Nup2p dynamically associates with the distal regions of the yeast nuclear pore complex

Abstract. Nucleocytoplasmic transport is mediated by the interplay between soluble transport factors and nucleoporins resident within the nuclear pore complex (NPC). Understanding this process demands knowledge of components of both the soluble and stationary phases and the interface between them. Here, we provide evidence that Nup2p, previously considered to be a typical yeast nucleoporin that binds import- and exportbound karyopherins, dynamically associates with the NPC in a Ran-facilitated manner. When bound to the NPC, Nup2p associates with regions corresponding to the nuclear basket and cytoplasmic fibrils. On the nucleoplasmic face, where the Ran–GTP levels are predicted to be high, Nup2p binds to Nup60p. Deletion o

Determining the architectures of macromolecular assemblies

To understand the workings of a living cell, we need to know the architectures of its macromolecular assemblies. Here we show how proteomic data can be used to determine such structures. The process involves the collection of sufficient and diverse high-quality data, translation of these data into spatial restraints, and an optimization that uses the restraints to generate an ensemble of structures consistent with the data. Analysis of the ensemble produces a detailed architectural map of the assembly. We developed our approach on a challenging model system, the nuclear pore complex (NPC). The NPC acts as a dynamic barrier, controlling access to and from the nucleus, and in yeast is a 50 MDa assembly of 456 proteins. The resulting structure, presented in an accompanying paper, reveals the configuration of the proteins in the NPC, providing insights into its evolution and architectural principles. The present approach should be applicable to many other macromolecular assemblies.

The molecular architecture of the nuclear pore complex

Nuclear pore complexes (NPCs) are proteinaceous assemblies of approximately 50 MDa that selectively transport cargoes across the nuclear envelope. To determine the molecular architecture of the yeast NPC, we collected a diverse set of biophysical and proteomic data, and developed a method for using these data to localize the NPC’s 456 constituent proteins (see the accompanying paper). Our structure reveals that half of the NPC is made up of a core scaffold, which is structurally analogous to vesicle-coating complexes. This scaffold forms an interlaced network that coats the entire curved surface of the nuclear envelope membrane within which the NPC is embedded. The selective barrier for transport is formed by large numbers of proteins with disordered regions that line the inner face of the scaffold. The NPC consists of only a few structural modules that resemble each other in terms of the configuration of their homologous constituents, the most striking of these being a 16-fold repetition of ‘columns’. These findings provide clues to the evolutionary origins of the NPC.