Domain topology of nucleoporin Nup98 within the nuclear pore complex

Abstract

Nuclear pore complexes (NPCs) facilitate selective transport of macromolecules across the nuclear envelope in interphase eukaryotic cells. NPCs are composed of roughly 30 different proteins (nucleoporins) of which about one third are characterized by the presence of phenylalanine-glycine (FG) repeat domains that allow the association of soluble nuclear transport receptors with the NPC. Two types of FG (FG/FxFG and FG/GLFG) domains are found in nucleoporins and Nup98 is the sole vertebrate nucleoporin harboring the GLFG-type repeats. By immuno-electron microscopy using isolated nuclei from Xenopus oocytes we show here the localization of distinct domains of Nup98. We examined the localization of the C- and N-terminal domain of Nup98 by immunogold-labeling using domain-specific antibodies against Nup98 and by expressing epitope tagged versions of Nup98. Our studies revealed that anchorage of Nup98 to NPCs through its C-terminal autoproteolytic domain occurs in the center of the NPC, whereas its N-terminal GLFG domain is more flexible and is detected at multiple locations within the NPC. Additionally, we have confirmed the central localization of Nup98 within the NPC using super resolution structured illumination fluorescence microscopy (SIM) to position Nup98 domains relative to markers of cytoplasmic filaments and the nuclear basket. Our data support the notion that Nup98 is a major determinant of the permeability barrier of NPCs.

From Hypothesis to Mechanism: Uncovering Nuclear Pore Complex Links to Gene Expression

“…However, this does not exclude possible functions for Nups in gene gating. In fact, accumulating evidence has also identified transcriptional roles for Nup98 in the nucleoplasm (61)(62)(63)(64). The dynamic shuttling of Nup98 between the NPC and nucleoplasm requires active transcription (64).…”

Molecular organization of the desmosome as revealed by direct stochastic optical reconstruction microscopy

“…Measuring desmosomal protein organization requires a technique with high resolution coupled with minimal sample manipulation to minimize structural artifacts and preserve physiological relevance. Super-resolution microscopy has revealed sub-resolution protein organization in numerous macromolecular complexes including the nuclear pore, ciliary transition zone, kinetochore, neuronal synapse, focal adhesion and hemidesmosome (Chatel et al, 2012;Loschberger et al, 2014Loschberger et al, , 2012Nahidiazar et al, 2015;Ribeiro et al, 2010;van Hoorn et al, 2014;Yang et al, 2015;Zhong, 2015). Here, we use direct stochastic optical reconstruction microscopy (dSTORM) (Rust et al, 2006) to elucidate protein organization within the desmosome at the molecular level both in vitro and in vivo.…”

Nucleoporins NPP-10, NPP-13 and NPP-20 are required for HCP-4 nuclear import to establish correct centromere assembly

“…On the cytoplasmic side, it interacts directly with Nup188 and on the nuclear side it directly binds to Nup96, indicating a polarized orientation that may be functional for directed transport (Griffis et al, 2003). However, more recently, this view was challenged by data placing Nup98 at the center of the NPC, where it interacts with Nup96/NPP-10C in the Nup107 complex (Chatel et al, 2012). This supports the argument that NPP-10C, NPP-10N and NPP-20 are mediators of a common pore function that is required for HCP-4 import.…”