Analysis of the initiation of nuclear pore assembly by ectopically targeting nucleoporins to chromatin.
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.
['Schwartz|Michal|M|', 'Travesa|Anna|A|', 'Martell|Steven W|SW|', 'Forbes|Douglass J|DJ|']
D021581:Active Transport, Cell Nucleus D001145:Arrhythmias, Cardiac D045744:Cell Line, Tumor D002843:Chromatin D006801:Humans D056968:Lac Repressors D009154:Mutation D009685:Nuclear Envelope D028861:Nuclear Pore Complex Proteins D017434:Protein Structure, Tertiary
nuclear pore assembly LacI Nup133 Nup153 LacO array cardiac arrhythmia ELYS FG, phenylalanine-glycine GFP, green fluorescent protein CFP, cyan fluorescent protein LacO, Lac operon LacI, Lac repressor gene Nup155 R391H Nup160/107 complex nuclear rim localization