The flexible composition, structure, and evolution of the yeast nuclear pore complex

The movement of macromolecules between the eukaryotic cytoplasm and nucleus is regulated by protein machines termed nuclear pore complexes (NPCs). NPCs are embedded in the nuclear envelope, the double membrane layer separating the nucleus and cytoplasm. As the only aqueous conduits connecting these compartments, NPCs serve two functions in the nucleo-cytoplasmic trafficking of macromolecules. First, the NPC creates a permeability barrier that restricts the movement of molecules larger than its size exclusion limit (∼30--40 kDa). Secondly, the NPC facilitates the movement of macromolecules (cargo) with signal sequences through the permeability barrier via soluble adapter proteins (karyopherins). NPCs are massive (60 to 120 MDa), consisting of ∼30 different proteins (nucleoporins), each of which is found in multiple copies per NPC.; Due to its fixed location in the nuclear envelope, the NPC is referred to as the "stationary phase" of transport, whereas the karyopherin-cargo complexes that move through it are the "mobile phase". In reality, the NPC is more dynamic than the "stationary" label suggests, and in this dissertation we describe NPC dynamics and flexibility in three ways. First, we demonstrate that the nucleoporin composition of the NPC changes in response to the abundance of specific transport factors. This indicates that the NPC is sensitive to the cellular environment, altering its architecture and its transport capacity to accommodate changes in the cell. Secondly, we use structural analyses to show that the nucleoporins involved directly in transport are unstructured in situ at the NPC and lack significant contributions of secondary structure. The nucleoporins represent the first example of a family of proteins that function collectively in an unstructured conformation. We propose that the NPC permeability barrier consists of a flexible meshwork of disordered proteins, implying a transport mechanism in which karyopherin-cargo complexes must bind and deform the meshwork in transit. Finally, we show that the amino acid sequences of disordered nucleoporins evolve rapidly and are permissive of amino acid substitutions. Together, these studies demonstrate that the NPC is a dynamic machine, adaptable to cellular conditions, structurally capable of accommodating different karyopherin-cargo transport complexes, and suited for the rapid evolution of expanded transport capacities.