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Vol. 14, Issue 5, 1923-1940, May 2003
Department of Cell and Molecular Biology, Medical Nobel Institute, Karolinska Institutet, S-17177 Stockholm, Sweden
Submitted September 30, 2002;
Revised December 13, 2002;
Accepted January 23, 2003
Monitoring Editor: Jennifer Lippincott-Schwartz
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.
Abbreviations used: aa, amino acid; AD, activation domain; AL, annulate lamellae; BD, binding domain; FA, formaldehyde; IF, immunofluorescence microscopy; NE, nuclear envelope; NBD, nuclear pore complex binding domain; NPC, nuclear pore complex; nt, nucleotide; RNAi, RNA interference; siRNA, small interfering RNA.
* Corresponding author. E-mail address: volker.cordes{at}cmb.ki.se.
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