Tail-anchored proteins of the inner nuclear membrane (INM) are a unique class of transmembrane proteins with poorly understood biosynthesis, targeting, and degradation. The INM is a specialized extension of the endoplasmic reticulum (ER) membrane, the major site of membrane protein synthesis. Shortly after translation, tail-anchored proteins are embedded by their C-terminal transmembrane domains (TMDs) into the ER (Rapaport and Herrmann, 2023). Unlike cytoplasmic tail-anchored proteins, which rapidly traffic out of the ER toward their target membranes, INM proteins must remain in the ER long enough to diffuse through the nuclear pore complex and be retained by binding to nuclear structures (Boni et al., 2015; Ungricht et al., 2015). This process repeats each time the nuclear envelope reforms at the end of mitosis (Ellenberg et al., 1997). INM proteins thus spend considerable time in the ER, but how they interact with the ER’s secretory or proteostatic functions remains unclear.

Our previous work identified emerin (EMD) as a short-lived INM protein in C2C12 mouse myoblasts (Buchwalter et al., 2019). While investigating how emerin is degraded, we found that C-terminally GFP-tagged emerin travels by vesicular transport to the Golgi, plasma membrane (PM), and then lysosome during ER stress (Buchwalter et al., 2019) – an itinerary that closely tracks with the path taken by cargoes of the rapid ER stress-induced export pathway (Satpute-Krishnan et al., 2014). This unexpected secretory trafficking was prevented by the removal of EMD-GFP’s N-terminal LEM domain, suggesting that this domain played a role in stress-induced lysosomal degradation. We hypothesized that the INM might dynamically respond to ER stress by shunting emerin through the secretory pathway to the lysosome. However, we now find that N-terminally GFP-tagged emerin traffics at a lower rate than C-terminally tagged emerin and find no conclusive evidence that untagged emerin traffics to the lysosome. We also show that the secretory trafficking of tagged emerin depends on its unusually hydrophobic TMD. In isolation, the TMDs of both emerin and the related tail-anchored protein LAP2β readily traffic to the PM, but trafficking of the full-length tagged proteins is limited by a conserved RXR-type ER retention motif. Our experiments indicate that emerin’s trafficking is artifactually expedited by C-terminal GFP tagging. However, these studies identify sequence elements that target emerin to the contiguous nuclear envelope/endoplasmic reticulum (NE/ER) network and highlight the interdependence between protein biogenesis, topology, and localization.

After our initial discovery, we aimed to understand the mechanism of emerin’s secretory trafficking and its conservation across species, cell types, and expression levels. As our earlier experiments used C2C12 mouse myoblasts, we first tested whether EMD-GFP trafficking is conserved in human cells. We stably expressed tet-inducible mouse and human GFP fusions in human osteosarcoma (U2OS) cells. When treated with the lysosome blocking agent bafilomycin A1 (BafA1), we found that U2OS cells, unlike C2C12 myoblasts (Buchwalter et al., 2019), do not rely on ER stress to initiate secretory trafficking of EMD-GFP. Instead, BafA1 alone caused EMD-GFP to accumulate in lysosomal puncta. We used this high secretory flux to differentiate between emerin constructs that could and could not traffic (Figure 1A). Consistent with our C2C12 findings, we verified that WT EMD-GFP could traffic to the lysosome of U2OS cells, while a construct lacking the N-terminal LEM domain (ΔLEM, Figure 1B) could not. Our constructs were tagged with a C-terminal/lumenal GFP, which allowed us to quantify the proportion of EMD-GFP in the secretory pathway using cell surface antibody labeling and flow cytometry (Figure 1C and D). We calculated the trafficking level of each construct by dividing the surface anti-GFP antibody signal by the total EMD-GFP fluorescence. In line with our microscopy experiments, the anti-GFP antibody did not detect any ΔLEM construct at the PM of U2OS cells, while the WT EMD-GFP PM signal was always above background (Figure 1E).

Figure 1

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Emerin’s hydrophobic TMD is necessary and sufficient for trafficking of EMD-GFP

We further leveraged this U2OS system to examine how emerin’s protein sequence facilitates trafficking. Emerin contains three major domains: an N-terminal LEM domain that interacts with the chromatin cross-linker BANF1/BAF, an intrinsically disordered region that binds the nuclear lamina, and a C-terminal TMD (Lee et al., 2001; Figure 1B). We tested each domain’s ability to traffic through the secretory pathway with a series of domain truncation experiments. Having established that removing the LEM domain abolished emerin’s ability to traffic, we next deleted the disordered region between the LEM domain and the TMD. This construct (termed LEM-TMD) accumulated at the PM 10-fold more than the WT protein, indicating that the TMD, LEM domain, or both together are sufficient for trafficking (Figure 1F). To distinguish between these possibilities, we tested whether emerin’s TMD alone can travel to the PM. We removed all but the TMD alpha helix and its surrounding 26 residues (mEMD 212–259), which we found to be required for membrane insertion of the truncated construct (not shown), and appended this TMD-only construct to either the C- or N-terminus of eGFP. Both TMD-GFP and GFP-TMD localized prominently to the ER and PM, and both were evident in the lysosome after BafA1 treatment (Figure 1F and G). These experiments reveal that emerin’s TMD alone can traffic to the lysosome, regardless of GFP tag orientation.

We wondered what properties of emerin’s TMD mediate its secretion. Tail-anchored proteins can sort via their TMDs along the secretory pathway (Ronchi et al., 2008; Borgese, 2016; Bulbarelli et al., 2002); long and hydrophobic sequences partition to the thicker, more hydrophobic membranes of the late secretory organelles (Sharpe et al., 2010; Figure 2—figure supplement 1B and C), while tail-anchored proteins with shorter, less hydrophobic TMDs preferentially remain in the ER. We noticed that emerin’s TMD is particularly hydrophobic among other NE/ER TMDs (Figure 2—figure supplement 1A and B), which led us to predict that emerin’s TMD is required for its exit from the ER. To test this, we replaced emerin’s TMD with that of cytochrome B5, a model ER protein with a modestly hydrophobic TMD (Pedrazzini et al., 2000). This substitution ablated trafficking through the secretory pathway (Figure 2A and B), indicating that emerin’s TMD is indeed necessary for trafficking. We tested whether this depended on the hydrophobic content of the alpha helix by sequentially mutating its aromatic residues to alanine. We divided the TMD into N- and C-terminal halves, mutating three aromatic amino acids in each half (Figure 2C). The N-terminal TMD mutant was slightly less hydrophobic and had a greater effect on secretory trafficking than the C-terminal mutant (Figure 2D and E). Combining the N- and C-terminal mutations additively reduced the surface ratio of EMD-GFP. While these mutations decreased the overall protein expression (Figure 2—figure supplement 1E), they did not affect the protein’s topology (Figure 2—figure supplement 1F), confirming that the observed loss of surface exposure was due to diminished secretory trafficking and not to defects in membrane insertion. These experiments indicate that manipulating the hydrophobicity and/or aromaticity of the TMD influences EMD-GFP’s transit to the cell surface.

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Emerin contains a conserved ER retention signal

The autonomous trafficking of emerin’s TMD challenged our initial hypothesis that emerin’s localization relies on the LEM domain. Because EMD ΔLEM-GFP cannot traffic despite containing the TMD, we wondered whether a sequence within the ΔLEM construct inhibits trafficking. We noted that removing the LEM domain exposed a highly charged and conserved motTif (QRRR) on the N-terminus of the construct (Figure 2—figure supplement 1D). Poly-arginine (RXR) motifs are known negative regulators of ER-to-Golgi transit (Michelsen et al., 2005) we therefore tested whether this unprotected RXR could explain the ER-restricted localization of ΔLEM-GFP. Indeed, further truncating the ΔLEM construct by deleting the QRRR (ΔLEMΔQRRR) freed the protein from the ER (Figure 2F–H).

To test whether the RXR motif limits the trafficking of full-length EMD-GFP, we mutated the QRRR to AAAA (designated RA) and detected more flux to the PM compared to WT EMD-GFP (Figure 2F and G). Both ΔLEMΔQRRR-GFP and EMD RA-GFP were less abundant at steady state and were more rapidly degraded after removal of doxycycline from the medium (Figure 2I and J), suggesting increased flux to the lysosome. These data indicate that the RXR motif constitutes an ER retention sequence in EMD-GFP that is strengthened by the removal of the LEM domain. We infer that the presence of the LEM domain modulates the accessibility of the RRR motif, while the LEM domain itself is in fact dispensable for trafficking, its removal constitutively exposes the RRR motif, enhancing emerin’s ER localization.

We next investigated the interplay between emerin’s pro-trafficking TMD and its anti-trafficking RXR motif by cloning the RA mutation into our TMD mutant constructs (see Figure 2C–E). The RA mutation generally increased the surface expression of the TMD mutants except for the construct with the lowest hydrophobicity score (TMD^m), which was not significantly altered by the RA mutation (Figure 3A and B). We infer from this that the TMD acts upstream of the RXR motif in emerin’s secretory trafficking, fitting with the hypothesis that low TMD hydrophobicity suffices to stably retain proteins in the ER (Ronchi et al., 2008).

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Emerin is not the only tail-anchored INM protein with a particularly hydrophobic TMD. Another such protein is LAP2β (Lomize et al., 2017), which shares several sequence features with emerin (Berk et al., 2013; Figure 2—figure supplement 1A, Figure 3C). We identified three RXR motifs within the LAP2β N-terminal domain, one of which (designated RXR1) is just downstream of the LEM-like fold – a striking similarity to emerin’s RXR motif (Figure 3C). To test whether the same localization mechanisms govern LAP2β, we tagged its C-terminus with GFP and expressed it in U2OS cells. LAP2β-GFP was detected at the cell surface, albeit at much lower levels than EMD-GFP (Figure 3D and E, Figure 3—figure supplement 1A and B). Interestingly, replacing most of the LAP2β N-terminal domain with the soybean ascorbate peroxidase (APEX2) enzyme caused a 20-fold increase in PM expression (Figure 3D, Figure 3—figure supplement 1B), suggesting that the LAP2β TMD has a similar proclivity for secretory trafficking. Finally, mutating the LAP2β RXR1 to AAA led to a 70% increase in PM accumulation (Figure 3D), mirroring the effect of the EMD RA mutation. Together, these data indicate that the emerin and LAP2β TMDs are insufficient for NE/ER targeting and that both proteins contain retention sequences which counteract TMD-dependent secretion.

RXR motifs are thought to bind COPI subunits to return escaped proteins to the ER (Michelsen et al., 2005; Michelsen et al., 2007), though this mechanism is not understood. To test whether the increased trafficking of the RA mutant results from reduced COPI interaction, we immunoprecipitated N-terminally FLAG-tagged mouse emerin constructs from U2OS cells (Figure 3—figure supplement 1C). Mass spectrometry revealed a robust interaction between emerin and the COPI coat, with several COPI subunits ranking among the top hits (Figure 3F). Surprisingly, however, this interaction was not weakened by the RXR mutation (Figure 3G). We noted that the mouse EMD bait also co-immunoprecipitated endogenous human EMD (Figure 3G), opening the possibility that COPI might indirectly interact with both FLAG constructs via endogenous emerin. Alternatively, the RXR motif’s function could depend on protein topology. Importantly, while we were able to detect FLAG-EMD in the lysosome by immunofluorescence (IF) (Figure 3—figure supplement 1D), FLAG-RA construct was not poorly expressed relative to FLAG-WT (Figure 3—figure supplement 1C) in contrast to RA-GFP compared to WT-GFP (Figure 2I). We also noted that the lysosomal accumulation of N-terminally FLAG-tagged emerin was more subtle than the C-terminally GFP-tagged version (Figures 1A and 2B), indicating that epitope tagging and/or tag orientation influences the extent of emerin protein turnover.

C-terminal tagging destabilizes emerin via secretory trafficking

To determine whether tagging and/or overexpression increase the extent of emerin trafficking, we needed a system to better control emerin expression. To this end, we generated a human iPSC line with a dual integrase cassette exchange (DICE) landing pad (Zhu et al., 2014) in the AAVS1 safe harbor locus (Figure 4A), then knocked emerin out of these cells with CRISPR/Cas9 (Figure 4B). Integrating GFP-tagged constructs into the parental and EMD knockout (KO) DICE landing pads enabled us to measure EMD-GFP trafficking in the presence or absence of the endogenous copy, respectively (Figure 4C). We included an mCherry-P2A upstream of the EMD-GFP to control for minor differences in construct expression (Figure 4A). The GFP-tagged emerin constructs mirrored the trafficking pattern we observed in U2OS cells; the EMD RA-GFP construct accumulated more at the PM, while the TMD mutant did not traffic (Figure 4D and E). The steady-state abundance reflected these trafficking patterns. The GFP fluorescence of the TMD mutant accumulated to higher levels, indicating that it was stabilized by its inability to traffic. In contrast, the RA mutant fluorescence was slightly lower, indicating that it was modestly less stable than the WT protein, although this difference is not statistically significant (Figure 4F and G). There was no difference in total or surface expression between integrants in the EMD KO and WT DICE backgrounds, suggesting that the trafficking of EMD-GFP is comparable both when moderately overexpressed and when expressed at endogenous levels (Figure 4D and E).

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Tracking tail-anchored (i.e. untagged/N-terminally tagged) emerin through the secretory pathway is challenging because it lacks a lumenal domain amenable to surface labeling. However, after discovering ways to limit lysosomal flux (via the TMD) or accelerate it (via the RXR motif), we reasoned that the trafficking patterns of tail-anchored mutants could be inferred from their steady-state abundance when expressed from the DICE landing pad. To test whether the GFP tag orientation affects protein expression, we compared the steady-state fluorescence of N- and C-terminally tagged mutants. Surprisingly, the N-terminally tagged (tail-anchored) GFP fusions were four to five times more abundant than the C-terminally tagged (single pass) fusions (Figure 4F and G). C-terminal fusions were also more completely relocalized to the lysosome after BafA1 than N-terminal fusions, where NE/ER EMD signal was still apparent (Figure 4—figure supplement 1A). Unlike the C-terminal fusions, the N-terminally tagged TMD mutant did not accumulate more than the N-terminally tagged WT protein, suggesting that N-terminally tagged emerin is not destabilized by trafficking. We interpret this finding with caution, as we observed a slight decrease in the level of the N-terminally tagged TMD mutant compared to WT (Figure 4G). Since the insertion of tail-anchored proteins relies on TMD hydrophobicity (Rapaport and Herrmann, 2023; Shao and Hegde, 2011), improper targeting to the ER membrane due to TMD mutation would confound our conclusion that N-terminally tagged constructs undergo similarly low levels of trafficking. Interestingly, upon treatment with BafA1, the N-terminally tagged WT and RA constructs formed GFP+ lysosomal puncta, while the N-terminally tagged TMD mutant did not (Figure 4—figure supplement 1A). This suggests that N-terminally tagged emerin does undergo some level of TMD-dependent trafficking. Further, the N-terminal RA fusion was slightly less fluorescent at steady state than WT (Figure 4G), indicating that the RXR motif also stabilizes tail-anchored GFP-EMD.

Because of the profound differences in the distribution of N- and C-terminally tagged constructs, we investigated how lumenal GFP affects emerin’s early secretory trafficking by analyzing its enrichment in ER exit sites (ERES). ER cargoes that are actively packaged into ERES can be distinguished from bulk flow secretory cargoes by incubating cells at 10°C, where ERES form and cargoes accumulate but cannot progress toward the Golgi (Mezzacasa and Helenius, 2002). We compared the co-localization of endogenous untagged emerin, WT-GFP, and TMD^m-GFP with the ERES marker SEC31 at 37°C and 10°C (Figure 4—figure supplement 1B and C). We found that GFP-tagged emerin co-localized with SEC31 at steady state, while this was not the case for endogenous emerin or the tagged TMD mutant. ERES co-localization was not increased at 10°C for any of the emerin variants analyzed, indicating that emerin does not contain specific ER exit signals.

Collectively, these data indicate that N- and C-terminally tagged emerin constructs travel to the lysosome via their hydrophobic TMDs, although C-terminal tags accelerate this trafficking by encouraging bulk flow ER exit.

Finally, we integrated untagged WT, RA, and TMD mutant constructs into EMD KO landing pad cells, then fixed and stained cells for confocal microscopy and flow cytometry. We saw no apparent differences in the localization or intensity of any of the emerin mutants, though the data were considerably noisier than the steady-state GFP measurements (Figure 4H). We were also unable to detect untagged emerin in the lysosome after BafA1 treatment (Figure 4—figure supplement 1D), suggesting it does not traffic through the secretory pathway. However, the sensitivity of these assays may be limited by fixation, permeabilization, and antibody staining; therefore, we cannot rule out the possibility that small differences do exist between untagged emerin variants.

In this work, we dissected how exogenous epitope tags and intrinsic sequence elements influence the secretory trafficking of the INM-resident protein emerin. We discovered that the aromatic amino acids in emerin’s 23-residue TMD facilitate its travel to the lysosome. In the absence of other signals, TMD length and hydrophobicity are sufficient to partition tail-anchored proteins along the secretory pathway (Ronchi et al., 2008; Borgese, 2016). The biophysical properties of the emerin and LAP2β TMDs resemble PM, rather than ER, resident TMDs (Figure 2—figure supplement 1, Sharpe et al., 2010). However, this notable hydrophobicity is likely important for the proteins’ biogenesis. Tail-anchored proteins with very hydrophobic TMDs are posttranslationally targeted by the TRC40/GET complex, which captures C-terminal alpha helices after they emerge from the ribosome and delivers them to the ER membrane (Rapaport and Herrmann, 2023). Emerin and Lap2β are established TRC40 client proteins (Coy-Vergara et al., 2019; Pfaff et al., 2016), so their hydrophobic TMDs are likely required for TRC40 recognition. However, adding a C-terminal GFP converts a tail-anchored protein to a single-pass transmembrane protein, which will instead be co-translationally targeted to the ER by the SRP pathway (Shao and Hegde, 2011; Walter et al., 1981). Our results indicate that this change in topology displaces emerin, making C-terminally tagged emerin less stable and more prone to lysosomal trafficking than its N-terminally tagged counterpart (Figure 4F and G, Figure 4—figure supplement 1A). Consistently, we found that only lumenally tagged emerin sampled ERES, and that this was reduced by manipulating the TMD hydrophobicity (Figure 4—figure supplement 1B and C). This suggests that hydrophobic TMDs are less energetically favored within the ER membrane when attached to a lumenal domain, or that different biogenesis pathways bias cargoes toward ER exit.

We hypothesize that active ER retention plays a role in INM protein targeting. In interphase, emerin is not entirely sequestered at the INM by the nuclear lamina; a pool of emerin protein remains in the peripheral ER (Nastały et al., 2020; Salpingidou et al., 2007). The intrinsic ability of emerin’s TMD to traffic from the ER implies that a limiting mechanism must exist to bias the protein toward diffusion to the INM. Emerin could stably reside in the ER in part by binding to other ER-resident proteins. Interestingly, emerin was reported to interact with the ER tethering protein MOSPD3 via its YEESY (95-99) motif (Cabukusta et al., 2020), which we previously found to limit trafficking (Buchwalter et al., 2019). While both TMDs traffic to the PM, full-length LAP2β-GFP is more retained in the NE/ER than EMD-GFP (Figure 3D, Figure 3—figure supplement 1A and B), indicating that the N-terminal domains limit trafficking to different extents.

We identified the RXR motif as a shared negative regulator of TMD-dependent trafficking, prompting us to revise our original model: the exposure of this motif, not the absence of the LEM domain, prevents ER exit of EMD-GFP (Figure 2F and G). This suggests that the sequences around the RXR motif influence its activity by steric occlusion or other mechanisms. RXR motifs prevent the ER exit of some PM-resident proteins until they are masked by oligomerization, thus coupling ER exit to folding and/or assembly (Michelsen et al., 2005). Like the canonical ER retrieval motif KKXX, RXR motifs are thought to engage COPI subunits to return escaped ER membrane proteins from the early Golgi (Michelsen et al., 2005; Marcello et al., 2010). Yeast-2-hybrid experiments predicted RXR signals to interact with the beta and delta subunits of the COPI coat (Michelsen et al., 2007), though the molecular details are still unclear. Contrary to KKXX motifs, found only on C-termini, RXR motifs may be on N-termini or within a polypeptide chain (Michelsen et al., 2005). Truncated, lumenally tagged variants of Sun2, another tail-anchored INM protein, require a poly-arginine motif to prevent mislocalization to the Golgi (Turgay et al., 2010). Turgay et al., 2010, observed purified Sun2 RXR mutants losing interaction with COPI subunits in GST pull-down assays. In contrast, we did not observe loss of COPI binding in our RXR mutant immunoprecipitations. This discrepancy may be explained by differences in methods (purified recombinant protein binding assays vs native IP) or by oligomerization of tagged emerin with endogenous emerin to indirectly bind COPI in lysate (Fernandez et al., 2021). Emerin’s interaction with the COPI coat was recently detected by proximity biotinylation in mouse P19 cells (Zhao et al., 2021). Interestingly, emerin was the only non-Golgi-resident protein identified in the gamma-COP subunit interactome, indicating that endogenous emerin might be a COPI cargo. While we predict this interaction occurs via emerin’s RXR motif, we cannot exclude the possibility of other COPI-interacting mechanisms.

Our previous work identified EMD as an unstable protein subject to significant lysosomal flux (Buchwalter et al., 2019). However, we have now discovered that emerin’s trafficking is markedly promoted by C-terminal GFP tagging, likely influencing many of our initial observations. While we used C-terminal tags to avoid disrupting emerin’s N-terminal interactions with chromatin and lamins, this approach inadvertently altered its localization. We did detect trafficking of N-terminally tagged emerin but found that it occurs at a much lower rate. We conclude that hydrophobic tail-anchored TMDs are readily dislocated by C-terminal tags and echo the warnings raised by generations of cell biologists regarding tag-induced localization artifacts (Stadler et al., 2013; Montecinos-Franjola et al., 2020; Margolin, 2012). We did not detect trafficking of the untagged emerin constructs. Nonetheless, because trafficking is detectable at or below endogenous levels with different tags and orientations, we cannot rule out the possibility that emerin traffics under specific conditions. We note poor correlation of emerin transcript and protein across tissues (Wang et al., 2019; Figure 4—figure supplement 1E), which suggests protein-level regulation that may dynamically regulate emerin levels. While we still do not know whether emerin’s ability to travel beyond the NE/ER influences its function, our experiments reveal an unanticipated connection between the topology and localization of INM proteins and raise new questions about the balance between secretion and retention of INM-destined proteins.

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Author details

1. Jessica Mella

   Cardiovascular Research Institute, University of California, San Francisco, San Francisco, United States

   Contribution

   Conceptualization, Data curation, Formal analysis, Funding acquisition, Validation, Investigation, Methodology, Writing – original draft, Writing – review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-7541-6789

2. Regan F Volk

   Cardiovascular Research Institute, University of California, San Francisco, San Francisco, United States

   Contribution

   Formal analysis, Investigation, Methodology

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-6748-719X

3. Balyn W Zaro

   1. Cardiovascular Research Institute, University of California, San Francisco, San Francisco, United States
   2. Department of Pharmaceutical Chemistry, University of California, San Francisco, San Francisco, United States

   Contribution

   Resources, Formal analysis, Investigation, Methodology

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-8938-9889

4. Abigail Buchwalter

   1. Cardiovascular Research Institute, University of California, San Francisco, San Francisco, United States
   2. Department of Physiology, University of California, San Francisco, San Francisco, United States

   Contribution

   Conceptualization, Resources, Formal analysis, Supervision, Funding acquisition, Investigation, Visualization, Methodology, Project administration, Writing – review and editing

   For correspondence

   abigail.buchwalter@ucsf.edu

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-7181-6961

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