Regulated degradation of the inner nuclear membrane protein SUN2 maintains nuclear envelope architecture and function
- Sir William Dunn School of Pathology, University of Oxford, United Kingdom
- Nuffield Department of Medicine, Target Discovery Institute, University of Oxford, United Kingdom
Figures
Figure 1 with 1 supplement
SUN2 stability depends on a non-canonical recognition motif for the SCFβTrCP ubiquitin ligase.
(A) SUN2 nucleoplasmic regions containing non-canonical recognition motifs for the SCFβTrCP hereafter called Sites 1 and 2. In each case, the residues mutated to Alanine (purple) or to Aspartate …
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Figure 1—source data 1
File contains original immunoblots for Figure 1E.
- https://cdn.elifesciences.org/articles/81573/elife-81573-fig1-data1-v2.zip
Figure 1—figure supplement 1
Evolutionary conservation analysis of SUN2 protein sequence.
(A) Schematic of human SUN2 domain organization. (B) Multiple sequence analysis of SUN2 Site 1 (top) and Site 2 (bottom) among primates (chimpanzee, rhesus, and green monkeys), other mammals (mouse …
Figure 2 with 1 supplement
Inner nuclear membrane (INM) degradation of SUN2 by an ERAD-like process.
(A) Flow cytometry analysis of tetracycline-induced expression of SUN2 WT, Sites 2A and 2D in HEK TF cells. Analysis was performed 24 hr post-induction in cells left untreated, incubated 4 hr with …
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Figure 2—source data 1
File contains original immunoblots for Figure 2B.
- https://cdn.elifesciences.org/articles/81573/elife-81573-fig2-data1-v2.zip
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Figure 2—source data 2
File contains original immunoblots for Figure 2C.
- https://cdn.elifesciences.org/articles/81573/elife-81573-fig2-data2-v2.zip
Figure 2—figure supplement 1
Depletion of β-TrCP1/2 stabilizes SUN2 Site 2D.
(A) Western blot analysis of SUN2 Site 2D steady-state levels in HEK TF cells transfected with an empty vector (EV), sgRNAs against βTrCP1 (three independent gRNAs), βTrCP2 (two independent gRNAs), …
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Figure 2—figure supplement 1—source data 1
File contains original immunoblots for Figure 2—figure supplement 1A.
- https://cdn.elifesciences.org/articles/81573/elife-81573-fig2-figsupp1-data1-v2.zip
Figure 3
Site 2 is critical for SUN2 recognition and ubiquitination by SCFβTrCP.
(A) Binding of WT and the indicated SUN2 mutants to subunits of the ubiquitin ligase SCFβTrCP and the ATPase p97 analyzed by immunoprecipitation. Expression of SUN2 derivatives was induced with Dox …
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Figure 3—source data 1
File contains original immunoblots for Figure 3A.
- https://cdn.elifesciences.org/articles/81573/elife-81573-fig3-data1-v2.zip
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Figure 3—source data 2
File contains original immunoblots for Figure 3B.
- https://cdn.elifesciences.org/articles/81573/elife-81573-fig3-data2-v2.zip
Figure 4 with 1 supplement
Genome-wide screening identifies Casein Kinase 2 as a positive regulator of SUN2 degradation.
(A) Workflow of the CRISPR-Cas9 genome-wide screen. (B) Significance score of the genes analyzed in the screen calculated by the MAGeCK algorithm. The x-axis represents the genes in alphabetical …
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Figure 4—source data 1
File contains original immunoblots for Figure 4D.
- https://cdn.elifesciences.org/articles/81573/elife-81573-fig4-data1-v2.zip
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Figure 4—source data 2
File contains original immunoblots for Figure 4E.
- https://cdn.elifesciences.org/articles/81573/elife-81573-fig4-data2-v2.zip
Figure 4—figure supplement 1
Analysis of genome-wide CRISPR/Cas9 genetic screening.
(A) Significance score of the genes analyzed in the genome-wide CRISPR/Cas9 genetic screen calculated by the MAGeCK algorithm as done in Figure 5 for additional timepoints at days 13 (top) and 17 …
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Figure 4—figure supplement 1—source data 1
File contains original immunoblots for Figure 4—figure supplement 1.
- https://cdn.elifesciences.org/articles/81573/elife-81573-fig4-figsupp1-data1-v2.zip
Figure 5 with 1 supplement
The phosphatase CTDNEP1 is a negative regulator of SUN2 degradation.
(A) Steady-state levels of endogenous SUN2 in HEK TF transfected with an empty vector (EV) or sgRNAs targeting CTDNEP1. Cell extracts were analyzed by sodium dodecyl sulfate–polyacrylamide gel …
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Figure 5—source data 1
File contains original immunoblots for Figure 5A.
- https://cdn.elifesciences.org/articles/81573/elife-81573-fig5-data1-v2.zip
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Figure 5—source data 2
File contains original immunoblots for Figure 5C.
- https://cdn.elifesciences.org/articles/81573/elife-81573-fig5-data2-v2.zip
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Figure 5—source data 3
File contains original immunoblots for Figure 5E.
- https://cdn.elifesciences.org/articles/81573/elife-81573-fig5-data3-v2.zip
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Figure 5—source data 4
File contains original immunoblots for Figure 5G.
- https://cdn.elifesciences.org/articles/81573/elife-81573-fig5-data4-v2.zip
Figure 5—figure supplement 1
Characterization of the CTDNEP1 role in SUN2 degradation.
(A) Immunofluorescence in parental and CTDNEP1 KO HeLa cells. Expression of SUN2 Site 2D was induced with Dox for 24 hr. Endogenous Lamin-B receptor (LBR) was detected with anti-LBR antibody. DNA …
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Figure 5—figure supplement 1—source data 1
File contains original immunoblots for Figure 5—figure supplement 1B.
- https://cdn.elifesciences.org/articles/81573/elife-81573-fig5-figsupp1-data1-v2.zip
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Figure 5—figure supplement 1—source data 2
File contains original immunoblots for Figure 5—figure supplement 1C.
- https://cdn.elifesciences.org/articles/81573/elife-81573-fig5-figsupp1-data2-v2.zip
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Figure 5—figure supplement 1—source data 3
File contains original immunoblots for Figure 5—figure supplement 1D.
- https://cdn.elifesciences.org/articles/81573/elife-81573-fig5-figsupp1-data3-v2.zip
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Figure 5—figure supplement 1—source data 4
File contains immunoblots for Figure 5—figure supplement 1F.
- https://cdn.elifesciences.org/articles/81573/elife-81573-fig5-figsupp1-data4-v2.zip
Figure 6 with 1 supplement
Accumulation of non-degradable SUN2 results in aberrant nuclear architecture and function.
(A) Micrographs of parental HeLa or cells expressing SUN2 WT or Site 2A. Cells were analyzed 24 hr after inducing SUN2 expression (left) or 24 hr after turning off SUN2 expression (right). SUN2 was …
Figure 6—figure supplement 1
Analysis of the accumulation of non-degradable SUN2 in mitotic progression.
(A) Flow cytometry analysis of transgenic SUN2 steady-state levels in HEK TF-expressing SUN2 WT (left) or Site 2A (right) either without (0 hr) or upon shut-off of the transgenes with washing out …
Figure 7
A kinase-phosphatase balance regulates ER-associated degradation of SUN2 from the inner nuclear membrane (INM).
The scheme illustrates the opposing effects of the kinase CK2 and the phosphatase CTDNEP1 on SUN2 degradation. Phosphorylation of SUN2 Site 2 promotes the binding of SCFβTrCP ubiquitin ligase and …
Author response image 1
