Bleyet al., Science 376 , eabm9129 (2022) 10 June 2022 2of18
Fig. 1. Reconstitution of a 16-proteinC. thermophilumcomplex composed of
coat and CF nups.(A) Cross-sectional schematic of the fungal NPC architecture.
NE, nuclear envelope. (B) Domain structures of the coat and CF nups.
(C) Schematic representation summarizing our biochemical reconstitution and
dissection experiments with purified recombinantC. thermophilumnups,
illustrating the CFNC architecture and its attachment to the CNC. The CNC harbors
two assembly sensors, Nup37CTEand Nup145CNTE,eachanchoringaCFNCvia
its central hub, with Nup37CTEexhibiting tighter binding than Nup145CNTE.
(D to F) SEC-MALS interaction analyses, showing the stepwise biochemical
reconstitution starting with (D) the CFNC (green) from Nup82•Nup159•Nsp1 (blue),
Gle2•Nup145N (cyan), and Dbp5 (red); then (E) CFNC•Gle1•Nup42GBM(green) from
CFNC (blue) and Gle1•Nup42GBM(red); and culminating with (F) the 16-protein
CNC•CFNC•Gle1•Nup42GBMcomplex (green) from CNC (red), CFNC (blue), and
Gle1•Nup42GBM(cyan). SDS-PAGE gel strips of peak fractions are shown. Measured
molecular masses are indicated, with respective theoretical masses in parentheses.
(G andH) LLPS interaction assays, assessing (G) CFNC (red) and Gle1•Nup42GBM
(cyan) incorporation into CNC-LLPS (green), and (H) CFNC incorporation into
CNC-LLPS, lacking either one or both Nup37CTEand Nup145CNTEassembly sensors.
N-terminally fluorescently labeled CNC (Bodipy), CFNC (Alexa Fluor 647), and
Gle1•Nup42GBM(Coumarin) were visualized by fluorescence microscopy. Pelleted
CNC condensate phase (P) and soluble (S) fractions were analyzed by SDS-PAGE
and visualized by Coomassie brilliant blue staining. Scale bars, 10mm.RESEARCH | STRUCTURE OF THE NUCLEAR PORE