Structure and Function of the Nuclear Pore Complex
The nuclear pore complex (NPC) is a supramolecular protein structure in the nuclear envelope that creates a forty-nanometer channel connecting the cytoplasm and nucleoplasm of eukaryotic cells. Its main function is to regulate the vital flow of proteins and RNA between these two major compartments. Small metabolites diffuse freely across the NPC, but the flow of most proteins and RNA is selective and requires specific transit signals. The signals are recognized by mobile receptors termed karyopherins (also called importins, exportins and transportins), which interact with proteins of the NPC (nucleoporins; Nups) to shuttle cargo across the NPC. The process requires thermal energy to operate the NPC machinery, and chemical energy to impart directionality to the transport process via the Ran GTPase.
Research in my lab focuses on two fundamental aspects of nucleocytoplasmic transport:
I. The mechanics of karyopherin movement across the NPC
II. The structure of the NPC transport conduit
We are addressing these topics using a combination of cell biological, biochemical, biophysical, structural, genetic, and molecular modeling techniques in the model eukaryote S. cerevisiae.
I. Mechanics of karyopherin transport across the nuclear pore complex
Many karyopherins and their cargo have been identified but a mechanistic description of how they are mobilized within the NPC is lacking. Each NPC contains more than 200 potential docking sites for karyopherins (provided by nucleoporins that contain FG repeats), so movement of karyopherin-cargo complexes across the NPC is envisioned to be a stochastic process that operates via repeated association-dissociation reactions of karyopherins with FG nucleoporins. Currently, we are charting the path of transport used by karyopherins within the NPC through the identification of nucleoporins that physically contact them in situ within the NPC transport conduit. We are also addressing the kinetics of karyopherin transport across the NPC by characterizing the dynamics of association and dissociation between karyopherins, cargos and nucleoporins, and by identifying factors (KaRFs) that function to accelerate the dissociation rate of the most stable, long-lived intermediate complexes in the transport processes.
II. Architecture of the NPC transport conduit
Although the mechanics of karyopherin-mediated transport across the NPC are still poorly understood, it is clear that interactions between karyopherins and FG Nups are central to the translocation process. Thus, knowledge of the structural characteristics of FG nucleoporins may explain how karyopherin-cargo complexes of different shapes and sizes can translocate across the NPC while its permeability barrier remains intact. We are characterizing the structure of individual FG nucleoporins using biophysical, structural and molecular modeling techniques. We find that FG repeat regions of Nups are largely devoid of secondary structure and are mostly random coils 200-700 amino acids in length. Thus, the ~200 FG Nups present in each NPC likely form a flexible and highly amorphous meshwork of filaments at its center, which captures and engulfs karyopherin-cargo complexes of different shapes and sizes as they move across the NPC. We are currently testing the notion that FG Nups self-assemble into a meshwork of filaments held together by weak hydrophobic interactions between FG repeats.