An amphipathic helix in Brl1 is required for nuclear pore complex biogenesis in S. cerevisiae
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Evaluation Summary:
This work advances our understanding of the nuclear pore complex (NPC) biogenesis pathway by providing much needed additional insight into the function of one of the few NPC assembly factors, Brl1. It thus addresses a long-standing and fundamental question relevant to individuals interested in nuclear transport, nuclear cell biology, and membrane-protein interactions more generally.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #3 agreed to share their name with the authors.)
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Abstract
The nuclear pore complex (NPC) is the central portal for macromolecular exchange between the nucleus and cytoplasm. In all eukaryotes, NPCs assemble into an intact nuclear envelope (NE) during interphase, but the process of NPC biogenesis remains poorly characterized. Furthermore, little is known about how NPC assembly leads to the fusion of the outer and inner NE, and no factors have been identified that could trigger this event. Here, we characterize the transmembrane protein Brl1 as an NPC assembly factor required for NE fusion in budding yeast. Brl1 preferentially associates with NPC assembly intermediates and its depletion halts NPC biogenesis, leading to NE herniations that contain inner and outer ring nucleoporins but lack the cytoplasmic export platform. Furthermore, we identify an essential amphipathic helix in the luminal domain of Brl1 that mediates interactions with lipid bilayers. Mutations in this amphipathic helix lead to NPC assembly defects, and cryo-electron tomography analyses reveal multilayered herniations of the inner nuclear membrane with NPC-like structures at the neck, indicating a failure in NE fusion. Taken together, our results identify a role for Brl1 in NPC assembly and suggest a function of its amphipathic helix in mediating the fusion of the inner and outer nuclear membranes.
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Author Response
Reviewer #1 (Public Review):
This manuscript by Kralt et al. provides insight into the enigmatic process of nuclear pore complex (NPC) biogenesis. By taking advantage of their recent development of an isotope labeling/affinity purification/quantitative mass spectrometry pipeline called KARMA, the authors convincingly demonstrate that Brl1, a double pass transmembrane protein, associates with early NPC assembly intermediates but not mature NPCs. A combination of auxin-inducible degradation of Brl1 coupled with an extensive analysis of nup localization (including the use of RITE technology) and cryo-electron tomography provides compelling evidence of the importance of Brl1 in NPC biogenesis at a step that correlates with inner and outer nuclear membrane fusion. Overall, the strengths of the work are that the experiments …
Author Response
Reviewer #1 (Public Review):
This manuscript by Kralt et al. provides insight into the enigmatic process of nuclear pore complex (NPC) biogenesis. By taking advantage of their recent development of an isotope labeling/affinity purification/quantitative mass spectrometry pipeline called KARMA, the authors convincingly demonstrate that Brl1, a double pass transmembrane protein, associates with early NPC assembly intermediates but not mature NPCs. A combination of auxin-inducible degradation of Brl1 coupled with an extensive analysis of nup localization (including the use of RITE technology) and cryo-electron tomography provides compelling evidence of the importance of Brl1 in NPC biogenesis at a step that correlates with inner and outer nuclear membrane fusion. Overall, the strengths of the work are that the experiments are innovative, the data are of the highest quality, and the conclusions are on a solid footing.
A potential weakness is that there is already convincing published data that implicates Brl1 as an NPC biogenesis factor. Although there is no doubt that the current work extends these findings to implicate a lumenal amphipathic helix as a key element of Brl1 function, there remains considerable uncertainty over the ultimate mechanism by which the amphipathic helix contributes to inner and outer nuclear membrane fusion.
We thank the reviewer for the overall positive evaluation.
We agree that several recent papers have pointed towards a role for Brl1 in NPC assembly. However, the literature was not conclusive on whether Brl1 directly acts in this process and did not provide any mechanistic insight into the function of Brl1. For example it was suggested that Brl1 might play a role in nuclear transport [1,2] or affect NPC assembly by regulating lipid homeostasis [3,4]. Especially the effect of Brl1 on lipid homeostasis and whether Brl1 indirectly affects NPC assembly by regulating the lipid composition remained controversial [3-5].
In this respect, we would like to highlight that figures 1-3 provide, to our knowledge, the first direct evidence that Brl1 acts as an NPC assembly factor, and our results clearly go beyond previously published data. First, we identify Brl1 as an NPC-interacting factor in an unbiased analysis of a previously published MS dataset [6]. Second, we show that Brl1 has a preference for binding to young, premature NPCs, which means it binds to NPC assembly intermediates but is not part of the mature complex. This is evident from our metabolic labeling assays and also seen in vivo using the RITE approach. Finally, by in-depth characterization of the associated NPC assembly defects and localization of Brl1 we show that (i) the activity of Brl1 is required for membrane fusion during new NPC assembly and (ii) the lipid binding of its luminal AH is essential for this activity. We agree that we do not have any direct evidence that Brl1 displays fusogenic activity, but such a direct proof would likely require biochemical reconstitutions, which are outside the scope of this study. However, our data provide a solid ground for further work in this direction.
Reviewer #2 (Public Review):
In this study, Kralt et al. investigate the mechanisms of nuclear pore complex (NPC) biogenesis in budding yeast, which only relies on interphase NPC assembly. By combining metabolic labeling and microscopy, they show that Brl1, a nuclear envelope (NE) transmembrane protein previously reported to partake in NPC biogenesis, associates with early NPC assembly intermediates. They further report that Brl1 depletion triggers NPC biogenesis defects, as revealed by (i) the characterization of NPC species lacking a subset of nucleoporins in fluorescence microscopy and metabolic labeling assays, and (ii) the detection of NE abnormalities (i.e. herniations) by cryo-electron tomography. In search of the underlying mechanisms, they identify an essential Brl1 motif predicted to fold as an amphipathic helix (AH), which exhibits liposome-binding activity in vitro and supports NE targeting in vivo. Finally, they demonstrate that overexpression of an AH-deficient Brl1 version blocks NPC assembly at a stage likely preceding the fusion of the inner and outer nuclear membranes. Based on these observations, they suggest that Brl1 AH is required for the membrane fusion step in de novo pore biogenesis.
Overall, the conclusions of the authors are supported by the large panel of high-resolution, quantitative data provided. This study provides an unprecedented characterization of Brl1 recruitment and function during the early steps of NPC maturation, although it was already reported that Brl1 contributes to pore assembly (e.g. Zhang et al., 2018). In this view, the involvement of an AH-containing factor in the fusion step represents the main conceptual advance here. Yet, although the featured results support a role for Brl1 AH in membrane fusion, they do not actually prove that Brl1 acts as a fusogen during nuclear pore formation. Additional characterization of Brl1 AH properties, in particular through in vitro experiments, will be required to understand the underlying mechanisms and their relationships with the other NE proteins proposed to contribute to this process (i.e. Brr6/Apq12).
Of note, this work also validates the utilization of the KARMA workflow (metabolic labeling coupled to affinity purification and mass spectrometry), previously published by the same authors, for the characterization of NPC assembly factors. While this methodological framework could thereby prove useful to assess the biogenesis of multiprotein complexes, beyond NPCs, some potential limitations also emerge, as highlighted here by the necessity to control for post-lysis intermixing.
We appreciate the very positive evaluation of our manuscript. We would like to highlight that the main conceptual advance of this study is not only the identification and characterization of the ahBrl1 but also the direct evidence and characterization of Brl1 as an NPC assembly factor (see also the general response to reviewer #1). Investigating in detail the mechanism of membrane fusion and the role of Brl1, Brr6 and Apq12 in this process will unquestionably be very interesting, but is beyond the scope of this study.
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Evaluation Summary:
This work advances our understanding of the nuclear pore complex (NPC) biogenesis pathway by providing much needed additional insight into the function of one of the few NPC assembly factors, Brl1. It thus addresses a long-standing and fundamental question relevant to individuals interested in nuclear transport, nuclear cell biology, and membrane-protein interactions more generally.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #3 agreed to share their name with the authors.)
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Reviewer #1 (Public Review):
This manuscript by Kralt et al. provides insight into the enigmatic process of nuclear pore complex (NPC) biogenesis. By taking advantage of their recent development of an isotope labeling/affinity purification/quantitative mass spectrometry pipeline called KARMA, the authors convincingly demonstrate that Brl1, a double pass transmembrane protein, associates with early NPC assembly intermediates but not mature NPCs. A combination of auxin-inducible degradation of Brl1 coupled with an extensive analysis of nup localization (including the use of RITE technology) and cryo-electron tomography provides compelling evidence of the importance of Brl1 in NPC biogenesis at a step that correlates with inner and outer nuclear membrane fusion. Overall, the strengths of the work are that the experiments are innovative, …
Reviewer #1 (Public Review):
This manuscript by Kralt et al. provides insight into the enigmatic process of nuclear pore complex (NPC) biogenesis. By taking advantage of their recent development of an isotope labeling/affinity purification/quantitative mass spectrometry pipeline called KARMA, the authors convincingly demonstrate that Brl1, a double pass transmembrane protein, associates with early NPC assembly intermediates but not mature NPCs. A combination of auxin-inducible degradation of Brl1 coupled with an extensive analysis of nup localization (including the use of RITE technology) and cryo-electron tomography provides compelling evidence of the importance of Brl1 in NPC biogenesis at a step that correlates with inner and outer nuclear membrane fusion. Overall, the strengths of the work are that the experiments are innovative, the data are of the highest quality, and the conclusions are on a solid footing. A potential weakness is that there is already convincing published data that implicates Brl1 as an NPC biogenesis factor. Although there is no doubt that the current work extends these findings to implicate a lumenal amphipathic helix as a key element of Brl1 function, there remains considerable uncertainty over the ultimate mechanism by which the amphipathic helix contributes to inner and outer nuclear membrane fusion.
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Reviewer #2 (Public Review):
In this study, Kralt et al. investigate the mechanisms of nuclear pore complex (NPC) biogenesis in budding yeast, which only relies on interphase NPC assembly. By combining metabolic labeling and microscopy, they show that Brl1, a nuclear envelope (NE) transmembrane protein previously reported to partake in NPC biogenesis, associates with early NPC assembly intermediates. They further report that Brl1 depletion triggers NPC biogenesis defects, as revealed by (i) the characterization of NPC species lacking a subset of nucleoporins in fluorescence microscopy and metabolic labeling assays, and (ii) the detection of NE abnormalities (i.e. herniations) by cryo-electron tomography. In search of the underlying mechanisms, they identify an essential Brl1 motif predicted to fold as an amphipathic helix (AH), which …
Reviewer #2 (Public Review):
In this study, Kralt et al. investigate the mechanisms of nuclear pore complex (NPC) biogenesis in budding yeast, which only relies on interphase NPC assembly. By combining metabolic labeling and microscopy, they show that Brl1, a nuclear envelope (NE) transmembrane protein previously reported to partake in NPC biogenesis, associates with early NPC assembly intermediates. They further report that Brl1 depletion triggers NPC biogenesis defects, as revealed by (i) the characterization of NPC species lacking a subset of nucleoporins in fluorescence microscopy and metabolic labeling assays, and (ii) the detection of NE abnormalities (i.e. herniations) by cryo-electron tomography. In search of the underlying mechanisms, they identify an essential Brl1 motif predicted to fold as an amphipathic helix (AH), which exhibits liposome-binding activity in vitro and supports NE targeting in vivo. Finally, they demonstrate that overexpression of an AH-deficient Brl1 version blocks NPC assembly at a stage likely preceding the fusion of the inner and outer nuclear membranes. Based on these observations, they suggest that Brl1 AH is required for the membrane fusion step in de novo pore biogenesis.
Overall, the conclusions of the authors are supported by the large panel of high-resolution, quantitative data provided. This study provides an unprecedented characterization of Brl1 recruitment and function during the early steps of NPC maturation, although it was already reported that Brl1 contributes to pore assembly (e.g. Zhang et al., 2018). In this view, the involvement of an AH-containing factor in the fusion step represents the main conceptual advance here. Yet, although the featured results support a role for Brl1 AH in membrane fusion, they do not actually prove that Brl1 acts as a fusogen during nuclear pore formation. Additional characterization of Brl1 AH properties, in particular through in vitro experiments, will be required to understand the underlying mechanisms and their relationships with the other NE proteins proposed to contribute to this process (i.e. Brr6/Apq12).
Of note, this work also validates the utilization of the KARMA workflow (metabolic labeling coupled to affinity purification and mass spectrometry), previously published by the same authors, for the characterization of NPC assembly factors. While this methodological framework could thereby prove useful to assess the biogenesis of multiprotein complexes, beyond NPCs, some potential limitations also emerge, as highlighted here by the necessity to control for post-lysis intermixing.
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Reviewer #3 (Public Review):
The data presented in this manuscript are generally of high quality, and they describe several new and interesting observations on the role of Brl1 in NPC formation. While a number of important studies have previously established a role for Brl1 in NPC assembly and the maintenance of nuclear envelope structure, little was known about the interactions of Brl1 with specific nucleoporins (Nups) during periods of NPC assembly or the function of these interactions. Using the MS-based KARMA approach, the authors present evidence that Brl1 binds preferentially to newly synthesized Nups. Their data show, for the first time, that Brl1 interacts transiently with a specific set of nups previously implicated by the authors, and others, as functioning in the early stages of NPC assembly. These results are a strength of …
Reviewer #3 (Public Review):
The data presented in this manuscript are generally of high quality, and they describe several new and interesting observations on the role of Brl1 in NPC formation. While a number of important studies have previously established a role for Brl1 in NPC assembly and the maintenance of nuclear envelope structure, little was known about the interactions of Brl1 with specific nucleoporins (Nups) during periods of NPC assembly or the function of these interactions. Using the MS-based KARMA approach, the authors present evidence that Brl1 binds preferentially to newly synthesized Nups. Their data show, for the first time, that Brl1 interacts transiently with a specific set of nups previously implicated by the authors, and others, as functioning in the early stages of NPC assembly. These results are a strength of the manuscript. Consistent with these observations, the authors use a degron system to show that depletion of cellular levels of Brl1 leads to the formation of structures associated with the inner nuclear membrane, which exhibit features of NPC substructures. Moreover, in combination with KARMA analysis of labeled Nups, their data suggest Brl1 functions prior to, or coincident with, membrane fusion and the incorporation of cytoplasmic structures and Mlp1 into the forming NPC. These data are consistent with previously published observations on brl1 mutants. Finally, in examining the structural features of Brl1, the authors identified an amphipathic helix positioned within a NE lumenal domain of Brl1 that was essential for its function. They provide reasonable evidence that the AH can bind to membranes. Moreover, they show that the overexpression of various AH point mutations induces a dominant-negative growth phenotype. They also provide reasonable evidence that overproduced AH mutants bind NPC substructures and induce adjacent membrane proliferation. For the most part, the data presented in the manuscript support the authors' conclusions. An interesting addition to the manuscript would have been the analysis of interactions of an ahBrl1 point mutant with Nups to further assess the consequences of AH loss of function on Brl1 interactions with the assembling NPC.
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