Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2015;11(8):1216-29.
doi: 10.1080/15548627.2015.1017180.

Mitochondrial outer-membrane E3 ligase MUL1 ubiquitinates ULK1 and regulates selenite-induced mitophagy

Jie Li  1 , Wei Qi, Guo Chen, Du Feng, Jinhua Liu, Biao Ma, Changqian Zhou, Chenglong Mu, Weilin Zhang, Quan Chen, Yushan Zhu
Affiliations

Mitochondrial outer-membrane E3 ligase MUL1 ubiquitinates ULK1 and regulates selenite-induced mitophagy

Jie Li et al. Autophagy. 2015.

Abstract

Mitochondria serve as membrane sources and signaling platforms for regulating autophagy. Accumulating evidence has also shown that damaged mitochondria are removed through both selective mitophagy and general autophagy in response to mitochondrial and oxidative stresses. Protein ubiquitination through mitochondrial E3 ligases plays an integrative role in mitochondrial outer membrane protein degradation, mitochondrial dynamics, and mitophagy. Here we showed that MUL1, a mitochondria-localized E3 ligase, regulates selenite-induced mitophagy in an ATG5 and ULK1-dependent manner. ULK1 partially translocated to mitochondria after selenite treatment and interacted with MUL1. We also demonstrated that ULK1 is a novel substrate of MUL1. These results suggest the association of mitochondria with autophagy regulation and provide a new mechanism for the beneficial effects of selenium as a chemopreventive agent.

Keywords: E3 ligase; MUL1; ULK1; mitophagy; selenite.

PubMed Disclaimer

Figures

Figure 1
Figure 1
(See previous page). Identification of MUL1 in selenite-induced mitophagy. (A) HeLa cells transfected with siRNAs specifically _targeting RNF185, AMFR, MARCH5 or MUL1 were treated with 5 μM of selenite for 12 h, followed by western blot analysis of the indicated proteins. (B) Quantitative analysis of the TOMM20 protein level described in (A). TOMM20 protein level was determined by dividing the intensity of TOMM20 with the intensity of tubulin on the blot. (The intensity of bands was measured with ImageJ software; mean ±SEM, from 3 independent experiments, 2-way ANOVA, ***P < 0.001, N.S., not significant.) (C) HeLa cells transfected with siMUL1 or scrambled RNA (Scr) were treated with the indicated stresses (5 μM FCCP, 6 h; EBSS, 6 h; hypoxia with 1% O2, 12 h), followed by the analysis of the indicated protein levels. (D) Quantitative analysis of the TOMM20 protein level described in (C). TOMM20 protein level was determined by dividing the intensity of TOMM20 with the intensity of tubulin on the blot (the intensity of bands was measured with ImageJ software; mean ±SEM, from 3 independent experiments, 2-way ANOVA, ***P < 0.001). (E) HeLa cells expressing GFP-LC3 were transfected with scrambled RNA or siMUL1-CY5, followed by treatment with 5 μM of selenite for 12 h and immunofluorescence microscopy to detect GFP-LC3 puncta. (F) Quantification of GFP-LC3 punctate structures associated with the mitochondria (TOMM20) described in (E) (mean ±SEM; n = 50 cells from 3 independent experiments, one-way ANOVA, **P < 0.01, ***P < 0.001). (G) HeLa cells transfected with MUL1-MYC were treated with 5 μM of selenite for 12 h with or without 10 nM BAF, 5 μM MG132 or 10 mg/mL pepstatin for 6 h and subjected to western blotting analysis of the indicated protein levels. (H) Quantitative analysis of the TOMM20 protein level described in (G). TOMM20 protein level was determined by dividing the intensity of TOMM20 with the intensity of tubulin on the blot (The intensity of bands was measured with Image J software. mean ±SEM, from 3 independent experiments, 2-way ANOVA, *P < 0.05, N.S., not significant). (I) HeLa cells were overexpressed with MUL1-MYC for 24 h, and then the samples were analyzed by electron microscopy. Arrows indicate that mitochondria are enclosed within autophagosomes.
Figure 2
Figure 2
(See previous page). Both ULK1 and ATG5 are required for selenite-induced mitophagy. (A) HeLa cells with MUL1 was knocked down were rescued by exogenous wild-type MYC-tagged MUL1, followed by treatment with selenite for 12 h before western blotting analysis of the indicated proteins. (B) Wild-type MEFs, Atg5−/−, Ulk1−/− and park2−/− MEFs transfected with GFP-LC3 were treated with or without 5 μM of selenite for 12 h, followed by staining with MitoTracker Red for mitochondria and immunofluorescence microscopy to visualize GFP-LC3 puncta. (C) Quantification of GFP-LC3 punctate structures associated with mitochondria (TOMM20) described in (B) (mean ±SEM; n = 50 cells from 3 independent experiments, one-way ANOVA, ***P < 0.001, N.S., not significant). (D) The MEFs cells described in (B) were treated with or without selenite for 12 h, followed by subjected to western blotting analysis of the indicated proteins.
Figure 3
Figure 3
(See previous page). MUL1 interacts with the ULK1 kinase in mammalian cells. (A) HeLa cells transfected with GFP-ULK1 (Green) were treated with 5 μM of selenite for the indicated time followed by staining with MitoTracker Red for mitochondria and DAPI (blue) for the nucleus. (B) HeLa cells were treated with 5 μM of selenite for the indicated times. Mitochondria were isolated and subsequently subjected to western blotting analysis of the indicated proteins. (C) Immunoprecipitation was performed with an ULK1 antibody. Coimmunoprecipitated endogenous MUL1 was detected through western blotting with an anti-MUL1 antibody. (D) ULK1-MYC was transfected into HEK 293T cells, and immunoprecipitation was performed with an anti-MYC antibody. Coimmunoprecipitated endogenous MUL1 was detected through western blotting with an anti-ULK1 antibody. (E) MUL1-MYC was transfected into HEK 293T cells, and immunoprecipitation was performed with an anti-MYC antibody. Coimmunoprecipitated endogenous ULK1 was detected through western blotting with an anti-ULK1 antibody. (F) HeLa cells transfected with MYC-vector or MUL1-MYC were treated with selenite for the indicated time and immunoprecipitation was performed with an anti-MYC antibody. Coimmunoprecipitated endogenous ULK1 level was detected through western blotting with an anti-ULK1 antibody. (G) Purified GST and GST-tagged MUL1 protein were used for the GST affinity isolation of endogenous ULK1, and blotted with an anti-ULK1 antibody. (H) GFP-MUL1 and ULK1-MYC were cotransfected with Flag-ATG13 or Flag vector, and immunoprecipitation was performed with an anti-Flag antibody. Coimmunoprecipitated ULK1 and MUL1 were detected through western blotting with anti-GFP and anti-MYC antibodies respectively. (I) Truncated forms of ULK1-MYC were constructed based on its functional domains. GFP-MUL1 was cotransfected with full-length or truncated forms of ULK1-MYC, and immunoprecipitation was performed with an anti-MYC antibody. Coimmunoprecipitated MUL1 was detected using an anti-GFP antibody. (J) Ulk1−/− MEFs were transfected with exogenous wild-type ULK1 or truncation ULK1Δ CTD, followed by treatment with selenite for 12 h before western blotting analysis of the indicated proteins.
Figure 4.
Figure 4.
Overexpression of MUL1 and treatment with selenite promotes ULK1 degradation through the proteasome pathway. (A) HeLa cells were treated with CHX (10 μM, 12 h) and selenite (5 μM) for the indicated time, with or without MG132, and subjected to western blotting analysis of the ULK1. (B) Quantification of ULK1 protein levels in (A) (mean ±SEM, from 3 independent experiments). (C) HeLa cells were treated with the indicated agents (FCCP 5 μM; hypoxia with 1% O2; selenite 5 μM), and then subjected to western blotting analysis of ULK1 (The intensity of indicated bands was measured with ImageJ software). (D) After transfection with plasmids as indicated, HeLa cells were treated with BAF (10 nM, 6 h) or MG132 (5 μM, 6 h) prior to harvesting, followed by western blotting analysis of the GFP-ULK1 level. (E) After transfection with MUL1-MYC as indicated, HeLa cells were treated with BAF (10 nM, 6 h) or MG132 (5 μM, 6 h), followed by western blotting analysis of the ULK1 protein level. (F) HeLa cells were transfected with MUL1-MYC or MUL1ΔR-MYC (MUL1 with absence of the RING finger domain) for 24 h, and subjected to western blotting analysis of the indicated protein levels (The intensity of indicated bands was measured with ImageJ software). (G) HeLa cells transfected with MUL1-MYC for 24 h, followed by treatment with CHX (10 μM) for the indicated time, with or without MG132, and subjected to western blotting analysis of the ULK1 protein level. (H) Quantification of ULK1 protein levels in (G). Mean ±SEM, from 3 independent experiments.
Figure 5
Figure 5
(See previous page). MUL1 promotes ubiquitination of ULK1. (A) HEK 293T cells were transfected with MUL1-MYC, MUL1ΔR-MYC, or the empty MYC-vector together with GFP-ULK1 and HA-Ub. Ubiquitination assays were performed as described in Materials and Methods. The ubiquitination level of GFP-ULK1 was detected using an anti-HA antibody. (B) In vitro ubiquitination assays were performed as described in Materials and Methods. The ubiquitinated form of ULK1 was immunoblotted using an anti-ULK1 antibody. (C) MUL1 knockdown cells transfected with GFP-ULK1 were subjected to ubiquitination assays for analysis with an anti-Ub antibody. (D) Quantitative analysis of ubiquitinated GFP-ULK1 level as described in (C) (The intensity of bands was measured with Image J software. mean±SEM; from 3 independent experiments). (E) HEK 293T cells were transfected with the indicated plasmids for 24 h, and subsequently a ubiquitination assay was performed for analysis with an anti-Ub antibody. (F) HeLa cells transfected with GFP-ULK1 were treated with 5 μM of selenite for the indicated time, followed by ubiquitination assays for analysis with an anti-Ub antibody. (G) HeLa cells were treated with 5 μM of selenite for the indicated time, followed by ubiquitination assays for analysis with an anti-Ub antibody. (H) Scrambled RNA- or siMUL1-transfected cells were treated with or without selenite for 12 h, followed by ubiquitination assays for analysis with an anti-Ub antibody. (I) NIH-3T3 cells were treated with 5 μM of selenite for the indicated time, followed by ubiquitination assays for analysis with an anti-Ub antibody. (J) NIH-3T3 cells were treated with selenite for the indicated time, with or without MG132, and subjected to western blotting analysis of the ULK1 protein level.
Figure 6.
Figure 6.
MUL1 response to selenite-induced ROS stress depending on conserved cysteines 62 and 87. (A) HeLa cells treated with selenite as indicated were stained with MitoSox Red and subjected to flow cytometry. (B) HeLa cells were pretreated with NAC (10 mM) or GSH-EE (10 mM) for 30 min, followed by treatment with 5 μM of selenite for 12 h. Cell lysates were subjected to western blotting analysis of the indicated proteins. (C) HeLa cells transfected with GFP-ULK1 were pretreated with NAC (10 mM) or GSH-EE (10 mM) for 30 mins, followed by treatment of selenite (12 h). The ubiquitination assays were performed as described in Materials and Methods. (D) Alignment of the MUL1 amino acids in different species. (E) HeLa cells with MUL1 knockdown were transfected with exogenous MYC-tagged wild-type MUL1 or MUL1 with cysteine mutations (C62S, C87S), followed by treatment with selenite (5 μM, 12 h) before western blotting analysis of the indicated proteins (The intensity of the indicated bands was measured with ImageJ software.).
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Wallace DC, Brown MD, Melov S, Graham B, Lott M. Mitochondrial biology, degenerative diseases and aging. BioFactors 1998; 7:187-90; PMID:9568243; http://dx.doi.org/10.1002/biof.5520070303 - DOI - PubMed
    1. Green DR, Reed JC. Mitochondria and apoptosis. Science 1998; 281:1309-12; PMID:9721092; http://dx.doi.org/10.1126/science.281.5381.1309 - DOI - PubMed
    1. Zhang H, Bosch-Marce M, Shimoda LA, Tan YS, Baek JH, Wesley JB, Gonzalez FJ, Semenza GL. Mitochondrial autophagy is an HIF-1-dependent adaptive metabolic response to hypoxia. J Biol Chem 2008; 283:10892-903; PMID:18281291; http://dx.doi.org/10.1074/jbc.M800102200 - DOI - PMC - PubMed
    1. Russell RC, Yuan HX, Guan KL. Autophagy regulation by nutrient signaling. Cell Res 2014; 24:42-57; PMID:24343578; http://dx.doi.org/10.1038/cr.2013.166 - DOI - PMC - PubMed
    1. Egan DF, Shackelford DB, Mihaylova MM, Gelino S, Kohnz RA, Mair W, Vasquez DS, Joshi A, Gwinn DM, Taylor R, et al. . Phosphorylation of ULK1 (hATG1) by AMP-activated protein kinase connects energy sensing to mitophagy. Science 2011; 331:456-61; PMID:21205641; http://dx.doi.org/10.1126/science.1196371 - DOI - PMC - PubMed

Publication types

MeSH terms

  NODES
Association 1
twitter 2