Effect of FG Motifs on Ordering Proteins within the Nuclear Pore Complex

Abstract

Transport across the nuclear membrane occurs through the nuclear pore complex (NPC). The transport through the NPC is both selective and rapid, and involves the phenylalanine glycine repeat nucleoporins (FG nups). The FG motifs can bind cargo and may also be able to interact with each other but the mechanism of transport remains unclear. We have developed a theoretical framework which exploits the symmetry of the NPC and its organization in the nuclear envelope to resolve the order and disorder of individual protein domains within the complex. Specific domains of individual nucleoporins (nups) were tagged with GFP and examined using fluorescence polarization microscopy to determine their organization and dynamics. We characterized the degree to which the FG domains are ordered in vivo. This approach revealed different degrees of organization within a single FG domain: the tips and middle of the FG domains are less ordered than NPC-anchored domains, but still display a surprising degree of organization. We examined three mammalian nups: Nup54, Nup62, and Nup98. We observe that of these, Nup54 is the most ordered within the NPC and Nup62 is the least ordered. We then investigated the contribution of the FG repeats to the organization of these nucleoporins by mutating the FG motifs to AG. We show that these mutations have no effect on the organization of Nup62, decrease but do not eliminate the order shown by Nup54, and prevent Nup98 from localizing correctly to the NPC. We therefore propose that multiple factors contribute to the organization of FG nups in vivo, and that the influence of the FG repeats on order varies between different FG nups.