TY - JOUR
T1 - An amphipathic helix in Brl1 is required for nuclear pore complex biogenesis in S. cerevisiae
AU - Kralt, Annemarie
AU - Wojtynek, Matthias
AU - Fischer, Jonas S.
AU - Agote-Aran, Arantxa
AU - Mancini, Roberta
AU - Dultz, Elisa
AU - Noor, Elad
AU - Uliana, Federico
AU - Tatarek-Nossol, Marianna
AU - Antonin, Wolfram
AU - Onischenko, Evgeny
AU - Medalia, Ohad
AU - Weis, Karsten
N1 - Funding Information:
We are grateful to all the Weis lab members for many fruitful discussions and helpful suggestions. We want to thank E Fatti for the purification of Brl1 constructs. AK thanks AC Meinema for valuable discussions on and help with image analyses. We thank P Picotti and A Leitner for the access to MS instruments, and the Center of Microscopy and Image Analysis (ZMB), University of Zürich, and ScopeM, ETH Zürich, for microscopy support. We would like to acknowledge the contributions of S Steiger and J Vailliant to early stages of this work during their student projects. AK was a recipient of a Swiss National Science Foundation Marie Heim-Voegtlin Fellowship (grant number: PMPDP3_171317) and AAA acknowledges the support from an EMBO postdoctoral fellowship (grant number: ALTF 910-2021). This study was supported by project grants from the Swiss National Science Foundation (31003A_179275 to KW and 31003A_179418 to OM), a grant from the Research Council of Norway (NFR 315615 to EO, KW, and EN), and a grant from the German Research Foundation (DFG AN377/7-1 to WA).
Publisher Copyright:
© Kralt, Wojtynek,.
PY - 2022/1/1
Y1 - 2022/1/1
N2 - 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 trans-membrane 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 amphipa-thic helix in mediating the fusion of the inner and outer nuclear membranes.
AB - 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 trans-membrane 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 amphipa-thic helix in mediating the fusion of the inner and outer nuclear membranes.
UR - http://www.scopus.com/inward/record.url?scp=85137125242&partnerID=8YFLogxK
U2 - 10.7554/eLife.78385
DO - 10.7554/eLife.78385
M3 - Article
C2 - 36000978
AN - SCOPUS:85137125242
VL - 11
JO - eLife
JF - eLife
SN - 2050-084X
M1 - e78385
ER -