The yeast nucleoporin Nup53p specifically interacts with Nic96p and is directly involved in nuclear protein import.

The bidirectional nucleocytoplasmic transport of macromolecules is mediated by the nuclear pore complex (NPC) which, in yeast, is composed of approximately 30 different proteins (nucleoporins). Pre-embedding immunogold-electron microscopy revealed that Nic96p, an essential yeast nucleoporin, is located about the cytoplasmic and the nuclear periphery of the central channel, and near or at the distal ring of the yeast NPC. Genetic approaches further implicated Nic96p in nuclear protein import. To more specifically explore the potential role of Nic96p in nuclear protein import, we performed a two-hybrid screen with NIC96 as the bait against a yeast genomic library to identify transport factors and/or nucleoporins involved in nuclear protein import interacting with Nic96p. By doing so, we identified the yeast nucleoporin Nup53p, which also exhibits multiple locations within the yeast NPC and colocalizes with Nic96p in all its locations. Whereas Nup53p is directly involved in NLS-mediated protein import by its interaction with the yeast nuclear import receptor Kap95p, it appears not to participate in NES-dependent nuclear export.

Comparative spatial localization of protein-A-tagged and authentic yeast nuclear pore complex proteins by immunogold electron microscopy.

The nuclear pore complex (NPC) mediates protein and RNP import in and RNA and RNP export out of the nucleus of eukaryotic cells. Due to its genetic tractability, yeast offers a versatile system for investigating the chemical composition and molecular architecture of the NPC. In this context, protein A tagging is a commonly used tool for characterizing and localizing yeast NPC proteins (nucleoporins). By preembedding anti-protein A immunogold electron microscopy (immunogold EM), we have localized two yeast nucleoporins, Nsp1p and Nic96p, in mutant yeast strains recombinantly expressing these nucleoporins tagged with four (Nsp1p) or two (Nic96p) IgG binding domains of protein A (i.e., ProtA-Nsp1p and ProtA-Nic96p). We have compared the location of the recombinant fusion proteins ProtA-Nsp1p and ProtA-Nic96p (i.e., as specified by their protein A tag) to the location of authentic Nsp1p and Nic96p (i.e., as defined by the epitopes recognized by corresponding nucleoporin antibodies) and found all of them to reside at the same three NPC sites. Hence, recombinant expression and protein A tagging of the nucleoporins Nsp1p and Nic96p have not caused any significant mislocation of the fusion proteins and thus enabled mapping of these two yeast nucleoporins at the ultrastructural level in a faithful manner.

The RNA export factor Gle1p is located on the cytoplasmic fibrils of the NPC and physically interacts with the FG-nucleoporin Rip1p, the DEAD-box protein Rat8p/Dbp5p and a new protein Ymr 255p.

Gle1p is an essential, nuclear pore complex (NPC)-associated RNA export factor. In a screen for high copy suppressors of a GLE1 mutant strain, we identified the FG-nucleoporin Rip1p and the DEAD-box protein Rat8p/Dbp5p, both of which have roles in RNA export; we also found Ymr255p/Gfd1p, a novel inessential protein. All three high copy suppressors interact with the C-terminal domain of Gle1p; immunoelectron microscopy localizations indicate that Gle1p, Rip1p and Rat8p/Dbp5p are present on the NPC cytoplasmic fibrils; Rip1p was also found within the nucleoplasm and on the nuclear baskets. In vivo localizations support the hypothesis that Rip1p contributes to the association of Gle1p with the pore and that Gle1p, in turn, provides a binding site for Rat8p/Dbp5p at the NPC. These data are consistent with the view that Gle1p, Rip1p, Rat8p/Dbp5p and Ymr255p/Gfd1p associate on the cytoplasmic side of the NPC to act in a terminal step of RNA export. We also describe a human functional homologue of Rip1p, called hCG1, which rescues Rip1p function in yeast, consistent with the evolutionary conservation of this NPC-associated protein.

The nuclear pore complex: from molecular architecture to functional dynamics.

Toward dissecting the molecular composition and architecture of the nuclear pore complex (NPC), over the past 18 months novel nucleoporins and NPC subcomplexes were identified and characterized. The three-dimensional structure of isolated yeast NPCs was determined by electron cryomicroscopy. New specimen preparation and labeling protocols localized a number of nucleoporins and NPC subcomplexes within the three-dimensional architecture of the yeast NPC. Structural changes of native NPCs mediated by physiological effectors such as calcium or ATP were monitored by time-lapse atomic force microscopy, thus revealing a first glimpse of the NPC's functional dynamics.

Molecular architecture of the yeast nuclear pore complex: localization of Nsp1p subcomplexes.

The nuclear pore complex (NPC), a supramolecular assembly of approximately 100 different proteins (nucleoporins), mediates bidirectional transport of molecules between the cytoplasm and the cell nucleus. Extensive structural studies have revealed the three- dimensional (3D) architecture of Xenopus NPCs, and eight of the approximately 12 cloned and characterized vertebrate nucleoporins have been localized within the NPC. Thanks to the power of yeast genetics, 30 yeast nucleoporins have recently been cloned and characterized at the molecular level. However, the localization of these nucleoporins within the 3D structure of the NPC has remain elusive, mainly due to limitations of preparing yeast cells for electron microscopy (EM). We have developed a new protocol for preparing yeast cells for EM that yielded structurally well-preserved yeast NPCs. A direct comparison of yeast and Xenopus NPCs revealed that the NPC structure is evolutionarily conserved, although yeast NPCs are 15% smaller in their linear dimensions. With this preparation protocol and yeast strains expressing nucleoporins tagged with protein A, we have localized Nsp1p and its interacting partners Nup49p, Nup57p, Nup82p, and Nic96p by immuno-EM. Accordingly, Nsp1p resides in three distinct subcomplexes which are located at the entry and exit of the central gated channel and at the terminal ring of the nuclear basket.

Nuclear mRNA export requires complex formation between Mex67p and Mtr2p at the nuclear pores.

We have identified between Mex67p and Mtr2p a complex which is essential for mRNA export. This complex, either isolated from yeast or assembled in Escherichia coli, can bind in vitro to RNA through Mex67p. In vivo, Mex67p requires Mtr2p for association with the nuclear pores, which can be abolished by mutating either MEX67 or MTR2. In all cases, detachment of Mex67p from the pores into the cytoplasm correlates with a strong inhibition of mRNA export. At the nuclear pores, Nup85p represents one of the targets with which the Mex67p-Mtr2p complex interacts. Thus, Mex67p and Mtr2p constitute a novel mRNA export complex which can bind to RNA via Mex67p and which interacts with nuclear pores via Mtr2p.