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Cited by 247Ubiquitin and proteasome pathwaysAutophagy in Disease and TherapyHeat shock proteins research

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The AAA-ATPase p97 is essential for outer mitochondrial membrane protein turnover

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Xu, Shan, Peng, Guihong, Wang, Yang, Fang, Shengyun, Karbowski, Mariusz·2011·Molecular Biology of the Cell
DOI 10.1091/mbc.e10-09-0748PMC PMC3031461
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Abstract

Recent studies have revealed a role for the ubiquitin/proteasome system in the regulation and turnover of outer mitochondrial membrane (OMM)-associated proteins. Although several molecular components required for this process have been identified, the mechanism of proteasome-dependent degradation of OMM-associated proteins is currently unclear. We show that an AAA-ATPase, p97, is required for the proteasomal degradation of Mcl1 and Mfn1, two unrelated OMM proteins with short half-lives. A number of biochemical assays, as well as imaging of changes in localization of photoactivable GFP-fused Mcl1, revealed that p97 regulates the retrotranslocation of Mcl1 from mitochondria to the cytosol, prior to, or concurrent with, proteasomal degradation. Mcl1 retrotranslocation from the OMM depends on the activity of the ATPase domain of p97. Furthermore, p97-mediated retrotranslocation of Mcl1 can be recapitulated in vitro, confirming a direct mitochondrial role for p97. Our results establish p97 as a novel and essential component of the OMM-associated protein degradation pathway.

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Figures

FIGURE 1: Proteasome- and p97-dependent regulation of Mcl1 and Mfn1 stability. HeLa cells were treated with the protein synthesis inhibitor CHX (20 μg/ml) (A) or a proteasome inhibitor, MG132 (50 μM) (C), for 0, 5, or 10 h followed by Western blotting as indicated in the figure. In (B), relative protein levels in cells treated as described in (A) were quantified and plotted against the time of treatment with CHX [data represent the mean ± SD of three (VDAC, Bak, BclXL) or four (Mfn1, Mcl1, Tom20, Tom40) independent experiments]. In (D), the levels of high-molecular-weight species of Mfn1 were quantified in control cells and cells treated with MG132 for 5 or 10 h (along blue lines as shown in C; Mfn1-long exp.; B, bottom; T, top). The data shown represent fold increases of Mfn1 levels in different points of intensity plots versus the respective values in control samples. The data were normalized with control values at the respective points of intensity plots taken as 1. In panel E, total cell lysates obtained from cells transfected with 3 different p97 shRNAi constructs (#1, #2, and #3) or with a GFP shRNAi construct (Control) were analyzed by Western blot as indicated in the figure. In panel F, changes in the protein levels of p97, Mcl1, Mfn1, and Tom20 in p97 RNAi cells (clones #1, #2, and #3) were quantified and plotted as the percentage of the protein levels in control RNAi cells.
FIGURE 1: Proteasome- and p97-dependent regulation of Mcl1 and Mfn1 stability. HeLa cells were treated with the protein synthesis inhibitor CHX (20 μg/ml) (A) or a proteasome inhibitor, MG132 (50 μM) (C), for 0, 5, or 10 h followed by Western blotting as indicated in the figure. In (B), relative protein levels in cells treated as described in (A) were quantified and plotted against the time of treatment with CHX [data represent the mean ± SD of three (VDAC, Bak, BclXL) or four (Mfn1, Mcl1, Tom20, Tom40) independent experiments]. In (D), the levels of high-molecular-weight species of Mfn1 were quantified in control cells and cells treated with MG132 for 5 or 10 h (along blue lines as shown in C; Mfn1-long exp.; B, bottom; T, top). The data shown represent fold increases of Mfn1 levels in different points of intensity plots versus the respective values in control samples. The data were normalized with control values at the respective points of intensity plots taken as 1. In panel E, total cell lysates obtained from cells transfected with 3 different p97 shRNAi constructs (#1, #2, and #3) or with a GFP shRNAi construct (Control) were analyzed by Western blot as indicated in the figure. In panel F, changes in the protein levels of p97, Mcl1, Mfn1, and Tom20 in p97 RNAi cells (clones #1, #2, and #3) were quantified and plotted as the percentage of the protein levels in control RNAi cells.CC BY-NC-SA · PubMed Central
FIGURE 2: p97 is required for the degradation of Mcl1 in an ATPase domain activity-dependent manner. Control and p97 RNAi cells (clone #1) were treated with CHX for 0, 30, 60, or 90 min, followed by Western blot analyses to detect protein levels of Mcl1 (two exposures of the same Mcl1 blot are shown) and p97. Tom20 served as a loading control (A). In panel B, relative Mcl1 protein levels in control RNAi and p97 RNAi cells were quantified and plotted as a function of the length of CHX treatment. Mcl1 levels detected in untreated samples (time, 0 min) were set at 100%. Control and p97- and p97QQ-expressing cells were treated with CHX (C) or MG132 (E) for 0, 30, 60, or 90 min, followed by Western blot analysis to detect protein levels of Mcl1. Expression levels of p97 and p97QQ were detected with anti-MYC antibody (MYC). Tom20 was used as a loading control. Relative protein levels in cells treated with CHX (D) and MG132 (F) as described in panels C and E were quantified (data represent the mean ± SD of at least three independent experiments). Mcl1 levels detected in untreated samples (time, 0 min) were set at 100%. In panel G, the total cell lysates (TCL) and mitochondria-enriched heavy membrane fractions (HM) obtained from control RNAi and p97 RNAi cells were analyzed by Western blot for the levels of Mcl1, Mfn1, and p97. Tom20 was used as a loading control. Note the mitochondrial accumulation of Mcl1 and Mfn1 in p97 RNAi cells.
FIGURE 2: p97 is required for the degradation of Mcl1 in an ATPase domain activity-dependent manner. Control and p97 RNAi cells (clone #1) were treated with CHX for 0, 30, 60, or 90 min, followed by Western blot analyses to detect protein levels of Mcl1 (two exposures of the same Mcl1 blot are shown) and p97. Tom20 served as a loading control (A). In panel B, relative Mcl1 protein levels in control RNAi and p97 RNAi cells were quantified and plotted as a function of the length of CHX treatment. Mcl1 levels detected in untreated samples (time, 0 min) were set at 100%. Control and p97- and p97QQ-expressing cells were treated with CHX (C) or MG132 (E) for 0, 30, 60, or 90 min, followed by Western blot analysis to detect protein levels of Mcl1. Expression levels of p97 and p97QQ were detected with anti-MYC antibody (MYC). Tom20 was used as a loading control. Relative protein levels in cells treated with CHX (D) and MG132 (F) as described in panels C and E were quantified (data represent the mean ± SD of at least three independent experiments). Mcl1 levels detected in untreated samples (time, 0 min) were set at 100%. In panel G, the total cell lysates (TCL) and mitochondria-enriched heavy membrane fractions (HM) obtained from control RNAi and p97 RNAi cells were analyzed by Western blot for the levels of Mcl1, Mfn1, and p97. Tom20 was used as a loading control. Note the mitochondrial accumulation of Mcl1 and Mfn1 in p97 RNAi cells.CC BY-NC-SA · PubMed Central
FIGURE 3: p97 inhibits apoptosis-induced degradation of Mcl1. Control HeLa cells or cells transfected with p97 or p97QQ were treated with ActD (20 μM; A) or STS (1 μM; C), two unrelated inducers of mitochondria-dependent apoptosis, for 0, 1, or 2 h, followed by Western blotting, as indicated. In panels B and D, relative protein levels in cells treated as described in panels A and C were quantified and plotted against the length of treatment with ActD (B) or STS (D). Data represent the mean ± SD of three independent experiments. Mcl1 levels detected in untreated cells (time, 0 min) were set at 100%.
FIGURE 3: p97 inhibits apoptosis-induced degradation of Mcl1. Control HeLa cells or cells transfected with p97 or p97QQ were treated with ActD (20 μM; A) or STS (1 μM; C), two unrelated inducers of mitochondria-dependent apoptosis, for 0, 1, or 2 h, followed by Western blotting, as indicated. In panels B and D, relative protein levels in cells treated as described in panels A and C were quantified and plotted against the length of treatment with ActD (B) or STS (D). Data represent the mean ± SD of three independent experiments. Mcl1 levels detected in untreated cells (time, 0 min) were set at 100%.CC BY-NC-SA · PubMed Central
FIGURE 4: p97 regulates the retrotranslocation of Mcl1 from the mitochondria to the cytosol. HeLa cells transfected with PAGFP-Mcl1 (A) or cotransfected with PAGFP-Mcl1 and p97QQ (B) were imaged over time, after whole imaging fields were photoactivated with a short pulse of UV light. Note the gradual decrease in mitochondrial PAGFP-Mcl1 fluorescence in cells expressing only PAGFP-Mcl1 (A) and the relatively stable mitochondrial PAGFP-Mcl1 fluorescence in cells expressing p97QQ (B). Cells are outlined in red. Bars in panels A and B: 20 μm. In panel C, the averages of mitochondrial PAGFP-Mcl1 fluorescence changes in numerous imaging experiments (see panel D) were plotted as a function of time. In panel D, mitochondrial PAGFP-Mcl1 fluorescence changes were quantified, and the mean ± SD and P values obtained with unpaired, two-tailed t-tests are shown (see figure for details).
FIGURE 4: p97 regulates the retrotranslocation of Mcl1 from the mitochondria to the cytosol. HeLa cells transfected with PAGFP-Mcl1 (A) or cotransfected with PAGFP-Mcl1 and p97QQ (B) were imaged over time, after whole imaging fields were photoactivated with a short pulse of UV light. Note the gradual decrease in mitochondrial PAGFP-Mcl1 fluorescence in cells expressing only PAGFP-Mcl1 (A) and the relatively stable mitochondrial PAGFP-Mcl1 fluorescence in cells expressing p97QQ (B). Cells are outlined in red. Bars in panels A and B: 20 μm. In panel C, the averages of mitochondrial PAGFP-Mcl1 fluorescence changes in numerous imaging experiments (see panel D) were plotted as a function of time. In panel D, mitochondrial PAGFP-Mcl1 fluorescence changes were quantified, and the mean ± SD and P values obtained with unpaired, two-tailed t-tests are shown (see figure for details).CC BY-NC-SA · PubMed Central
FIGURE 5: In vitro recapitulation of p97-dependent retrotranslocation of Mcl1. (A, B) Mitochondria-enriched heavy membrane (left panels) and cytosolic fractions (right panels) obtained from control and p97- and p97QQ-expressing cells treated with CHX for 0, 30, 60, or 90 min (A) or ActD for 0, 60, or 120 min were analyzed for levels of endogenous Mcl1 protein. Expression of exogenous p97 and p97QQ was detected with anti-MYC tag monoclonal antibody (MYC). Tom20 was used as a loading control. (C) Mitochondrial fractions isolated from p97 RNAi cells (lines 1–5) were combined with cytosolic fractions isolated from control RNAi (lines 1, 4, 5) or p97 RNAi cells (lines 2 and 3) and incubated at 35°C for 30 min (lines 2 and 4) or for 60 min (lines 3 and 5) or left on ice for 60 min (line 1). Following the incubation, samples were centrifuged (20,000 × g for 30 min), supernatants were removed, and pellets (15 μg of protein) were analyzed by Western blotting for levels of Mcl1 and p97. Tom20 was used as a loading control. (D) Cytosolic fractions isolated from control RNAi cells were incubated with ATP (lines 1 and 2), ATP-γ-S (lines 3), or MG132 (lines 4), and then combined with mitochondrial fractions obtained from p97 RNAi cells and incubated at 35°C for 60 min (lines 2–4) or left on ice for 60 min (line 1). Following incubation, samples were centrifuged (20,000 × g for 30 min), and supernatants (cytosolic fractions; 45 μg) and pellets (mitochondrial fractions; 15 μg) were analyzed by Western blotting for levels of Mcl1 and p97. Tom20 was used as a loading control. In panel E, the amount of p97 in cytosolic fractions obtained from control RNAi and p97 RNAi cells, which was used in experiments described in panel C, is shown. In panel F, quantification of Mcl1 levels in two independent experiments described in panels C and D is shown (data represent the mean ± SD). *, unknown/cross-reactive band detectable in cytosolic fractions incubated with anti-Mcl1 antibody.
FIGURE 5: In vitro recapitulation of p97-dependent retrotranslocation of Mcl1. (A, B) Mitochondria-enriched heavy membrane (left panels) and cytosolic fractions (right panels) obtained from control and p97- and p97QQ-expressing cells treated with CHX for 0, 30, 60, or 90 min (A) or ActD for 0, 60, or 120 min were analyzed for levels of endogenous Mcl1 protein. Expression of exogenous p97 and p97QQ was detected with anti-MYC tag monoclonal antibody (MYC). Tom20 was used as a loading control. (C) Mitochondrial fractions isolated from p97 RNAi cells (lines 1–5) were combined with cytosolic fractions isolated from control RNAi (lines 1, 4, 5) or p97 RNAi cells (lines 2 and 3) and incubated at 35°C for 30 min (lines 2 and 4) or for 60 min (lines 3 and 5) or left on ice for 60 min (line 1). Following the incubation, samples were centrifuged (20,000 × g for 30 min), supernatants were removed, and pellets (15 μg of protein) were analyzed by Western blotting for levels of Mcl1 and p97. Tom20 was used as a loading control. (D) Cytosolic fractions isolated from control RNAi cells were incubated with ATP (lines 1 and 2), ATP-γ-S (lines 3), or MG132 (lines 4), and then combined with mitochondrial fractions obtained from p97 RNAi cells and incubated at 35°C for 60 min (lines 2–4) or left on ice for 60 min (line 1). Following incubation, samples were centrifuged (20,000 × g for 30 min), and supernatants (cytosolic fractions; 45 μg) and pellets (mitochondrial fractions; 15 μg) were analyzed by Western blotting for levels of Mcl1 and p97. Tom20 was used as a loading control. In panel E, the amount of p97 in cytosolic fractions obtained from control RNAi and p97 RNAi cells, which was used in experiments described in panel C, is shown. In panel F, quantification of Mcl1 levels in two independent experiments described in panels C and D is shown (data represent the mean ± SD). *, unknown/cross-reactive band detectable in cytosolic fractions incubated with anti-Mcl1 antibody.CC BY-NC-SA · PubMed Central
FIGURE 6: Spatial relationship between p97 and the OMM. HeLa cells were immunostained to detect p97 (A and green fluorescence on overlay images shown in panel D) and Tom20, a marker of the OMM (B and red fluorescence on overlay images shown in panel D), followed by image acquisition and analysis using Zeiss AxioVision Colocalization software (Zeiss MicroImaging). The colocalizing pixels, representing the subcellular areas within a representative z-section containing both p97 and Tom20, are shown in panel C. The bottom images in panels A–D show magnified areas (bounded by squares). In panel E, line scans along the lines marked with I and II in the magnified areas taken from panels A–C are plotted (red lines represent Tom20 fluorescence pattern, green lines represent p97 fluorescence patterns, and blue represents colocalization of the green and red channels in pixels along lines I and II). Bar in D: 20 μm.
FIGURE 6: Spatial relationship between p97 and the OMM. HeLa cells were immunostained to detect p97 (A and green fluorescence on overlay images shown in panel D) and Tom20, a marker of the OMM (B and red fluorescence on overlay images shown in panel D), followed by image acquisition and analysis using Zeiss AxioVision Colocalization software (Zeiss MicroImaging). The colocalizing pixels, representing the subcellular areas within a representative z-section containing both p97 and Tom20, are shown in panel C. The bottom images in panels A–D show magnified areas (bounded by squares). In panel E, line scans along the lines marked with I and II in the magnified areas taken from panels A–C are plotted (red lines represent Tom20 fluorescence pattern, green lines represent p97 fluorescence patterns, and blue represents colocalization of the green and red channels in pixels along lines I and II). Bar in D: 20 μm.CC BY-NC-SA · PubMed Central

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  1. Bar-Nun S, 2005, Curr Top Microbiol Immunol, 300, 95

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Metadata

Authors
Xu, S., Peng, G., Wang, Y., Fang, S., & Karbowski, M.
Affiliations
University of Maryland, Baltimore (US)
Corresponding author
Karbowski, Mariusz
ORCID
Venue
Molecular Biology of the Cell
Publisher
American Society for Cell Biology (ASCB)
Published
2011-02
Volume
22
Issue
3
Pages
291-300
ISSN
  • 1059-1524 (ISSN-L)
  • 1939-4586
DOI
10.1091/mbc.e10-09-0748
PMC
PMC3031461
OpenAlex
W2082069715
Cited by
243
References
46
Citations by year
2026: 5 · 2025: 6 · 2024: 12 · 2023: 14 · 2022: 12 · 2021: 16 · 2020: 21 · 2019: 16 · 2018: 17 · 2017: 15 · 2016: 23 · 2015: 15 · 2014: 23 · 2013: 19 · 2012: 16
FWCI
6.26
Type
journal article
Language
en
Open access
Licence
cc-by-nc-sa
Locations
  • Molecular Biology of the Cell (publishedVersion)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by-nc-sa)
  • PubMed Central (publishedVersion, cc-by-nc-sa)
  • PubMed (publishedVersion)
Funders
National Institute of General Medical Sciences (R01GM083131)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Ubiquitin and proteasome pathways
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › Autophagy in Disease and Therapy
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Heat shock proteins research
Keywords
VCP, AAA+ ATPase, mitochondrial outer membrane, protein turnover, proteasomal degradation, Mcl-1, Mfn1, retrotranslocation, ubiquitin-proteasome system, mitochondrial protein degradation, ATPase domain
MeSH terms
Adenosine Triphosphatases, Cytosol, HeLa Cells, Humans, Nuclear Proteins, Recombinant Fusion Proteins, Protein Structure, Tertiary, Proto-Oncogene Proteins c-bcl-2, GTP Phosphohydrolases, Protein Transport, Mitochondrial Proteins, Membrane Transport Proteins, Mitochondrial Membrane Transport Proteins, Proteasome Endopeptidase Complex, Green Fluorescent Proteins, Mitochondrial Membranes, Protein Stability, Myeloid Cell Leukemia Sequence 1 Protein

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