C-terminal tagging, transmembrane domain hydrophobicity, and an ER retention motif influence the secretory trafficking of the inner nuclear membrane protein emerin
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Curated by eLife
eLife Assessment
This solid work, a Research Advance linked to Buchwalter et al., 2019, demonstrates that epitope tagging influences protein fate, serving as a cautionary example of how different tagging and imaging strategies may alter the pattern of endogenous protein trafficking. The information presented will be useful for researchers in the field of membrane trafficking, particularly in guiding their experimental designs. That being said, the study offers limited new insights into the biogenesis or disposal of endogenous Emerin.
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Abstract
The inner nuclear membrane (INM), a subdomain of the endoplasmic reticulum (ER), sequesters hundreds of transmembrane proteins within the nucleus. We previously found that one INM protein, emerin, can evade the INM by secretory transport to the lysosome, where it is degraded. In this work, we used targeted mutagenesis to identify intrinsic sequences that promote or inhibit emerin’s secretory trafficking. By manipulating these sequences across several tag and expression level combinations, we now find that emerin’s localization is sensitive to C-terminal GFP tagging. While emerin’s long, hydrophobic C-terminal transmembrane domain facilitates trafficking to the lysosome, extending its lumenal terminus with a GFP tag biases the protein toward this pathway. In contrast, we identify a conserved ER retention sequence that stabilizes N- and C-terminally tagged emerin by limiting its lysosomal flux. These findings underscore long-standing concerns about tagging artifacts and reveal novel determinants of tail-anchored INM protein targeting.
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eLife Assessment
This solid work, a Research Advance linked to Buchwalter et al., 2019, demonstrates that epitope tagging influences protein fate, serving as a cautionary example of how different tagging and imaging strategies may alter the pattern of endogenous protein trafficking. The information presented will be useful for researchers in the field of membrane trafficking, particularly in guiding their experimental designs. That being said, the study offers limited new insights into the biogenesis or disposal of endogenous Emerin.
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Reviewer #1 (Public review):
Summary:
The authors revisit the specific domains/signals required for the redirection of an inner nuclear membrane protein, emerin, to the secretory pathway. They find that epitope tagging influences protein fate, serving as a cautionary tale for how different visualisation methods are used. Multiple tags and lines of evidence are used, providing solid evidence for the altered fate of different constructs.
Strengths:
This is a thorough dissection of domains and properties that confer INM retention vs secretion to the PM/lysosome, and will serve the community well as a caution regarding the placement of tags and how this influences protein fate.
Weaknesses:
Biogenesis pathways are not explored experimentally: it would be interesting to know if the lysosomal pool arrives there via the secretory pathway (eg by …
Reviewer #1 (Public review):
Summary:
The authors revisit the specific domains/signals required for the redirection of an inner nuclear membrane protein, emerin, to the secretory pathway. They find that epitope tagging influences protein fate, serving as a cautionary tale for how different visualisation methods are used. Multiple tags and lines of evidence are used, providing solid evidence for the altered fate of different constructs.
Strengths:
This is a thorough dissection of domains and properties that confer INM retention vs secretion to the PM/lysosome, and will serve the community well as a caution regarding the placement of tags and how this influences protein fate.
Weaknesses:
Biogenesis pathways are not explored experimentally: it would be interesting to know if the lysosomal pool arrives there via the secretory pathway (eg by engineering a glycosylation site into the lumenal domain) or by autophagy, where failed insertion products may accumulate in the cytoplasm and be degraded directly from cytoplasmic inclusions.
It would be helpful if the topology of constructs could be directly demonstrated by pulse-labelling and protease protection. It's possible that there are mixed pools of both topologies that might complicate interpretation.
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Reviewer #2 (Public review):
In this manuscript, Mella et al. investigate the effect of GFP tagging on the localization and stability of the nuclear-localized tail-anchored (TA) protein Emerin. A previous study from this group showed that C-terminally GFP-tagged Emerin protein traffics to the plasma membrane and reaches lysosomes for degradation. It is suggested that the C-terminal tagging of tail-anchored proteins shifts their insertion from the post-translational TRC/GET pathway to the co-translational SRP-mediated pathway. The authors of this paper found that C-terminal GFP tagging causes Emerin to localize to the plasma membrane and eventually reach lysosomes. They investigated the mechanism by which Emerin-GFP moves to the secretory pathway. By manipulating the cytosolic domain and the hydrophobicity of the transmembrane domain …
Reviewer #2 (Public review):
In this manuscript, Mella et al. investigate the effect of GFP tagging on the localization and stability of the nuclear-localized tail-anchored (TA) protein Emerin. A previous study from this group showed that C-terminally GFP-tagged Emerin protein traffics to the plasma membrane and reaches lysosomes for degradation. It is suggested that the C-terminal tagging of tail-anchored proteins shifts their insertion from the post-translational TRC/GET pathway to the co-translational SRP-mediated pathway. The authors of this paper found that C-terminal GFP tagging causes Emerin to localize to the plasma membrane and eventually reach lysosomes. They investigated the mechanism by which Emerin-GFP moves to the secretory pathway. By manipulating the cytosolic domain and the hydrophobicity of the transmembrane domain (TMD), the authors identify that an ER retention sequence and strong TMD hydrophobicity contribute to Emerin trafficking to the secretory pathway. Overall, the data are solid, and the knowledge will be useful to the field. However, the authors do not fully answer the question of why C-terminally GFP-tagged Emerin moves to the secretory pathway. Importantly, the authors did not consider the possible roles of GFP in the ER lumen influencing Emerin trafficking to the secretory pathway.
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