One of the great hallmarks of evolution is the enclosure of genetic information in the nucleus. This spatial separation creates the necessity for efficient communication between the nucleus and the cytoplasm, which is achieved through the selective transport of folded proteins and of protein/nucleic acid complexes across the double membrane of the nuclear envelope (NE). The nuclear pore complex (NPC) is the sole gateway that allows passage of macromolecules through the NE, making this transport organelle an essential machine for eukaryotic life. NPCs are embedded in circular pores permeating the NE and can accomplish the bidirectional transport of particles of up to ~40 nm in diameter and at a rate of several hundred events per second. Electron microscopic studies have revealed that the NPC consists of a central core with an 8-fold rotational symmetry across a nucleo-cytoplasmic axis and a two-fold rotational symmetry across the plane of the NE. This symmetric core links to "cytoplasmic filaments" and a "nuclear basket" structure. The NPC is built from approximately 30 distinct proteins, termed nucleoporins (nups) that are organized into six distinct subcomplexes. Each nup is present in the NPC in multiple copies such that the entire assembly reaches the extraordinary molecular mass of ~60 MDa in yeast and even more in vertebrates. The NPC functions not just as a transport channel, but has a comprehensive role in other modes of gene regulation, for example through direct interaction with the transcription and mRNA export machineries. As such, it is less surprising that NPC dysfunction has been observed in a diverse set of human illnesses, such as neoplastic or retroviral disease. These associations as well as the NPC's existential role in eukaryotic cell biology have motivated investigations into its detailed architecture. The NPC's size and flexibility along with the unavailability of sufficient quantities of suitable material presently preclude the crystallographi determination of the structure of the entire intact NPC in one piece. An alternative approach proposed herein seeks to elucidate the atomic architecture of the NPC through recombinant reconstitution and crystallographic characterization of NPC subcomplexes, which constitute the physiological building blocks of the intact NPC in vivo. Combined with electron microscopic reconstruction, biochemical protein-protein interaction maps and cellular assays, this strategy is designed to lead to a composite pseudo-atomic model for the entire NPC and provide a roadmap for comprehensive structure-function analyses. As such, the outcome of the proposed research is expected to further our understanding of the molecular mechanisms that govern the involvement of the NPC in nucleocytoplasmic transport and other cellular processes, while at the same time creating a mechanistic basis for currently untreatable "nucleoporin diseases." Furthermore, the methodologies developed herein will challenge the current boundaries of structural cell biology and serve as a paradigm for other large macromolecular assemblies with essential cellular roles whose functional mechanism has remained elusive due to lack of structural insight.
In human cells, the hereditary material is sequestered in a separate compartment, which is accessible only through so-called nuclear pore complexes (NPCs). NPCs constitute massive transport channels through which traffic is tightly regulated and changes to NPC components have been linked to a diverse set of human diseases, such as aggressive forms of leukemia. Our research seeks to enhance our understanding of the NPC in health and disease by creating a molecular snapshot of the NPC that depicts its atomic architecture.