Production of inositol hexakisphosphate (IP 6 ) by Ipk1, the inositol-1,3,4,5,6-pentakisphosphate 2-kinase, is required for Gle1-mediated mRNA export in Saccharomyces cerevisiae cells. To examine the network of interactions that require IP 6 production, an analysis of fitness defects was conducted in mutants harboring both an ipk1 null allele and a mutant allele in genes encoding nucleoporins or transport factors. Enhanced lethality was observed with a specific subset of mutants, including nup42, nup116, nup159, dbp5, and gle2, all of which had been previously connected to Gle1 function. Complementation of the nup116⌬ipk1⌬ and nup42⌬ipk1⌬ double mutants did not require the Phe-Gly repeat domains in the respective nucleoporins, suggesting that IP 6 was acting subsequent to heterogeneous nuclear ribonucleoprotein targeting to the nuclear pore complex. With Nup42 and Nup159 localized exclusively to the nuclear pore complex cytoplasmic side, we speculated that IP 6 may regulate a cytoplasmic step in mRNA export. To test this prediction, the spatial requirements for the production of IP 6 were investigated. Restriction of Ipk1 to the cytoplasm did not block IP 6 production. Moreover, coincident sequestering of both Ipk1 and Mss4 (an enzyme required for phosphatidylinositol 4,5-bisphosphate production) to the cytoplasm also did not block IP 6 production. Given that the kinase required for inositol 1,3,4,5,6-pentakisphosphate production (Ipk2) is localized in the nucleus, these results indicated that soluble inositides were diffusing between the nucleus and the cytoplasm. Additionally, the cytoplasmic production of IP 6 by plasma membrane-anchored Ipk1 rescued a gle1-2 ipk1-4 synthetic lethal mutant. Thus, cytoplasmic IP 6 production is sufficient for mediating the Gle1-mRNA export pathway.The nucleus is the defining structure of a eukaryotic cell and houses the genetic information that characterizes the organism. The nuclear compartment is separated from the cytoplasm by the nuclear envelope (NE), 1 two lipid bilayer membranes that join to form pores containing nuclear pore complexes (NPCs). The 60-MDa NPC structure is assembled from multiple copies of ϳ30 distinct proteins designated nucleoporins (Nups) (1, 2). The central region of the NPC consists of inner and outer rings connected by a series of spokes. Fibrils extend from either face of the NPC, and the filaments on the nuclear side are joined at the distal end to form a basket structure (3). The NPCs create aqueous semipermeable portals across the NE that allow the passive diffusion of small molecules. However, translocation of macromolecules through the NPC requires facilitated transport mediated by Nups, other NPC-associated proteins, and shuttling transport factors (4 -6). One class of macromolecules that must traverse the NPC is mRNAs. The movement of mRNA through the NPC is a regulated process in the progression from DNA transcription in the nucleus to the production of a translated protein in the cytoplasm (7,8). To ensure proper gene expression, there are...