Tpr is a 267-kDa protein forming coiled coil-dominated homodimers that locate at 
the nucleoplasmic side of the nuclear pore complex (NPC). The proteins that 
tether Tpr to this location are unknown. Moreover, the question whether Tpr 
itself might act as a scaffold onto which other NPC components need to be 
assembled has not been answered to date. To assess Tpr's role as an 
architectural element of the NPC, we have studied the sequential disassembly and 
reassembly of NPCs in mitotic cells, paralleled by studies of cells depleted of 
Tpr as a result of posttranscriptional tpr gene silencing by RNA interference 
(RNAi). NPC assembly and recruitment of several nucleoporins, including Nup50, 
Nup93, Nup96, Nup98, Nup107, and Nup153, in anaphase/early telophase is shown to 
precede NPC association of Tpr in late telophase. In accordance, cellular 
depletion of Tpr by RNAi does not forestall binding of these nucleoporins to the 
NPC. In a search for proteins that moor Tpr to the NPC, we have combined the 
RNAi approach with affinity-chromatography and yeast two-hybrid interaction 
studies, leading to the identification of nucleoporin Nup153 as the binding 
partner for Tpr. The specificity of this interaction is demonstrated by its 
sensitivity to Tpr amino acid substitution mutations that abolish Tpr's ability 
to adhere to the NPC and affect the direct binding of Tpr to Nup153. 
Accordingly, cellular depletion of Nup153 by RNAi is shown to result in 
mislocalization of Tpr to the nuclear interior. Nup153 deficiency also causes 
mislocalization of Nup50 but has no direct effect on NPC localization of the 
other nucleoporins studied in this investigation. In summary, these results 
render Tpr a protein only peripherally attached to the NPC that does not act as 
an essential scaffold for other nucleoporins.