcripts and proteins based on the GO term for mitochondrial ribosome provided by the Saccharomyces genome database. d, SILAC-based strategy for the quantitative analysis of alterations in the proteome of yeast cells bearing the mia40-4int mutation. Derivatives of mia40-4int and wild type (mia40-4intS and WTS) were used for SILAC. The mia40-4intS cells grown at permissive temperature 19 °C in heavy medium containing Lys8 and Arg10 were mixed in equal ratio with WTS cells grown in the light medium containing Lys0 and Arg0 for set 1 (two biological replicates) or vice versa for set 2 (one additional biological replicate). e, The mia40-4intS strain and the parental mia40-4int strain exhibited a similar temperature-sensitive phenotype. Both mutants and corresponding wild-type strains were subjected to consecutive tenfold dilutions, spotted on YPD plates and grown at the indicated temperatures. f, The mia40-4intS strain showed no defect in the import of matrix-targeted Su9-DHFR as expected for mia40-4int. The [35S]-labelled precursor of Su9-DHFR was incubated with isolated mitochondria from mia40-4intS and WTS strains for the indicated time points. g, Import of the MIA substrate, Tim9, was decreased in mia40-4intS as expected for mia40-4int. [35S]-labelled precursor of Tim9 was incubated with the isolated mitochondria from mia40-4intS and WTS strains for the indicated time points. f, g, An excess of non-imported precursor was removed by the treatment of mitochondria with proteinase K. Samples were analysed by reducing and non-reducing SDS–PAGE followed by autoradiography. h, Sections of MS survey spectra of SILAC-encoded peptides for the MIA pathway substrates Cox19, Tim12 and Pet191 exhibiting decreased levels in mia40-4intS cells. MS survey spectra were acquired from the experimental set 1 as depicted in Extended Data Fig. 1d. i, Mitochondria were isolated from mia40-4intS and corresponding WTS grown at 19 °C and analysed by western blotting. j, WTS and mia40-4intS yeast were grown to the stationary phase at 19 °C. Cellular extracts for mitochondrial proteins were analysed. The changes in protein abundance in mia40-4intS were as expected for the mia40-4int strain. k, Proteins quantified in three independent biological replicates were plotted according to their P value (log10) against the log10-transformed mia40-4intS /WTS ratios. Proteins with a P value <0.05 and a fold-change in protein abundance >1.5 or <−1.5 were considered upregulated and downregulated and are marked in green and red, respectively. The MIA pathway substrates are highlighted by enlarged circles. WT, wild-type; p, precursor; m, mature; asterisk indicates unspecific band; IAA, iodoacetamide. f, g, i, j, Uncropped blots/gels are in Supplementary Information Fig. 1.

Extended Data Figure 2 Protein abundance in mia40-4int and mia40-4ints .

a, Distribution of proteins quantified by SILAC-MS analysis of WTS and mia40-4intS cells based on the GO terms for cellular components provided by the Saccharomyces genome database. Subcellular localizations are shown for fractions of proteins with a significant 1.5-fold change in abundance and a P value < 0.05 in the mia40-4intS cells. b, GO term enrichment analysis of proteins found to be significantly downregulated (top) or upregulated (bottom) in mia40-4intS. c, WTS and mia40-4intS yeast were grown to the stationary phase at 19 °C. Cellular extracts were analysed for non-mitochondrial proteins. d, WT and mia40-4int yeast were grown in the respiratory medium to the logarithmic phase at 19 °C and shifted for 6 h to 37 °C. Cellular extracts were analysed. No changes in protein abundance in mia40-4int in comparison to wild type were observed. c, d, Uncropped blots are in Supplementary Information Fig. 1. e, f, Yeast were cultured in the full medium with galactose to early logarithmic phase and shifted for 3 h to 37 °C (mia40-4ints ) (e) or 6 h 37 °C (mia40-4int) (f). Cells were treated with 100 µg ml−1 cycloheximide for 10 min. Yeast lysates were fractionated on a 10–50% linear sucrose gradient and absorbance was monitored at 254 nm. The retention of 40S, 60S ribosomal subunits, monosomes (80S) and polysomes is indicated. The monosomes versus polysomes ratio was quantified. Mean ± s.e.m., n = 3. WT, wild type.

Extended Data Figure 3 Characterization of the mia40-4int mutant.

a, Wild type and mia40-4int were grown in respiratory medium and their growth was compared upon shift to the restrictive temperature of 37 °C. D600nm (OD600) was measured at the indicated time points. b, The survival of wild type and mia40-int grown to the logarithmic phase at 19 °C, heat-shocked for 6 h at 37 °C or heat-killed for 3 min at 80 °C was assessed by propidium iodide (PI) staining. c, The mia40-4int and wild-type cells transformed or not transformed with the plasmid encoding Mia40 were grown in respiratory medium at 19 °C and shifted to 37 °C for the indicated times. Protein levels were analysed. The mia40-4int-dependent defect in protein levels was complemented by MIA40. Uncropped blots are in Supplementary Information Fig. 1. d, mRNA levels of IRC25 or POC4 in mia40-4intS, mia40-4int and the corresponding wild type. Cultures were grown in respiratory medium at 19 °C. The heat stress was conducted at 37 °C for the indicated time. Transcript levels of ALG9, FBA1 and TUB2 were used for normalization. Wild type was set to 1. Mean ± s.e.m., n = 3. WT, wild type.

Extended Data Figure 4 Proteasomal activity in mia40 mutants.

a, Wild-type cells were grown in respiratory medium at 19 °C and shifted to 37 °C for 6 h. Where indicated, cell lysates were incubated with 50 µM MG132 for 2 min before addition of caspase-like or chymotrypsin-like proteasome substrate. The activity was inhibited upon MG132 addition, confirming the proteasomal specificity of the assay. b, Wild-type cells and cells deleted for POC1, POC2, IRC25, POC4, UMP1 and PRE9 were grown in respiratory medium to stationary phase at 24 °C. The proteasome caspase-like and chymotrypsin-like activities were reduced upon the compromised proteasome demonstrating the specificity of the assay. Mean ± s.e.m., n = 3. *P value <0.05. c, Cells were grown in respiratory medium to logarithmic phase at 19 °C and shifted to 37 °C for 6 h. Proteasomal activities were analysed over time. Left, mean of 7 biological replicates; right, mean of 4 biological replicates. d, Wild-type cells were grown at 24 °C and were shifted for 6 h at 37 °C or for 4 h at 42 °C. Proteasome activities and the levels of proteasomal subunits, Pre10, Rpt1 and Rpt5 were not changed. Mean ± s.e.m., n = 3. e, Total protein extracts of plasmid-borne mia40 mutants were analysed by anti-ubiquitin immunoblotting and Coomassie staining. d, e, Uncropped blots/gels are in Supplementary Information Fig. 1. f, Proteasomal activity of wild type and mia40 mutants. Mean ± s.e.m., n = 4. *P < 0.05; **P < 0.03. WT, wild type; RFU, relative fluorescent units.

Extended Data Figure 5 Characterization of cells that overproduce Mia40.

a, Formation of a disulfide-bonded intermediate between Mia40 and Tim9 is accelerated in mitochondria with overproduced Mia40. Mitochondria were isolated from wild-type and Mia40 overproducing (Mia40↑) strains and incubated with [35S]-labelled Tim9 precursor. When indicated, iodoacetamide (IAA) was added as a control to block mitochondrial import. b, More efficient import of proteins in mitochondria with overproduced Mia40. Mitochondria from WT and Mia40↑ strains were incubated with [35S]-labelled precursors. The samples were treated with proteinase K to remove non-imported proteins. c, Quantification of [35S]radiolabelled Tim9 and Cox19 import. Mean ± s.e.m., n = 3. d, Cellular and mitochondrial protein levels were analysed in wild-type and Mia40↑ strains by western blotting. The overproduction of Mia40 did not change the protein levels. e, Quantification of Pet191 in mitochondria (M) in wild type and Mia40↑ after 6 h induction (Fig. 1i). f, The WT and Mia40↑ cells producing Pet191Flag were grown in fermentable medium with 2% glucose at 24 °C and shifted to galactose-containing medium for overnight induction. Protein levels in total (T), post-mitochondrial supernatant (S) and mitochondria (M) were analysed. The mitochondrial localization (M) of Pet191 in WT and Mia40↑ after overnight induction was quantified. WT, wild type. a, b, d, f, Uncropped gels/blots are in Supplementary Information Fig. 1.

Extended Data Figure 6 Characterization of mutants that affect the import via the TIM23 presequence pathway.

a, The wild-type strain was grown to stationary phase at 19 °C and treated with CCCP for 2 h to dissipate the electrochemical potential of the inner mitochondrial membrane. The CCCP treatment resulted in the accumulation of precursor proteins. b, Representation of the pulse-chase experiment. The wild type was treated for 30 min with CCCP (pulse) and chased in fresh medium without CCCP for 20 or 45 min for analysis. c, Non-processed precursor proteins accumulate in the tim17 mutants. The tim17 mutants and the corresponding wild type were grown in fermentative medium to stationary phase at 19 °C, shifted to 37 °C and analysed by western blotting. d, The tim17-5 mutant was grown to stationary phase at 19 °C and shifted to 37 °C for 90 min. The cells were fractionated and equal volumes of total (T), post-mitochondrial supernatant (S) and mitochondrial (M) fractions were analysed by western blotting. Precursor proteins were localized in the cytosol together with cytosolic proteins (Rpl17 and Pgk1), in contrast to mature mitochondrial proteins (Cyc3, Tim23, Tom70). e, The pam16 and pam18 mutants were grown to logarithmic growth phase and shifted to restrictive temperature. Total protein content was analysed by western blotting. No changes in protein levels were detected with the exception for a small decrease in the ribosomal proteins Rpl17 and Rpl24 was noticed. f, Ubiquitinated species decreased in the tim17-4 and tim17-5 mutants. The tim17 mutants and corresponding WT strain were grown in respiratory medium at 19 °C, shifted to 37 °C and analysed by anti-ubiquitin immunoblotting. g, The pam16-1 and pam18-1 mutants were grown in respiratory medium at 19 °C and shifted to 37 °C for proteasomal activity assays. Mean of 3 biological replicates. h, The tim17 mutants and corresponding wild-type strain were grown in respiratory medium at 19 °C and shifted to 37 °C for proteasomal activity assays. Mean ± s.e.m., n = 3 (caspase-like activity), n = 5 (chymotrypsin-like activity). ***P < 0.01. i, Northern blot of rRNA and quantification. The pam16-3 and wild-type cells were grown in respiratory medium and shifted to 37 °C. Mean ± s.e.m., n = 3. j, Incorporation of [35S]-labelled amino acids is decreased in tim17 mutants compared to wild type. Strains were grown in respiratory medium and shifted to 37 °C. Samples were taken after 1 or 2 h of [35S] labelling and analysed by SDS–PAGE and autoradiography. k, Representative gradient profiles of ribosomes in pam16-3 and wild type and quantification of the monosome versus polysome fractions. Mean ± s.e.m., n = 3. *P < 0.05. Cells were grown to logarithmic phase, shifted to 37 °C for 3 h and treated with 100 µg ml−1 cycloheximide for 10 min. Lysates were fractionated on a 10–50% linear sucrose gradient and absorbance was monitored at 254 nm. The monosomes versus polysomes ratio was quantified. WT, wild-type. RFU, relative fluorescent units. a, cf, i, j, Uncropped blots/gels are in Supplementary Information Fig. 1.

Extended Data Figure 7 Translation and proteasomal activity in the cells overproducing mitochondrial proteins.

a, Pet191Flag and Mix17Flag were expressed in WT cells at 24 °C. b, Flag-tagged proteins were expressed in WT and when indicated cells were treated with MG132 for 4 h. Fractions of total (T), aggregates (P) and soluble (S) proteins were analysed by western blotting. c, Wild type expressing Pet191Flag or Mix17Flag were grown at 24 °C and analysed for total protein content by western blotting. No changes in protein levels compared to wild type were found, including ribosomal or proteasome subunits. d, Northern blot analysis and quantification of rRNA in cells expressing Mix17Flag. Mean ± s.e.m., n = 3. e, Mistargeted mitochondrial proteins do not alter the rate of translation. Incorporation of [35S]-labelled amino acids in wild type expressing Pet191Flag or Mix17Flag. The expression of Flag-tagged proteins was induced for 6 h at 24 °C. Samples were taken 20 min after initiation of [35S] labelling and analysed by SDS–PAGE and audioradiography. f, Expression of Pet191Flag or Mix17Flag stimulates proteasomal activity. Mean of 3 biological replicates. g, Cox4Flag with (pCox4FLAG) or without mitochondrial presequence (mCox4Flag) was expressed in wild type. h, Mdj1Flag and mMdj1Flag proteins were expressed in WT cells. The presence of Flag-tagged proteins was confirmed by immunoblotting. i, Wild type expressing Mdj1Flag or mMdj1Flag were grown at 24 °C and analysed for total protein levels by western blotting. No changes in protein levels compared to control were found, including ribosomal and proteasome subunits. WT, wild type. RFU, relative fluorescent units. ae, gi, Uncropped blots are in Supplementary Information Fig. 1.

Extended Data Figure 8 Proteasomal activity in the cells overproducing non-mitochondrial proteins.

a, Expression of DHFRFlag and DHFRdsFlag was induced at 24 °C and Pex22Flag at 28 °C in wild type. The presence of Flag-tagged proteins was confirmed by immunoblotting. b, Ubc9ts–GFP was induced in galactose and subsequently wild-type cells were shifted to glucose medium at 37 °C to initiate unfolding. After indicated time points, samples were analysed by western blotting. c, Flag-tagged proteins were expressed in wild type for the indicated time and the proteasome was inhibited with MG132 for 3 h if indicated. Fractions of total (T), aggregates (P) and soluble (S) proteins were analysed and no aggregation was observed. d, e, Proteasomal activity in WT expressing DHFRFlag or DHFRdsFlag grown at 24 °C. Mean ± s.e.m. n = 6 (d). Mean of 6 biological replicates (e). f, Wild-type cells expressing DHFRFlag or DHFRdsFlag were grown at 24 °C and analysed for total protein content. No changes in protein levels were found compared to wild type, including ribosomal and proteasome subunits. g, Proteasomal activity in wild type expressing Pex22Flag grown at 28 °C and the abundance of proteins were not significantly changed. No significant change in proteasomal activity was detected upon expression of Ubc9ts–GFP. Mean ± s.e.m., n = 6. h, The proteasomal stimulation by CCCP and Mix17Flag is additive. Wild-type cells overproducing Mix17Flag were treated with CCCP to measure the chymotrypsin-like activity of the proteasome. In the case of Mix17Flag, proteasomal stimulation was less efficient than stimulation reported for Mix17Flag in Fig. 3a due to change in experimental conditions imposed by the CCCP treatment. Mean ± s.e.m., n = 3. *P < 0.05; **P < 0.03. Analysis of cellular protein content showed no difference in proteasome subunits (Rpt1, Rpt5) or ribosomal protein Rpl17. WT, wild type. RFU, relative fluorescent units. ac, fh, Uncropped blots are in Supplementary Information Fig. 1.

Extended Data Figure 9 Auxiliary factors are required to stimulate proteasome by mitochondrial precursor proteins.

a, Proteasomal activity in wild-type cells expressing simultaneously either Poc1Flag and Poc2Myc or Irc25Flag and Poc4Myc. Mean ± s.e.m., n = 3. *P < 0.05. The overexpression of POC1 and POC2 or IRC25 and POC4 was induced from one plasmid. The overexpression of Irc25Flag and Poc4Myc led to a small increase in the proteasomal activity, in spite of the inability to detect these proteins likely due to tight regulation of their abundance (not shown). b, Proteasomal activity in cells lacking Irc25 or Poc4, expressing Mix17Flag or Pet191FLAG and grown at 24 °C. Mean of 4 biological replicates. c, Affinity purification of the proteasome complex via Pre3TAP from cells grown at 28 °C. Load, 5%; eluate, 100%. Chymotrypsin-like activity of the proteasome bound to the column was measured. The specificity was checked by the treatment of the on-column fraction with proteasomal inhibitor MG132. Activity of the purified proteasome via Pre3TAP was measured upon addition of purified Mix17Flag or Pet191Flag (for 7.5% of the on-column fraction). Mean ± s.e.m., n = 3. Uncropped blots are in Supplementary Information Fig. 1. d, Representation of the proteasome complex affinity purification. The subunits of proteasome are assembled into the 20S catalytic core and the 19S regulatory particle. The core and regulatory particles joined together to form the 26S proteasome. Overexpression of an activator (that is, Mix17) stimulates the 26S proteasome assembly. Thus, upon affinity purification via a TAP-tagged proteasome subunit, more proteasomal subunits representing more assembled proteasomes are found in the eluate in the presence of an activator. WT, wild type. RFU, relative fluorescent units.

Extended Data Figure 10 The proteasome assembly heterodimer Irc25–Poc4 is required to protect cells against stress.

a, Representation of heat stress experiments. Cells overexpressing mitochondrial proteins were exposed to different heat shock conditions and subsequently subjected to lethality assessment (left panel). Cells were exposed to a gradual increase in temperature from 42 °C to 53 °C within 30 min or were incubated at 53 °C for 30 min. Mean lethality values of wild-type cells expressing empty plasmid increased with harsher stress conditions (9% for middle panel; 22% for right panel). The lethality of cells expressing empty plasmid was set to 1. Mean ± s.e.m., n = 5 (middle panel), n = 4 (right panel). **P < 0.03; ***P < 0.01. b, Wild type or cells deleted for the IRC25 or POC4 genes and overproducing Pet191Flag or Cox12Flag protein were cultured on agar plates with sucrose. Consecutive tenfold dilutions of cells were spotted on selective medium plates with either glucose or galactose. Cells were grown at the indicated temperatures. c, Model for cellular responses activated by the mitochondrial protein import and precursor over-accumulation stress. WT, wild type.

Supplementary information

Supplementary Information

This file contains full legends for Supplementary Tables 1-3 and Supplementary images. (PDF 13722 kb)

Supplementary Table 1

This file contains RNA-Seq analysis of the mia40-4int mutant versus wild-type strain – see Supplementary Information file for full legend. (XLSX 1323 kb)

Supplementary Table 2

This file contains SILAC-based proteomics analysis of mia40-4intS mutant versus wild-type - see Supplementary Information file for full legend. (XLSX 974 kb)

Supplementary Table 3

This file contains proteins quantified in SILAC-based proteomics in at least two biological replicates - see Supplementary Information file for full legend. (XLSX 1034 kb)

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Wrobel, L., Topf, U., Bragoszewski, P. et al. Mistargeted mitochondrial proteins activate a proteostatic response in the cytosol. Nature 524, 485–488 (2015). https://doi.org/10.1038/nature14951

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