Analysis of the initiation of nuclear pore assembly by ectopically targeting nucleoporins to chromatin

Abstract

Nuclear pore complexes (NPCs) form the gateway to the nucleus, mediating virtually all nucleocytoplasmic trafficking. Assembly of a nuclear pore complex requires the organization of many soluble sub-complexes into a final massive structure embedded in the nuclear envelope. By use of a LacI/LacO reporter system, we were able to assess nucleoporin (Nup) interactions, show that they occur with a high level of specificity, and identify nucleoporins sufficient for initiation of the complex process of NPC assembly in vivo. Eleven nucleoporins from different sub-complexes were fused to LacI-CFP and transfected separately into a human cell line containing a stably integrated LacO DNA array. The LacI-Nup fusion proteins, which bound to the array, were examined for their ability to recruit endogenous nucleoporins to the intranuclear LacO site. Many could recruit nucleoporins of the same sub-complex and a number could also recruit other sub-complexes. Strikingly, Nup133 and Nup107 of the Nup107/160 subcomplex and Nup153 and Nup50 of the nuclear pore basket recruited a near full complement of nucleoporins to the LacO array. Furthermore, Nup133 and Nup153 efficiently targeted the LacO array to the nuclear periphery. Our data support a hierarchical, seeded assembly pathway and identify Nup133 and Nup153 as effective "seeds" for NPC assembly. In addition, we show that this system can be applied to functional studies of individual nucleoporin domains as well as to specific nucleoporin disease mutations. We find that the R391H cardiac arrhythmia/sudden death mutation of Nup155 prevents both its subcomplex assembly and nuclear rim targeting of the LacO array.

A self-inhibitory interaction within Nup155 and membrane binding are required for nuclear pore complex formation

“…The R391H mutation was suggested to affect NPC localization of Nup155. Indeed, in a targeting assay, in which Nup155 R391H was ectopically localized to chromatin, its interaction partners Nup53 and POM121 showed less corecruitment compared with the wild-type Nup155 control (Schwartz et al, 2015). However, given that Nup155 is essential in C. elegans, Drosophila and mouse, most likely because of its crucial role in NPC assembly, it seems questionable whether organisms are viable without Nup155 being able to localize to assembling NPCs.…”

Leukemia-Associated Nup214 Fusion Proteins Disturb the XPO1-Mediated Nuclear-Cytoplasmic Transport Pathway and Thereby the NF-κB Signaling Pathway

“…The formation of RanGTP-XPO1-NES protein complexes is enhanced by Nup98 (64) and RanBP3 (65)(66)(67). Nup214 functions as a scaffold for the recruitment of several nucleoporins, such as Nup88, Nup358, Nup62, and Nup98 (38,40,68,69). These proteins may facilitate stable complex formation induced by SET-Nup214 and DEK-Nup214.…”

Lost in Transportation: Nucleocytoplasmic Transport Defects in ALS and Other Neurodegenerative Diseases

“…A single NPC is assembled from approximately 30 Nups, each present in multiple copies (Cronshaw et al, 2002; Davis and Blobel, 1986; Rout et al, 2000; Schmidt and Gorlich, 2016). Based on their approximate location within the NPC and their structural role, Nups may be organized into seven general categories (Figure 1): (1) cytoplasmic filament Nups, (2) nucleoplasmic and cytoplasmic ring Nups, (3) inner ring complex Nups, (4) transmembrane Nups, (5) central Nups containing phenylalanine-glycine (FG) repeats, (6) nuclear basket Nups, and (7) adaptor Nups (Hoelz et al, 2016; Schwartz et al, 2015; Schwartz, 2016). The role of the NPC in ALS has entered the spotlight due to several recent lines of evidence, including the finding that genes encoding NPC constituents are genetic modifiers of disease in ALS model systems, the demonstration that disease-related proteins may directly poison the NPC, and the identification of ALS-causing mutations in at least one component of the NPC, as described in detail below.…”