Final Activity Report Summary - NPC ASSEMBLY (The Nuclear Pore Complex: Assembly Required) The genome of human cells, plant, animals, and even some unicellular organisms is segregated from the rest of the cell and kept inside the nucleus. The nuclear pore complex (NPC) mediates all traffic between the nucleus and the rest of the cell. The regulation of this traffic affects every aspect of gene expression and a multitude of other cellular processes. The assembly and function of the NPC are therefore a major topic for biomedical research as well as future drug design. We are using a cell-free system derived from frog egg extracts to assemble functional nuclei in the test-tube and to study the mechanism of NPC assembly. To gain a better understanding of this process, we set out to molecularly define early steps in nuclear pore assembly, beginning with chromatin binding. The order of recruitment of the first few building blocks, including two key integral pore-membrane proteins, was established. We find that most of the soluble nuclear pore subunits are added only after the recruitment of these key membrane components. These findings strongly support one of the two conflicting mechanistic models that have been suggested thus far to explain NPC assembly. In addition, we have isolated native soluble nuclear pore subunits from egg extracts and we continued to investigate the regulatory role of the prototypic import receptor, importin, in NPC assembly. Overall, our results provide a detailed view of the early stages of NPC assembly, as well as the basis for mapping the exact molecular interfaces that determine this process.