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Research Projects

Structure, Mechanism and Evolution of the Nuclear Pore Complex

The nuclear transport cycle.
The nuclear transport cycle
An import-bound Kap binds to its NLS-bearing cargo in the cytoplasm and transits the NPC by the process of virtual gating. On the nucleoplasmic side, RanGTP binds to the Kap, causing a conformational change which releases the cargo. On the other hand, exporting Kaps bind their cargoes in the presence of RanGTP. The resulting nuclear complexes pass the NPC through the same mechanism of virtual gating. On the cytoplasmic side, RanGTP hydrolysis is stimulated by the cytoplasmically-disposed RanGAP resulting in the release of cargo. RanGDP is then recycled to the nucleoplasm by NTF2 and it is reloaded with GTP to begin another cycle.

Our lab focuses on aspects of the nuclear information pathway; how macromolecular complexes dynamically interact control the passage and transport of genetic information from intranuclear DNA via RNA through the nuclear pore complexes (NPCs). As the sole mediator of nucleocytoplasmic exchange across the nuclear envelope that encloses eukaryotic DNA, NPCs thus define the contents of the nucleus. The pivotal role of the NPC in controlling communication between the genetic material and the rest of the cell is reflected in the many oncogenic and developmental defects directly associated with alterations in nucleocytoplasmic transport. Nucleocytoplasmic transport depends on the interplay between transport cargoes (which carry NLS or NES targeting sequences), their cognate soluble transport factors (many termed kaps), and NPCs.
Rout & Aitchison 2001 (PDF)

As a full understanding of how the NPC mediates transport is needed to discern the nature of these defects, we have taken a comprehensive approach to defining the functional architecture of the NPC in the model eukaryote Saccharomyces (yeast). We have identified all the yeast NPC proteins (nups) and plotted their approximate relative positions, which has allowed us to propose a new mechanism for nuclear transport.
Rout et al., 2000 (PDF)
Rout et al., 2003 (PDF)

Proteomic data can be used to determine the architectures of macromolecular assemblies. 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. Thus, we have determined the position, shape and stoichiometry of each nup, and have systematically isolated nup subcomplexes and analyzed their composition by mass spectrometry in order to determine the network of interactions they make. Together, this wealth of information represents thousands of spatial restraints, allowing us to to localize the NPC's 456 constituent proteins.
Alber et al, 2007 A (PDF)

Diagram of NPC and associated transport factors.
Diagram of NPC and associated transport factors

The resulting map supports our hypothesis that the NPC is derived from an ancient membrane-coating complex (which we term the protocoatomer), related to the clathrin/adaptin and COP complexes.
Devos et al., 2004 (PDF)
Devos et al., 2006 (PDF)

Our structure reveals that half of the NPC is made up of a core scaffold; it is this that 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.
Alber et al, 2007 B (PDF)

Localization of major substructures and their component nucleoporins in the NPC.
Three-dimensional structure of the yeast NPC.
Protein-based substructures of various types — outer rings (yellow), inner rings (purple), membrane rings (brown), linker nucleoporins (blue and pink), and FG nucleoporins (green) — assemble to form the nuclear pore complex (center). The pore membrane is gray.
Predicted secondary structure maps of nups.
Predicted secondary structure maps of nups.
Thin horizontal lines represent the primary sequence of each protein; secondary structure predictions are shown as columns above each line for -strands (cyan) and -helices (magenta), with arrows showing proeolytically sensitive sites. Ribbon representation of nup models: -sheets ( -propellers), -helices ( -solenoids) are colored magenta, cadherin domains are dark blue, the autoproteolytic domain of Nup98 are yellow, the RRM is orange, predicted transmembrane helices are shown in green, coiled-coils in red, FG repeats in black, and unstructured regions are represented by an empty box.