Amino Acid Substitutions of Coiled-Coil Protein Tpr Abrogate Anchorage to the Nuclear Pore Complex but Not Parallel, In-Register Homodimerization

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Vol. 12, Issue 8, 2433-2452, August 2001

Amino Acid Substitutions of Coiled-Coil Protein Tpr Abrogate Anchorage to the Nuclear Pore Complex but Not Parallel, In-Register Homodimerization

Manuela E. Hase, Nikolai V. Kuznetsov, and Volker C. Cordes^*

Department of Cell and Molecular Biology, Medical Nobel Institute, Karolinska Institutet, S-17177 Stockholm, Sweden

Tpr is a protein component of nuclear pore complex (NPC)-attached intranuclear filaments. Secondary structure predictionssuggest a bipartite structure, with a large N-terminal domaindominated by heptad repeats (HRs) typical for coiled-coil-formingproteins. Proposed functions for Tpr have included roles as ahomo- or heteropolymeric architectural element of the nuclearinterior. To gain insight into Tpr's ultrastructural properties,we have studied recombinant Tpr segments by circular dichroismspectroscopy, chemical cross-linking, and rotary shadowing electronmicroscopy. We show that polypeptides of the N-terminal domainhomodimerize in vitro and represent $alpha$ -helical molecules of extendedrod-like shape. With the use of a yeast two-hybrid approach, arrangementof the coiled-coil is found to be in parallel and in register.To clarify whether Tpr can self-assemble further into homopolymericfilaments, the full-length protein and deletion mutants were overexpressedin human cells and then analyzed by confocal immunofluorescencemicroscopy, cell fractionation, and immuno-electron microscopy.Surplus Tpr, which does not bind to the NPC, remains in a solublestate of ~7.5 S and occasionally forms aggregates of entangledmolecules but neither self-assembles into extended linear filamentsnor stably binds to other intranuclear structures. Binding tothe NPC is shown to depend on the integrity of individual HRs;amino acid substitutions within these HRs abrogate NPC bindingand render the protein soluble but do not abolish Tpr's generalability to homodimerize. Possible contributions of Tpr to thestructural organization of the nuclear periphery in somatic cellsarediscussed.

^* Corresponding author. E-mail address: volker.cordes{at}cmb.ki.se.

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