Transport of macromolecules between the nucleus and cytoplasm is a critical cellular process for eukaryotes, and the machinery that mediates nucleocytoplasmic exchange is subject to multiple levels of control. Regulation is achieved by modulating the expression or function of single cargoes, transport receptors, or the transport channel. Each of these mechanisms has increasingly broad impacts on transport patterns and capacity, and this hierarchy of control directly affects gene expression, signal transduction, development, and disease.

The nuclear envelope is a physical barrier between the nucleus and cytoplasm and, as such, separates the mechanisms of transcription from translation. This compartmentalization of eukaryotic cells allows spatial regulation of gene expression; however, it also necessitates a mechanism for transport between the nucleus and cytoplasm. Macromolecular trafficking of protein and RNA occurs exclusively through nuclear pore complexes (NPCs), specialized channels spanning the nuclear envelope. A novel family of NPC proteins, the FG-nucleoporins (FG-Nups), coordinates and potentially regulates NPC translocation. The extensive repeats of phenylalanine-glycine (FG) in each FG-Nup directly bind to shuttling transport receptors moving through the NPC. In addition, FG-Nups are essential components of the nuclear permeability barrier. In this review, we discuss the structural features, cellular functions, and evolutionary conservation of the FG-Nups.Subcellular compartmentalization of eukaryotic cells into organelles imparts functional and spatial separation of essential cellular processes. Interorganellar communication, however, is required to coordinate activities within the cell. The movement of molecules between the cytoplasm and a given organelle is accomplished by the use of a regulatory transport pore(s) embedded in the organelle membrane. One of the most complex molecular translocons is the nuclear pore complex (NPC), which mediates all traffic of macromolecules in and out of the nucleus.NPCs are large, selective channels that regulate the nucleocytoplasmic transport of macromolecules but are permeable to the movement of ions, small metabolites, and small proteins by free diffusion. The ability of the NPC to rapidly transport specific macromolecules and coincidently selectively preclude other molecules from entering the nucleus is one of the mysteries of this biological machine. To overcome the permeability barrier, each cargo greater than ϳ40 kDa must display a nuclear localization sequence (NLS) or nuclear export sequence (NES). The respective NLS or NES is recognized and bound by a specific transport receptor, of which many exist in eukaryotic cells.Transport receptors interact with a subset of NPC proteins to mediate translocation and, as such, serve as a molecular bridge between NPC proteins and cargoes to allow efficient nuclear import and export. A unique family of NPC proteins is directly involved, and are designated the FG-nucleoporins (FG-Nups). The FG-Nups are characterized by domains with extensive repeats of phenylalanine-glycine (FG), and these proteins have specific and essential roles in transport through the NPC (discussed below). Recent work has offered many insights into the biophysical nature of the FG-Nups. Structural aspects of interactions between FG-Nups and transport receptors have been resolved, and regulatory roles of FG-Nups in transport, disease, and development have been discovered. Importantly, understanding the structural, functional, and regulatory properties of FG-Nups has provided new ...

Trafficking of nucleic acids and large proteins through nuclear pore complexes (NPCs) requires interactions with NPC proteins that harbor FG (phenylalanine-glycine) repeat domains. Specialized transport receptors that recognize cargo and bind FG domains facilitate these interactions. Whether different transport receptors utilize preferential FG domains in intact NPCs is not fully resolved. In this study, we use a large-scale deletion strategy in Saccharomyces cerevisiae to generate a new set of more minimal pore (mmp) mutants that lack specific FG domains. A comparison of messenger RNA (mRNA) export versus protein import reveals unique subsets of mmp mutants with functional defects in specific transport receptors. Thus, multiple functionally independent NPC translocation routes exist for different transport receptors. Our global analysis of the FG domain requirements in mRNA export also finds a requirement for two NPC substructures—one on the nuclear NPC face and one in the NPC central core. These results pinpoint distinct steps in the mRNA export mechanism that regulate NPC translocation efficiency.

Human cytomegalovirus (HCMV) modulates numerous cellular signaling pathways. Alterations in signaling are evident from the broad changes in cellular phosphorylation that occur during HCMV infection and from the altered activity of multiple kinases. Here we report a comprehensive RNAi screen, which predicts that 106 cellular kinases influence growth of the virus, most of which were not previously linked to HCMV replication. Multiple elements of the AMP-activated protein kinase (AMPK) pathway scored in the screen. As a regulator of carbon and nucleotide metabolism, AMPK is poised to activate many of the metabolic pathways induced by HCMV infection. An AMPK inhibitor, compound C, blocked a substantial portion of HCMV-induced metabolic changes, inhibited the accumulation of all HCMV proteins tested, and markedly reduced the production of infectious progeny. We propose that HCMV requires AMPK or related activity for viral replication and reprogramming of cellular metabolism.herpesvirus | siRNA V iruses are dependent on host cell signaling pathways for replication and spread. Infection with the prevalent β-herpes virus human cytomegalovirus (HCMV) induces increased levels of protein phosphorylation and markedly alters host cell signal transduction pathways (1). A portion of the phosphorylation changes induced by HCMV infection are attributed to the Ser/ Thr kinase encoded by the viral genome, pUL97 (2), and others may derive from cellular kinase(s) packaged into virions (3) or from cellular kinases known to be activated by HCMV (4).Here we sought to more completely delineate the effects of HCMV infection on kinase signaling by performing an siRNA screen of the entire cellular kinome. The screen identified 106 kinases predicted to influence the production of virus. The hits included the 5′-AMP-activated protein kinase (AMPK), a sensor of cellular energy homeostasis. AMPK is composed of three subunits: a catalytic subunit, AMPKα, and two regulatory subunits, AMPKβ and AMPKγ. Activation of the kinase requires cooperative AMP binding to AMPKγ, which occurs stochastically with shifts in the AMP:ATP ratio, and phosphorylation of AMPKα at Thr172 (5, 6). At least three different kinases are reported to phosphorylate Thr172 of AMPKα: Ca 2+ /calmodulindependent kinase kinase (CaMKK), TGF-β-activated kinase 1 (TAK1), and liver kinase B1 (LKB1) (7). Activated AMPK phosphorylates a number of substrates to effect changes in central carbon metabolism, lipid metabolism, physiological homeostasis, cell growth, apoptosis, and gene expression (5).HCMV induces glycolysis (8-10) and also causes increased levels of the glucose transporter GLUT4 at the plasma membrane increasing glucose uptake (11).AMPK controls GLUT4 relocalization to the plasma membrane (5), and this regulation likely links the kinase to altered metabolism in HCMV-infected cells. However, previous work indicates that pharmacological activation of AMPK during the early phase of HCMV infection can be deleterious to viral replication (12), yet CaMKK activity is required for ...

Directional export of messenger RNA (mRNA) protein particles (mRNPs) through nuclear pore complexes (NPCs) requires multiple factors. In Saccharomyces cerevisiae, the NPC proteins Nup159 and Nup42 are asymmetrically localized to the cytoplasmic face and have distinct functional domains: a phenylalanine-glycine (FG) repeat domain that docks mRNP transport receptors and domains that bind the DEAD-box ATPase Dbp5 and its activating cofactor Gle1, respectively. We speculated that the Nup42 and Nup159 FG domains play a role in positioning mRNPs for the terminal mRNP-remodeling steps carried out by Dbp5. Here we find that deletion (D) of both the Nup42 and Nup159 FG domains results in a cold-sensitive poly(A)+ mRNA export defect. The nup42DFG nup159DFG mutant also has synthetic lethal genetic interactions with dbp5 and gle1 mutants. RNA cross-linking experiments further indicate that the nup42DFG nup159DFG mutant has a reduced capacity for mRNP remodeling during export. To further analyze the role of these FG domains, we replaced the Nup159 or Nup42 FG domains with FG domains from other Nups. These FG "swaps" demonstrate that only certain FG domains are functional at the NPC cytoplasmic face. Strikingly, fusing the Nup42 FG domain to the carboxy-terminus of Gle1 bypasses the need for the endogenous Nup42 FG domain, highlighting the importance of proximal positioning for these factors. We conclude that the Nup42 and Nup159 FG domains target the mRNP to Gle1 and Dbp5 for mRNP remodeling at the NPC. Moreover, these results provide key evidence that character and context play a direct role in FG domain function and mRNA export. IN eukaryotes, messenger RNA (mRNA) export from the nucleus is an essential process that is highly regulated, with events sequentially coordinated to ensure proper RNA processing and cytoplasmic fate (reviewed in Moore 2005; MullerMcNicoll and Neugebauer 2013). In general, transcripts exit the nucleus through nuclear pore complexes (NPCs), large (60 MDa in yeast, 100 MDa in vertebrates) proteinaceous structures that provide an aqueous channel for ions, metabolites, proteins, and ribonuclear protein complexes (RNPs) to cross the nuclear envelope (reviewed in Wente and Rout 2010; Bilokapic and Schwartz 2012). For molecules .40 kDa, transport through the NPC is facilitated via transport receptors (TRs) that bind both the cargo and NPC proteins (nucleoporins, or Nups). For mRNA, binding to TRs is carefully coordinated to allow export only after the message is fully processed in the nucleus (reviewed in Muller-McNicoll and Neugebauer 2013).A key aspect of NPC function involves different NPC structures performing specific roles to ensure efficient transport. During NPC translocation, TRs interact with a class of Nups known as phenylalanine-glycine (FG) Nups (reviewed in Wente and Rout 2010). Each FG Nup has an unstructured domain enriched with FG, glycine-leucine-phenylalanineglycine (GLFG), or phenylalanine-any amino acid-phenylalanine-glycine (FxFG) repeats, which are flanked by characteristic...