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.