Molecular and ultrastructural analysis of Tpr, a nuclear pore complex-attached coiled-coil protein

Abstract

Anchored in the nuclear envelope, nuclear pore complexes (NPCs) are large multiprotein complexes which serve as the gateway for nucleocytoplasmic transport. In vertebrates, a 267kDa protein termed Tpr has been localized to the nucleoplasmic side of the NPCs. The function of Tpr has remained largely elusive. The main focus of this thesis has been to gain insight into Tprs ultrastructural properties and investigate its potential contribution to NPC and intranuclear architecture.

The Tpr protein is divided into a large, coiled-coil forming aminoterminal domain and a shorter, highly acidic carboxyterminal domain. By constructing expression vectors encoding various Tpr deletion mutants, we mapped the position of Tprs nuclear localization signal within the carboxyterminal domain, whereas a short segment of the aminoterminal domain was shown to be sufficient for NPC- binding. Individual amino acid substitutions introduced into this region were shown to abolish Tprs ability to bind to the NPC and instead rendered the protein soluble, resulting in its accumulation in the nuclear interior.

To gain insight into Tprs ultrastructural properties, we studied recombinant Tpr polypeptides by circular dichroism spectroscopy, chemical cross-linking, and rotary shadowing electron microscopy. We showed that Tprs aminoterminal domain forms coiled-coil homodimers in vitro, and has an extended rod-like shape. Using a yeast-two hybrid approach, the arrangement of the coiled-coil was shown to be in parallel and in register. However, Tpr was neither found to self-assemble into extended linear filaments nor stably bound to other intranuclear structures.

At this point it was still uncertain whether or not Tpr acts as a scaffold onto which other NPC proteins (termed as nucleoporins) need to assemble. Moreover, it was also unknown which nucleoporin is the binding partner for Tpr at the NPC. To assess these questions we studied the sequential disassembly and reassembly of NPCs in mitotic cells, paralleled by studies of cells depleted of Tpr by RNA interference. The loss of Tpr was shown to neither have an effect on the assembly of the NPC nor the recruitment of any of various other nucleoporins. Using two-hybrid approaches and affinity chromatography, the binding partner that tethers Tpr to the NPC was found to be a nucleoporin termed Nup 153. Whereas RNAi-promoted loss of Tpr had no effect on correct incorporation of Nup153 into the NPC, cellular depletion of Nup153 by RNAi abolished NPC binding of Tpr and caused mislocalization of Tpr to the nuclear interior.

In summary, this study outlines all basic structural characteristics of Tpr and provides insight into its architectural properties as a protein peripherally attached to the NPC.