Molecular architecture of the luminal ring of the Xenopus laevis nuclear pore complex

Abstract

The nuclear pore complex (NPC) mediates the flow of substances between the nucleus and cytoplasm in eukaryotic cells. Here we report the cryo-electron tomography (cryo-ET) structure of the luminal ring (LR) of the NPC from Xenopus laevis oocyte. The observed key structural features of the LR are independently confirmed by single-particle cryo-electron microscopy (cryo-EM) analysis. The LR comprises eight butterfly-shaped subunits, each containing two symmetric wings. Each wing consists of four elongated, tubular protomers. Within the LR subunit, the eight protomers form a Finger domain, which directly contacts the fusion between the inner and outer nuclear membranes and a Grid domain, which serves as a rigid base for the Finger domain. Two neighboring LR subunits interact with each other through the lateral edges of their wings to constitute a Bumper domain, which displays two major conformations and appears to cushion neighboring NPCs. Our study reveals previously unknown features of the LR and potentially explains the elastic property of the NPC.

Types of nuclear localization signals and mechanisms of protein import into the nucleus

“…The protein components of the NPC are known as nucleoporins (Nup). A single NPC contains 34 different Nup types, most of which are conserved among different organisms, and each Nup is represented in multiple copies [ 60 , 61 ]. Interaction of importin β1 with the NPC occurs through multiple phenylalanine-glycine (FG) repeats on the Nups, enabling the trimeric complex to translocate into the nucleus [ 62 ].…”

Structure of the cytoplasmic ring of the Xenopus laevis nuclear pore complex by cryo-electron microscopy single particle analysis

“…The NE of X. laevis oocytes were prepared as described. 21 Cryo-ET only allows a relatively low dose of electrons at each defined tilting angle, which may hamper the resolution due to decreased signal-to-noise ratio. To overcome this drawback, we took the cryo-EM SPA approach.…”

“…The protocol is similar to that as described. 21 Briefly, stage VI oocytes of Xenopus laevis (X. laevis) were sorted in the modified Barth's saline (MBS) (10 mM HEPES, pH 7.5, 88 mM NaCl, 1 mM KCl, 0.82 mM MgSO 4 , 0.33 mM Ca(NO 3 ) 2 and 0.41 mM CaCl 2 ). The nucleus was isolated and transferred into a low salt buffer (LSB) (10 mM HEPES, pH 7.5, 1 mM KCl, 0.5 mM MgCl 2 , 10 μg/mL aprotinin, 5 μg/mL leupeptin).…”

“…Within the lumen of the nuclear envelope (NE), there is a circular scaffold known as the luminal ring (LR). 8,21 The NPC displays an approximate eight-fold symmetry. The unusually large size of the NPC, together with its conformational plasticity, represents a challenge for structural determination at improved resolutions, particularly by cryo-ET.…”

The promise and the challenges of cryo‐electron tomography

“…Cryo-ET has provided insights into the structure and function of viruses [9][10][11][12][92][93][94][95][96][97], nuclear pore complexes [64,[98][99][100], ribosomes [101][102][103], coated vesicles [104][105][106][107][108][109], nucleosome arrangement in chromatin [110], cytoskeletal elements [111][112][113], bacterial secretion systems [114][115][116][117], chemotaxis [118,119], flagellar motors [120], and S-layers [121]. Obtaining information about the spatial arrangement of proteins by cryo-ET has also found use in assessing protein denaturation at the air-buffer interface for single-particle analysis [122].…”