A nuclear-based quality control pathway for non-imported mitochondrial proteins
- Department of Biochemistry, University of Utah School of Medicine, United States
Figures
Figure 1 with 1 supplement
Non-imported mitochondrial protein Ilv2 localizes to the nucleus upon import failure.
(A) Representative images of old and young yeast expressing the indicated Ilv2-GFP and nuclear marker Nup49-mCherry. Percentage of cells with Ilv2 in the nucleus (n = 30 cells) and age of representative cell (determined by bud scar counting) are indicated in bottom panels. Bud scars stained with calcofluor. (B) Yeast expressing Ilv2-GFP and Tom70-mCherry ± FCCP. (C) Indirect immunofluorescence for endogenous Ilv2 of yeast expressing Tom70-mCherry. (D) Western blots of yeast expressing Ilv2-GFP ± FCCP. (E) Western blots of endogenous Ilv2 in yeast treated ± FCCP. Pgk1 = loading control in all instances. (F) Western blot showing enrichment of the precursor form of Ilv2-HA in the nuclear fraction. Nup49-GFP and H2B = nuclear markers, Tom70 and Tim44 = mitochondrial markers. (G) RITE-tagged cells treated with β-estradiol at time of FCCP addition to initiate Cre/lox switching of Ilv2 epitope tag from GFP (old) to RFP (new). (B, C, and G) Nucleus stained with DAPI. P = precursor, M = mature. All scale bars = 2 μm. Arrows denote nucleus.
Figure 1—figure supplement 1
Mitochondrial protein Ilv2 localizes to the nucleus upon import failure.
(A) Fivefold serial dilutions of WT, Ilv2 KO, and endogenous C-terminally GFP, HA, or FLAG-tagged Ilv2 expressing yeast strains on SD complete or isoleucine and valine dropout agar plates. (B) Indirect immunofluorescence of yeast expressing Ilv2-FLAG and Tom70-mCherry. Nucleus in (B, C) stained with NucBlue and DAPI, respectively. Arrows indicate nucleus. (C) Tom70-mCherry wild type (WT) and tetp-TOM40 (TOM40 KD) yeast expressing Ilv2-GFP in the presence of doxycycline. (D) Western blot for Tom40 in wild-type (WT) and tetp-TOM40 (KD) strains in the presence of doxycycline. (E) Western blot of yeast expressing Ilv2-HA ± FCCP. P = precursor and M = mature. Scale bars = 2 μm.
Figure 2 with 2 supplements
The nucleus in one of several fates for non-imported mitochondrial proteins.
(A) Yeast expressing the indicated mCherry or GFP-tagged mitochondrial proteins ± FCCP. (B–D) Western blots of yeast expressing the indicated GFP-tagged mitochondrial proteins ± FCCP or ± FCCP ± MG-132. (E) Summary of non-imported mitochondrial protein fates. All scale bars = 2 μm. Arrows denote nucleus (A, class 1) or ER (A, class 4). P = precursor and M = mature.
Figure 2—figure supplement 1
Non-imported mitochondrial proteins have several distinct fates.
(A) Yeast expressing ER marker Sec61-mCherry and Mir1-GFP ± FCCP. (B) Indirect immunofluorescence of Ilv2, Tom20, and OM45 in yeast expressing Tom70-mCherry or Sec61-mCherry. (C) Indirect immunofluorescence of yeast expressing the indicated FLAG-tagged proteins and Tom70-mCherry. (D) Tom70-mCherry wild-type (WT) and tetp-TOM40 (TOM40 KD) yeast expressing the indicated GFP-tagged mitochondrial proteins in the presence of doxycycline. All bars = 2 μm. Nucleus in (B, C) stained with NucBlue. Arrows indicate nucleus (B–D, class 1) or ER (A–D, class 4).
Figure 2—figure supplement 2
Non-imported mitochondrial proteins have numerous fates.
(A) Western blots of yeast expressing the indicated GFP-tagged proteins ± FCCP. (B) Western blots against indicated native protein in yeast ± FCCP. (C–E) Western blots of yeast expressing the indicated HA-tagged mitochondrial proteins ± FCCP (C) or ± FCCP ± MG-132 (D, E). P = precursor form, M = mature form. Pgk1 = loading control.
Figure 3 with 2 supplements
Nuclear protein quality control degrades non-imported mitochondrial proteins.
(A) Western blot of yeast expressing Ilv2-GFP ± FCCP ± MG-132. (B) Western blot of yeast expressing Ilv2-GFP ± FCCP in wild-type (WT) and E3 KO strains. (C) Western blots showing cycloheximide (CHX) chase of Ilv2-GFP in WT and E3 KO strains in the presence of FCCP. (D) Western blot showing ubiquitylation of immunoprecipitated Ilv2-GFP ± FCCP in WT and E3 KO strains. Pgk1 = loading control. E3 KO = san1Δ ubr1Δ doa10Δ. P = precursor and M = mature.
Figure 3—figure supplement 1
Nuclear protein quality control promotes non-imported mitochondrial protein degradation.
(A) Western blot of yeast expressing Ilv2-HA ± FCCP ± MG-132. (B, C) Western blots of yeast expressing the Ilv2-HA (B) or endogenous Ilv2 (C) ± FCCP in WT and E3 KO strains. (D) Western blots of yeast expressing Ilv2-GFP ± FCCP in WT and the indicated mutant yeast strains. (E, F) Western blots showing the CHX chase of Ilv2-HA (E) or endogenous Ilv2 (F) in WT and E3 KO strains in the presence of FCCP. (G) Western blots of yeast expressing the Lat1-GFP ± FCCP in WT and E3 KO strains. P = precursor form, M = mature form. Pgk1 = loading control.
Figure 3—figure supplement 2
Nuclear protein quality control promotes non-imported mitochondrial protein degradation.
(A) Yeast expressing the indicated GFP and mCherry tagged mitochondrial proteins ± FCCP. (B) Quantification of (A). N > 99 cells per replicate, error bars = SEM of three replicates. (C) Tom70-mCherry wild-type (WT) and tetp-TOM40 (TOM40 KD) yeast expressing the indicated GFP-tagged mitochondrial proteins in the presence of doxycycline. (A, C) Nucleus stained with DAPI, Arrows = nucleus. Bar = 2 μm. (D) Western blots of yeast strains expressing indicated GFP-tagged mitochondrial proteins ± FCCP ± MG-132. (E) Western blots of yeast expressing the indicated GFP-tagged mitochondrial proteins ± FCCP in WT and E3 KO strains. (F) Western blot of yeast expressing indicated GFP-tagged mitochondrial proteins ± FCCP in WT and the indicated mutant yeast strains. Pgk1 = loading control. E3 KO = san1Δ ubr1Δ doa10Δ. P = precursor and M = mature.
Figure 4 with 1 supplement
Non-imported mitochondrial precursors are toxic and localize to nuclear-associated foci when clearance is impaired.
(A) Fivefold serial dilutions of WT and E3 KO strains on YPAD ± FCCP agar plates. (B) WT and E3 KO yeast expressing Ilv2-GFP and Tom70-mCherry ± FCCP. Nucleus stained with DAPI. Arrows = nuclear associated foci. Bar = 2 μm. (C) Quantification of (B). N > 99 cells per replicate, error bars = SEM of three replicates. (D) Quantification of average pixel intensity of Ilv2-GFP nuclear foci from (B). N = 20 cells, error bars = SD, p-value=0.0005. (E) Fivefold serial dilutions of WT and E3 KO strains expressing endogenous Ilv2-GFP (endo) ± mild overexpression of full-length Ilv2-GFP (FL) from pRS413-Ilv2-GFP on SD-His ± FCCP agar plates. (F) Yeast strain expressing WT Ilv2-GFP or Ilv2-NES-GFP and Tom70-mCherry ± FCCP. Nucleus stained with DAPI. Arrows = nucleus. Bar = 2 μm. (G) Quantification of (F). (H) Western blot of yeast expressing Ilv2-GFP or Ilv2-NES-GFP ± FCCP. (I) Fivefold serial dilutions of endogenously tagged WT Ilv2-GFP and Ilv2-NES-GFP strains on YPAD ± FCCP agar plates. P = precursor and M = mature.
Figure 4—figure supplement 1
Impaired clearance of non-imported mitochondrial proteins targets them to nuclear-associated foci.
(A) Fivefold serial dilutions of WT and the indicated mutant strains on YPAD ± FCCP agar plates. (B, C) WT and E3 KO (san1Δ ubr1Δ doa10Δ) yeast expressing Dld1-GFP or Dld2-GFP and Tom70-mCherry ± FCCP. Nucleus stained with DAPI, arrows = nuclear associated foci, and bar = 2 μm. (D, E) Quantification of (B) and (C), respectively. N > 99 cells per replicate, error bars = SEM of three replicates.
Figure 5 with 1 supplement
The mitochondrial targeting sequence (MTS) is required for non-imported precursor toxicity and quality control.
(A) Schematic of full-length GFP-tagged Ilv2 (FL), mitochondrial targeting sequence deleted (ΔMTS) GFP-tagged Ilv2, and MTSIlv2 GFP only (MTS). (B) Tom70-mCherry yeast expressing endogenous Ilv2-GFP ± the indicated Ilv2 variant ± FCCP. Nucleus stained with DAPI. Arrows = nuclear-associated foci. Bars = 2 μm. (C, D) Quantification of cells with diffuse Ilv2 nuclear localization (C) or Ilv2 nuclear foci (D) from (B). For (C, D), N > 99 cells per replicate, error bars = SEM of three replicates. (E) Western blot of strains expressing indicated Ilv2-GFP variants ± FCCP. Pgk1 = loading control. (F) Fivefold serial dilutions of WT and E3 KO strains expressing endogenous Ilv2-GFP (endo) ± mild overexpression of the indicated Ilv2-GFP variants on SD-His ± FCCP agar plates. (G) Western blot of strains expressing indicated Cox15-GFP or Lat1-GFP variants, respectively, ± FCCP. Pgk1 = loading control. (H) Fivefold serial dilutions of WT strains expressing endogenous Cox15-GFP or Lat1-GFP (endo) ± mild overexpression of the indicated variants on SD-His ± FCCP agar plates. P = precursor and M = mature.
Figure 5—figure supplement 1
The MTS is required for non-imported precursor toxicity and degradation.
(A) Fivefold serial dilutions of strains expressing endogenous Ilv2-GFP (endo) ± mild overexpression of the indicated Ilv2-GFP variants or untagged Ilv2 variants on SD-His ± FCCP agar plates. (B) Tom70-mCherry yeast expressing endogenous Dld2-GFP ± the indicated Dld2 plasmid-expressed variants ± FCCP. Nucleus stained with DAPI. Arrows = nuclear associated foci. Bars = 2 μm. (C, D) Quantification of cells with diffuse Dld2-GFP nuclear localization (C) or Dld2-GFP nuclear foci (D) from (B). For (C and D), N > 99 cells per replicate, error bars = SEM of three replicates. (E) Western blot of strains expressing indicated Dld2-GFP variants ± FCCP. Pgk1 = loading control. (F) Fivefold serial dilutions of WT and E3 KO strains expressing endogenous Dld2-GFP (endo) ± mild overexpression of the indicated Dld2-GFP variants on SD-His ± FCCP agar plates.
Figure 6
MTS-mediated toxicity of Ilv2 is not entirely linked to mitochondrial import clogging.
(A) 35S-labeled Su9-DHFR was imported into mitochondria isolated from yeast overexpressing the indicated Ilv2-GFP variants. (B) Western blot showing protein levels in mitochondria isolated from yeast overexpressing the indicated Ilv2-GFP variants. (C) 35S-labeled Su9-DHFR was imported into mitochondria isolated from yeast expressing endogenous Ilv2-GFP ± FCCP. (D) Western blot showing protein levels in mitochondria isolated from yeast expressing endogenous Ilv2-GFP ± FCCP. (E) Yeast expressing Idh1-GFP and Tom70-mCherry in WT or Ilv2 (untagged) overexpressing strains ± FCCP. Bars = 2 μm. (F) Quantification of (E). (G) 35S-labeled Su9-DHFR was imported into mitochondria isolated from yeast expressing endogenous Ilv2-GFP or Ilv2-NES-GFP ± FCCP. (H) Western blot showing protein levels in mitochondria isolated from yeast expressing endogenous Ilv2-GFP or Ilv2-NES-GFP. For (A), (C), and (G), a mixture of antimycin A, oligomycin, and valinomycin (AVO) was used to dissipate the membrane potential. Non-imported proteins were proteolytically removed with proteinase K and the import was analyzed by SDS–PAGE and autoradiography. P = precursor and M = mature.
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background Saccharomyces cerevisiae | Yeast GFP collection co-expressing Tom70-mCherry | PMID:27097106 | N/A | N/A |
| Strain, strain background (S. cerevisiae) | BY4741 | ATCC | Cat # 201388 | N/A |
| Strain, strain background (S. cerevisiae) | BY4743 | ATCC | Cat # 201390 | N/A |
| Strain, strain background (S. cerevisiae) | Other strains used in this study | This paper | Supplementary file 2 | N/A |
| Antibody | Anti-GFP (mouse monoclonal) | Millipore Sigma | Cat # 11814460001 RRID: AB_390913 | WB (1:2500) |
| Antibody | Anti-HA (mouse monoclonal) | Millipore Sigma | Cat # 11583816001 RRID: AB_514506 | WB (1:1000), IF (1:200) |
| Antibody | Anti-PGK1 (mouse monoclonal) | Abcam | Cat # 22C5D8 RRID: AB_2756444 | WB (1:1000) |
| Antibody | Anti-FLAG M2 (mouse monoclonal) | Millipore Sigma | Cat # F1804 RRID: AB_262044 | IF (1:200) |
| Antibody | Anti-H2B (rabbit polyclonal) | Active Motif | Cat # 39947 RRID: AB_2793403 | WB (1:1000) |
| Antibody | Anti-Idh1 (goat polyclonal) | ORIGENE | Cat # Ap31099PU-N | WB (1:500) |
| Antibody | Anti-Ilv2 (rabbit polyclonal) | Dr. Agnieszka Chacinska | N/A | WB (1:1000), IF (1:200) |
| Antibody | Anti-Tom70 (rabbit polyclonal) | Dr. Nikolaus Pfanner | N/A | WB (1:1000) |
| Antibody | Anti-Tom20 (rabbit polyclonal) | Dr. Nikolaus Pfanner | N/A | WB (1:1000), IF (1:200) |
| Antibody | Anti-Tim54 (rabbit polyclonal) | Dr. Nikolaus Pfanner | N/A | WB (1:1000) |
| Antibody | Anti-Tim50 (rabbit polyclonal) | Dr. Nikolaus Pfanner | N/A | WB (1:1000) |
| Antibody | Anti-Tim18 (rabbit polyclonal) | Dr. Nikolaus Pfanner | N/A | WB (1:1000) |
| Antibody | Anti-OM45 (rabbit polyclonal) | Dr. Carla Koehler | N/A | WB (1:1000), IF (1:200) |
| Antibody | anti-Tom40 (rabbit polyclonal) | Dr. Toshiya Endo | N/A | WB (1:1000) |
| Recombinant DNA reagent | pKT128 (plasmid) | Addgene | Plasmid # 8729 | N/A |
| Recombinant DNA reagent | pKT127-mCherry (plasmid) | Daniel Gottschling | N/A | N/A |
| Recombinant DNA reagent | pFA6a-5FLAG-KanMX6 (plasmid) | Addgene | Plasmid # 15983 | N/A |
| Recombinant DNA reagent | pFA6A-mCherry-HphMX (plasmid) | Addgene | Plasmid # 105156 | N/A |
| Recombinant DNA reagent | pVL015 (plasmid) | Addgene | Plasmid # 64766 | N/A |
| Recombinant DNA reagent | pFA6A-3HA-KanMX (plasmid) | Addgene | Plasmid # 39295 | N/A |
| Recombinant DNA reagent | Template plasmid for 35S-radiolabeled Su9-DHFR | Walter Neupert PMID:2884042 | N/A | N/A |
| Recombinant DNA reagent | Other plasmids generated in this study | This paper | Supplementary file 4 | N/A |
| Sequenced-based reagent | Oligos used in this study | This paper | Supplementary file 3 | N/A |
| Chemical compound, drug | -Estradiol | Millipore Sigma | Cat # E8875; CAS # 50-28-2 | N/A |
| Chemical compound, drug | Carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) | Millipore Sigma | Cat # C2920; CAS # 370-86-5 | N/A |
| Chemical compound, drug | cOmplete Protease Inhibitor Cocktail | Millipore Sigma | Cat# 11697498001 | N/A |
| Chemical compound, drug | Dimethyl sulfoxide | Millipore Sigma | Cat # D2650; CAS # 67-68-5 | N/A |
| Chemical compound, drug | Cycloheximide | Millipore Sigma | Cat # C1988; CAS # 66-81-9 | N/A |
| Chemical compound, drug | Doxycycline hyclate | Millipore Sigma | Cat # C9891; CAS # 24390-14-5 | N/A |
| Chemical compound, drug | Polyvinylpyrrolidone | Millipore Sigma | Cat # PVP40; CAS # 9003-39-8 | N/A |
| Chemical compound, drug | Pepstatin | Millipore Sigma | Cat # 10253286001; CAS # 26305-03-3 | N/A |
| Chemical compound, drug | Phenylmethylsulfonyl fluoride | Millipore Sigma | Cat # P7626; CAS # 329-98-6 | N/A |
| Chemical compound, drug | Calcofluor Fluorescent Brightener 28 | Millipore Sigma | Cat # F3543; CAS # 4404-43-7 | N/A |
| Chemical compound, drug | Diamidino-2-phenylindole dihydrochloride (DAPI) | Thermo Fisher | Cat # D1306 | N/A |
| Chemical compound, drug | Glass Antifade Mountant with NucBlue Stain (P36981) | Thermo Fisher | Cat # P36981 | N/A |
| Chemical compound, drug | (S)-MG-132 | Cayman Chemical | Cat # 10012628; CAS # 133407-82-6 | N/A |
| Chemical compound, drug | N-ethylmaleimide (NEM) | Sigma–Aldrich | Cat # S3876; CAS# 128-53-0 | N/A |
| Chemical compound, drug | IGEPAL NP-40-substitute | Sigma–Aldrich | Cat # CA-630; CAS # 9002-93-1 | N/A |
| Chemical compound, drug | Zymolyase 100T | US Biological Life Sciences | Cat # Z1004; CAS# 37340-57-1 | N/A |
| Chemical compound, drug | Triton X-100 | Bio-Rad | Cat # 1610407; CAS# 9002-93-1 | N/A |
| Chemical compound, drug | Formaldehyde 16% in aqueous solution, EM Grade | VWR | Cat # 100503–914; CAS# 50-00-0 | N/A |
| Chemical compound, drug | DTT (dithiothreitol) (>99% pure) protease free | GOLDBIO | Cat # DTT10; CAS# 27565-41−9/3483- 12–3 | N/A |
| Chemical compound, drug | Antimycin A | Sigma–Aldrich | Cat # A8674; CAS# 1397-94-0 | N/A |
| Chemical compound, drug | Phusion polymerase | New England Biolabs | Cat # M0530L | N/A |
| Chemical compound, drug | Valinomycin | Sigma–Aldrich | Cat # V0627; CAS# 2001-95-8 | N/A |
| Chemical compound, drug | Oligomycin | Sigma–Aldrich | Cat # 75351; CAS# 579-13-5 | N/A |
| Commercial assay or kit | Gibson Assembly Cloning Kit | New England Biolabs | Cat # E5510S | N/A |
| Commercial assay or kit | TNT SP6 Quick Coupled Transcription/Translation System | Promega | Cat # L2080 | N/A |
| Software, Algorithm | FIJI | PMID:22743772 | Version 1 | N/A |
| Software, Algorithm | Prism | Graphpad Software | Version 8 | N/A |
| Software, Algorithm | SnapGene | GSL Biotech | Version 4.2 | N/A |
| Software, Algorithm | Image Lab | Bio-Rad | Version 6 | N/A |
| Software, Algorithm | Zen Blue | Carl Zeiss | Version 2.6 | N/A |
| Software, Algorithm | Zen Black | Carl Zeiss | Version 2.3 | N/A |
| Software, Algorithm | Mitoprot | PMID:8944766 | N/A | N/A |
| Software, Algorithm | Illustrator CC | Adobe | Version 22.1 | N/A |
| Software, Algorithm | Photoshop CC | Adobe | Version 19.1 | N/A |
Additional files
-
Supplementary file 1
List of mitochondrial protein fates upon FCCP treatment.
- https://cdn.elifesciences.org/articles/61230/elife-61230-supp1-v2.xlsx
-
Supplementary file 2
List of yeast strains used in this study.
- https://cdn.elifesciences.org/articles/61230/elife-61230-supp2-v2.docx
-
Supplementary file 3
List of oligos used in this study.
- https://cdn.elifesciences.org/articles/61230/elife-61230-supp3-v2.docx
-
Supplementary file 4
List of plasmids generated in this study.
- https://cdn.elifesciences.org/articles/61230/elife-61230-supp4-v2.docx
-
Transparent reporting form
- https://cdn.elifesciences.org/articles/61230/elife-61230-transrepform-v2.docx
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A nuclear-based quality control pathway for non-imported mitochondrial proteins
eLife 10:e61230.
https://doi.org/10.7554/eLife.61230
