Selective clearance of the inner nuclear membrane protein emerin by vesicular transport during ER stress

  1. Abigail Buchwalter  Is a corresponding author
  2. Roberta Schulte
  3. Hsiao Tsai
  4. Juliana Capitanio
  5. Martin Hetzer  Is a corresponding author
  1. University of California, San Francisco, United States
  2. Chan Zuckerberg Biohub, United States
  3. The Salk Institute for Biological Studies, United States
9 figures, 1 table and 6 additional files

Figures

Figure 1 with 1 supplement
Dynamic proteomic analysis of inner nuclear membrane protein turnover.

(A) Diagram of the ER with associated ribosomes, the NE composed of the ONM and INM, the NPCs, and the underlying nuclear lamina. INM proteins are synthesized in the ER, pass through the NPC, and …

https://doi.org/10.7554/eLife.49796.002
Figure 1—figure supplement 1
Example half life fits.

Examples of half-life fits for proteins with predicted half-lives of 0.5 days (A), 1 day (B), 2 days (C), 4 days (D), 8 days (E), and 17 days (F).

https://doi.org/10.7554/eLife.49796.003
Figure 2 with 1 supplement
RITE analysis of INM proteins enables visualization of proteasome-dependent turnover.

RITE analysis of INM proteins corroborates protein turnover determined by proteomics. (A) Schematic of recombination-induced tag exchange (RITE) expression cassette for visualizing protein turnover …

https://doi.org/10.7554/eLife.49796.004
Figure 2—figure supplement 1
Identification of a disease-linked emerin mutant with normal NE localization.

(A) EMDΔ95–99-GFP (EMDΔ-GFP) has normal localization at the NE (B) and normal residence time at the NE, based on fluorescence recovery after photobleaching timecourse at the NE (C,D).

https://doi.org/10.7554/eLife.49796.005
Figure 3 with 3 supplements
Acute stressors destabilize mutant emerin protein levels.

(A) C2C12 cells stably expressing EMDΔ-GFP and treated with DMSO vehicle control, CHX alone, CHX and MG132, or MG132 alone for 8 hours. All images were acquired using the same laser power and …

https://doi.org/10.7554/eLife.49796.006
Figure 3—figure supplement 1
Localization and stability of a disease-linked emerin variant.

(A–B) C2C12 cells stably expressing EMDΔ-GFP (A) or EMD-WT-GFP (B) and treated with DMSO vehicle control, CHX alone, CHX and MG132, or MG132 alone for 8 hr. All images were acquired using the same …

https://doi.org/10.7554/eLife.49796.007
Figure 3—figure supplement 2
siRNA-mediated E2 or E3 ubiquitin ligase knockdowns do not stabilize EMDΔ-GFP.

(A–B) EMDΔ-GFP protein levels do not change in C2C12 cells stably expressing EMDΔ-GFP and transfected in duplicate with 50 nM RNAi targeting the E3 ubiquitin ligases Rnf26, CGRRF1, MARCH6, or a …

https://doi.org/10.7554/eLife.49796.008
Figure 3—figure supplement 3
Glycosylation reporter variants are destabilized by ER stress and recovered by BFA treatment.

Glycosylation reporter variant of EMDΔ-GFP localizes normally to the NE and responds to ER stress induced by THG and secretory pathway disruption caused by BFA (A–B). (C) Pattern of glycosylation …

https://doi.org/10.7554/eLife.49796.009
Figure 4 with 1 supplement
Stress-induced clearance of mutant emerin from the ER and NE involves the secretory pathway.

(A-C) Representative confocal slices of cells stably expressing EMDΔ-GFP, treated with DMSO or THG for the indicated times and costained for giantin to mark the Golgi (magenta). All images were …

https://doi.org/10.7554/eLife.49796.010
Figure 4—figure supplement 1
Colocalization of emerin with the Golgi.

(A–B) Representative confocal slices of cells stably expressing EMD-WT-GFP, treated with DMSO or THG for the indicated times and costained for giantin to mark the Golgi (magenta). All images were …

https://doi.org/10.7554/eLife.49796.011
Mutant emerin trafficking is dependent on lysosomal but not autophagosomal function.

(A) Representative confocal slices of cells stably expressing EMDΔ-GFP after 8 hr of treatment with DMSO vehicle control, THG, co-treatment with THG and BFA, or co-treatment with THG and KU55933. …

https://doi.org/10.7554/eLife.49796.012
Figure 6 with 1 supplement
Mutant emerin traffics through the plasma membrane upon ER stress.

(A) Schematic of antibody uptake assay experimental design. If emerin accesses the plasma membrane (PM), it will be detected by anti-GFP antibody (green), which will bind the surface-exposed GFP …

https://doi.org/10.7554/eLife.49796.013
Figure 6—figure supplement 1
Access of emerin to the plasma membrane.

(A) Uptake of anti-GFP antibody (magenta) by cells stably expressing EMD-WT-GFP and treated with DMSO vehicle control or THG for the indicated times. Cells were incubated with anti-GFP antibody for …

https://doi.org/10.7554/eLife.49796.014
Figure 7 with 1 supplement
Emerin, but not other INM proteins, undergoes stress-dependent clearance from the NE and ER and accesses lysosomes.

(A-C) Representative confocal slices of cells stably expressing NRM-GFP (A), Sun2-GFP (B), or EMD-GFP (C) after 16 hr of treatment with DMSO vehicle control, THG, or co-treatment with THG and BFA. …

https://doi.org/10.7554/eLife.49796.015
Figure 7—figure supplement 1
Glycosylation reporter variants are destabilized by ER stress and recovered by BFA treatment.

(A–B) Glycosylation reporter variant of EMD-WT-GFP localizes normally to the NE and responds to ER stress induced by THG and secretory pathway disruption caused by BFA.

https://doi.org/10.7554/eLife.49796.016
Figure 8 with 2 supplements
A signal within emerin’s.

LEM domain is required for stress-dependent clearance from the NE and ER (A) Diagram of emerin domain organization with N-terminal LEM domain deletion indicated (amino acids 1-45). (B-C) …

https://doi.org/10.7554/eLife.49796.017
Figure 8—figure supplement 1
Stability of EMDΔLEM-GFP over time of cycloheximide treatment.

Western blot detection of EMDΔLEM-GFP protein levels in cells treated with DMSO vehicle control or CHX for the indicated times.

https://doi.org/10.7554/eLife.49796.018
Figure 8—figure supplement 2
Emerin is mislocalized to the ER, but not degraded in lmna - /- MEFs.

(A) representative immunostaining of WT MEFs or of lmna - /- MEFs with lamin A antibody (red) and Hoechst (blue). (B) Representative immunofluorescence of EMD-GFP stably expressed in WT MEFs (top …

https://doi.org/10.7554/eLife.49796.019
Competition model for emerin sorting via its LEM domain competitively binding to BAF or to the ER export machinery.
https://doi.org/10.7554/eLife.49796.020

Tables

Key resources table
Reagent
type (species)
or resource
DesignationSource or
reference
IdentifiersAdditional
information
Gene (Mus musculus)emerinNCBI RefSeq NM_007927
Gene (Mus musculus)nurimNCBI RefSeq NM_134122
Gene (Mus musculus)Sun2NCBI RefSeq NM_001205346
Cell line (Mus musculus)C2C12ATCCCRL-1772
Cell line (Homo sapiens)U-2-OSATCCHTB-96
Recombinant DNA reagent (plasmid)pQCXIB vectorCampeau et al. (2009) AddgeneRetroviral construct for stable expression
Recombinant DNA reagent (plasmid)Myc/FLAG RITE vectorToyama et al. (2019)Lentiviral contruct for stable expression of RITE-tagged protein
Recombinant DNA reagent (plasmid)pQCXIB emerin-GFPThis paperRetroviral construct for stable expression
Recombinant DNA reagent (plasmid)pQCXIB emerin-D95-99-GFPThis paperRetroviral construct for stable expression
Recombinant DNA reagent (plasmid)pQCXIB emerin-DLEM-GFPThis paperRetroviral construct for stable expression
Recombinant DNA reagent (plasmid)pQCXIB emerin-GFP-SSNKTVDThis paperRetroviral construct for stable expression
Recombinant DNA reagent (plasmid)pQCXIB emerin-Δ95–99-GFP-SSNKTVDThis paperRetroviral construct for stable expression
Recombinant DNA reagent (plasmid)pQCXIB emerin-ΔLEM-GFP-SSNKTVDThis paperRetroviral construct for stable expression
Recombinant DNA reagent (plasmid)pQCXIB Sun2-GFPThis paperRetroviral construct for stable expression
Recombinant DNA reagent (plasmid)pQCXIB nurim-GFPThis paperRetroviral construct for stable expression
Recombinant DNA reagent (plasmid)Emerin-RITEThis paperLentiviral contruct for stable expression of RITE-tagged protein
Recombinant DNA reagent (plasmid)Nurim-RITEThis paperLentiviral contruct for stable expression of RITE-tagged protein
Recombinant DNA reagent (plasmid)Emerin-Δ95–99-RITEThis paperLentiviral contruct for stable expression of RITE-tagged protein
AntibodyRabbit polyclonal anti-emerinSanta Cruz BiotechnologySc-15378WB (1:1000)
AntibodyGFPAbcamab290Ab uptake (1:500); WB (1:1000)
AntibodyMouse monoclonal anti-FLAGSigma-AldrichF1804IF (1:1000)
AntibodyMouse monoclonal anti-MycCell Signaling2233IF (1:1000); Ab uptake (1:500)
AntibodyMouse monoclonal anti-tubulinSigma-AldrichT5168WB (1:2500)
AntibodygiantinBioLegendPRB-114CIF (1:1000)
AntibodyLAMP1Abcamab24170IF(1:100)
OtherAlexa-647 WGALife TechnologiesW32466IF (5 ug/ml)
Commercial assay or kitPNGase FNEBP0704
Commercial assay or kitEndo HNEBP0702
Chemical compound, drugThapsigarginThermo FisherT7459Used at 100 nM
Chemical compound, drugMG132Cayman Chemical1211877-36-9Used at10 uM
Chemical compound, drugBafilomycin A1BioVioticaBVT-0252Used at100 nM
Chemical compound, drugBrefeldin ATocris1231Used at2.5 uM
Chemical compound, drugLeupeptinSigma-AldrichL5793Used at 125 uM
Chemical compound, drugcycloheximideSigma-AldrichC-7698Used at200 ug/ml
Other13C6-LysineCambridge IsotopesCLM-2247
Other13C6, 15N4-ArginineCambridge IsotopesCNLM-539
OtherLysine/arginine free DMEMThermo Fisher88364
OtherDialyzed fetal bovine serumThermo Fisher26400044
OtherHoechst stainMolecular ProbesH1399Used at 10 ug/ml
Recombinant DNA reagent (plasmid)UBE2G1 miR-E LT3GEPIRKnott et al., 2014TGCTGTTGACAGTGAGCGAAAGACAGCTGGCAGAACTCAATAGTGAAGCCACAGATGTATTGAGTTCTGCCAGCTGTCTTCTGCCTACTGCCTCGGA
Recombinant DNA reagent (plasmid)UBE2G2 miR-E LT3GEPIRKnott et al., 2014TGCTGTTGACAGTGAGCGAACCGGGAGCAGTTCTATAAGATAGTGAAGCCACAGATGTATCTTATAGAACTGCTCCCGGTCTGCCTACTGCCTCGGA
Recombinant DNA reagent (plasmid)UBE2J1 miR-E LT3GEPIRKnott et al., 2014TGCTGTTGACAGTGAGCGAAAGGTTGTCTACTTCACCAGATAGTGAAGCCACAGATGTATCTGGTGAAGTAGACAACCTTCTGCCTACTGCCTCGGA
Recombinant DNA reagent (plasmid)MARCH6 miR-E LT3GEPIRKnott et al., 2014TGCTGTTGACAGTGAGCGACTGGATCTTCATTCTTATTTATAGTGAAGCCACAGATGTATAAATAAGAATGAAGATCCAGCTGCCTACTGCCTCGGA
Software, algorithmFijihttps://fiji.sc/
Software, algorithmRStudiohttps://rstudio.com/

Additional files

Source data 1

Filtered peptide data for half life calculations.

Peptide turnover data for all peptides passing quality control filters. See R script and Materials and methods for details.

https://doi.org/10.7554/eLife.49796.021
Source data 2

Filtered protein data for half life calculations.

Filtered and averaged protein turnover data. See R script and Materials and methods for details.

https://doi.org/10.7554/eLife.49796.022
Supplementary file 1

Results of half life fits passing quality filters.

https://doi.org/10.7554/eLife.49796.023
Supplementary file 2

Complete list of half life fits.

https://doi.org/10.7554/eLife.49796.024
Supplementary file 3

Half lives and protein topology data.

Selected data related to Figure 1G-H.

https://doi.org/10.7554/eLife.49796.025
Transparent reporting form
https://doi.org/10.7554/eLife.49796.026

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