Nuclear pore complexes (NPCs) control the passage of large biological molecules into and out of the nucleus. Dr. Rout is interested in how NPCs mediate this transport and in understanding the nature of cancer-causing and developmental defects associated with alterations to it. With collaborators, his lab is also working to develop technology to map and interpret the dynamic molecular interactions within a cell.
Using a variety of techniques and yeast as a model system, Dr. Rout and his colleagues are studying the structure of NPCs and relating the structure to sites of interactions and reactions with soluble nuclear transport factors. They hope to gain a better understanding of the part NPCs play in nuclear regulation and maintenance.
A full understanding of how NPCs mediate transport requires a comprehensive inventory of the molecular components of NPCs, a knowledge of how each component contributes to the overall structure of these large molecular translocation machines, and information on the interactions their proteins make with components of the soluble phase. Dr. Rout and his colleagues have catalogued the components of the yeast NPC and determined that it is composed of a surprisingly small number of proteins whose size and overall high degree of symmetry account for the NPC’s large mass. They have also determined the position, shape, fold type, and stoichiometry of each nuclear protein, or “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, which have been computationally integrated into a three-dimensional map of the NPC’s architecture sufficient to resolve the molecular organization of the entire NPC. This mapping has exposed similarities between structures in coated vesicles and those in the NPC, supporting a hypothesis for their common evolutionary origin in a progenitor protocoatomer. Moreover, the map reveals an extensive underlying modularity in the architecture of the NPC, suggesting that repeated duplication events led to the evolution of the NPC’s final architecture. Work continues to characterize the architecture of the NPC.
The lab is studying members of the mobile phase in a similar fashion, focusing on the kinetic behavior of nup-transport factor and transport factor-cargo interactions using a variety of in vitro and in vivo approaches. Already, these results have suggested a mechanism for nuclear transport. The lab’s eventual aim is to integrate ultrastructural and biochemical studies to understand the molecular basis of the translocation of different transport factors across the NPC. Dr. Rout aims to reconstitute key reactions of these processes in vitro, study the high-resolution structures mediating the transport processes, and test possible mechanistic models in vivo to understand the complete sequence of events during a transport cycle.
With support from the National Institutes of Health, Dr. Rout has formed the National Center for Dynamic Interactome Research (www.ncdir.org), where several laboratories at Rockefeller and at other institutions are collaborating to develop the methodology required to obtain a comprehensive map of protein interactions within any organism and to study their dynamic behaviors. This will allow researchers to explore the utility of the technology for functional elucidation of complex biological processes, with an initial focus on those processes related to cell cycle control, transcription, oncogenesis, and viral infection.