Shared and Distinctive Neighborhoods of Emerin and Lamin B Receptor Revealed by Proximity Labeling and Quantitative Proteomics

Abstract

Emerin and lamin B receptor (LBR) are abundant transmembrane proteins of the nuclear envelope that are concentrated at the inner nuclear membrane (INM). Although both proteins interact with chromatin and nuclear lamins, they have distinctive biochemical and functional properties. Here, we have deployed proximity labeling using the engineered biotin ligase TurboID (TbID) and quantitative proteomics to compare the neighborhoods of emerin and LBR in cultured mouse embryonic fibroblasts. Our analysis revealed 232 high confidence proximity partners that interact selectively with emerin and/or LBR, 49 of which are shared by both. These included previously characterized NE-concentrated proteins, as well as a host of additional proteins not previously linked to emerin or LBR functions. Many of these are TM proteins of the ER, including two E3 ubiquitin ligases. Supporting these results, we found that 11/12 representative proximity relationships identified by TbID also were detected at the NE with the proximity ligation assay. Overall, this work presents methodology that may be used for large-scale mapping of the landscape of the INM and reveals a group of new proteins with potential functional connections to emerin and LBR.

Keywords: TurboID; emerin; inner nuclear membrane; lamin B receptor; nuclear envelope; nuclear lamina; proximity labeling.

Figure 1

Proximity labeling strategy to investigate the neighborhoods of emerin and LBR using TbID fusions. (A) Schematic diagram of the NE, illustrating the continuity of the INM and ONM at the NPC, and the contiguity of the ONM with the peripheral ER. Ectopically expressed constructs with TbID fused to the N-terminus of emerin (Emd-TbID) or LBR (LBR-TbID) were concentrated at the INM as depicted but also were located in the peripheral ER and other endomembranes at a lower concentration (not shown). Unfused TbID lacking a TM domain, which served as a control, is distributed throughout the nucleoplasm and cytoplasm. The NL is indicated by green stipple. Both emerin and LBR are predicted to contain multiple intrinsically disordered regions in their N-terminal nucleoplasmic domains (emerin, aa 1–223; LBR, aa 1–221; UniProtKB). (B) Western blots of parental MEFs or MEFs stably expressing V5-tagged TbID, TbID-Emd, or TbID-LBR as indicated. Blots were probed with anti-V5 tag or anti-actin (left panels), anti-emerin (middle panel), or anti-LBR (right panel). (C) Immunofluorescence micrographs of MEFs stably expressing TbID constructs (panel B) that had been incubated with exogenous biotin for 2 h and stained as indicated to detect the V5 tag, biotin (streptavidin), or DNA (DAPI). Merged images, right panels. Bar, 1 μm. (D) Western blots of parental MEFs or MEFs stably transduced with TbID constructs as indicated. Cell samples were incubated without (−) or with (+) 500 uM biotin for 2 h prior to probing with streptavidin or anti-actin, as indicated.

Figure 2

Summary of results from analysis of proximity samples by TMT labeling and proteomics. (A) Workflow depicting steps in the analysis (see text for details). HCPP are streptavidin-enriched proteins from the bait-TbID samples that were detected with at least two unique peptides and that showed at least 3-fold enrichment with p < 0.05, in comparison to unfused TbID samples. (B) Venn diagrams illustrating HCPP selectively labeled by Emd-TbID or LBR-TbID and overlap between the groups. (C) Pie charts depicting the subcellular locations of HCPP labeled by Emd-TbID or LBR-TbID. (D) Gene annotation summaries (Biological Process and Molecular Function) of HCPP labeled by Emd-TbID or LBR-TbID.

Figure 3

HCPP proteins labeled by Emd-TbID or LBR-TbID. (A) Volcano plot describing preferential labeling of HCPP by Emd-TbID vs LBR-TbID. Prey analyzed in Figures 4 and 5 are labeled. (B) HCPP from a group of well-characterized NE proteins².^, Fold-enrichment of hits from Emd-TbID (purple) or LBR-TbID (green) samples, relative to unfused TbID is indicated on the bottom. Left: NPC proteins are depicted in blue; NL proteins in black.

Figure 4

Proximity ligation analysis (PLA) of representative HCPP among well-characterized NE-associated proteins. (A) Summary of proximity labeling data by Emd-TbID and LBR-TbID, and results of the PLA analysis, from the group of HCPP analyzed. FC, fold change. Yellow shading, HCPP for emerin and/or LBR. (B) Representative immunofluorescence images describing PLA obtained for cells stably expressing Myc-MBP, Myc-Emd, or Myc-LBR and transiently transduced with V5-tagged Cbx3 (left block of images) or Tmem43 (right block of images). First columns, anti-V5 staining; middle columns, PLA signal; right columns, merged imaged. Bars, 5 μm. (C) Graphs depicting specific PLA signal obtained for samples in (A) with either low V5 expression (lower 50th percentile) or high V5 expression (upper 50th percentile). *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 5

PLA of HCPP without known links to emerin or LBR. (A) Summary of proximity labeling data by Emd-TbID and LBR-TbID, and results of the PLA analysis, from HCPP analyzed. FC, fold change. Yellow shading, HCPP for emerin and/or LBR. (B) Representative immunofluorescence images describing PLA obtained for cells stably expressing Myc-MBP, Myc-Emd, or Myc-LBR and transiently transduced with V5-tagged Reep3 (left block of images) or Cgrrf1 (right block of images). First columns, anti-V5 staining; middle columns, PLA signal; right columns, merged imaged. Bars, 5 μm. (C) Graphs depicting specific PLA signals obtained for samples in panel (A) with either low V5 expression (lower 50th percentile) or high V5 expression (upper 50th percentile). *p < 0.05, **p < 0.01, ***p < 0.001. (D) Left panel, western blots documenting expression of V5-tagged Rnf185 and Cgrrf1 in stably transduced MEFs; right panels, quantification of the levels of endogenous emerin and LBR in these MEF strains, relative to untransduced cells (UTD), with normalization to α-tubulin. Original images used for quantification are in Figure S6.