Nup116p Associates with the Nup82p-Nsp1p-Nup159p Nucleoporin Complex

113

108

Our previous studies have focused on a family of Saccharomyces cerevisiae nuclear pore complex (NPC) proteins that contain domains composed of repetitive tetrapeptide glycine-leucine-phenylalanine-glycine (GLFG) motifs. We have previously shown that the GLFG regions of Nup116p and Nup100p directly bind the karyopherin transport factor Kap95p during nuclear protein import. In this report, we have further investigated potential roles for the GLFG region in mRNA export. The subcellular localizations of green fluorescent protein (GFP)-tagged mRNA transport factors were individually examined in yeast cells overexpressing the Nup116-GLFG region. The essential mRNA export factors Mex67-GFP, Mtr2-GFP, and Dbp5-GFP accumulated in the nucleus. In contrast, the localizations of Gle1-GFP and Gle2-GFP remained predominantly associated with the NPC, as in wild type cells. The localization of Kap95p was also not perturbed with GLFG overexpression. Coimmunoprecipitation experiments from yeast cell lysates resulted in the isolation of a Mex67p-Nup116p complex. Soluble binding assays with bacterially expressed recombinant proteins confirmed a direct interaction between Mex67p and the Nup116-GLFG or Nup100-GLFG regions. Mtr2p was not required for in vitro binding of Mex67p to the GLFG region. To map the Nup116-GLFG subregion(s) required for Kap95p and/or Mex67p association, yeast two-hybrid analysis was used. Of the 33 Nup116-GLFG repeats that compose the domain, a central subregion of nine GLFG repeats was sufficient for binding either Kap95p or Mex67p. Interestingly, the first 12 repeats from the fulllength region only had a positive interaction with Mex67p, whereas the last 12 were only positive with Kap95p. Thus, the GLFG domain may have the capacity to bind both karyopherins and an mRNA export factor simultaneously. Taken together, our in vivo and in vitro results define an essential role for a direct Mex67p-GLFG interaction during mRNA export.To move between the nuclear and cytoplasmic compartments of a eukaryotic cell, all molecules must pass through nuclear pore complexes (NPCs) 1 embedded in the nuclear envelope.Ions, metabolites, and small proteins may diffuse through an ϳ9-nm aqueous channel in the NPC. In contrast, the movement of large macromolecules, including proteins and RNA, is energy-dependent and facilitated (reviewed in Refs. 1-3). The central channel of the proteinaceous NPC is formed by a symmetrical, 8-fold assembly of spoke-like structures sandwiched between nuclear and cytoplasmic rings. Distinct filamentous structures extend from these rings on both the nuclear and cytoplasmic faces, with the filaments on the nuclear side culminating in a basket-like structure. Overall, a vertebrate NPC measures ϳ200 nm from the tips of the cytoplasmic filaments to the base of the nucleoplasmic basket (4, 5). Unraveling the mechanism for active transport between the cytoplasmic and nucleoplasmic NPC faces will require an in-depth understanding of both the NPC itself and soluble transport factors. The Saccharomyces cerev...

Section: Figmentioning

confidence: 97%

The GLFG Regions of Nup116p and Nup100p Serve as Binding Sites for Both Kap95p and Mex67p at the Nuclear Pore Complex

Strawn

Shen

Wente

2001

113

108

“…The middle domain harbors an extensive span of predominantly GLFG repeats and binds Mex67 and Kaps (13,14,60,63,64). Meanwhile, the C-terminal region, conserved with the C-terminal regions of Nup100 and Nup145N, interacts with Nup82 and mediates Nup116 assembly in the NPC (65,66). Using a panel of TRP1/CEN plasmids expressing deletion mutants of NUP116, we identified the region required for complementation of both the nup116⌬ gle1-1 and nup116⌬ ipk1⌬ phenotypes.…”

Section: The Glycine-leucine-phenylalanine-glycine (Glfg) Region Of Nmentioning

confidence: 99%

“…1A. Nup82 interacts with both Nup116 and in an Nsp1-Nup159 complex (65,66,69). Gle2 directly interacts with Nup116 (61, 62), whereas Dbp5 is connected to Nup159 (19,26).…”

Section: The Glycine-leucine-phenylalanine-glycine (Glfg) Region Of Nmentioning

confidence: 99%

Cytoplasmic Inositol Hexakisphosphate Production Is Sufficient for Mediating the Gle1-mRNA Export Pathway

Miller

Suntharalingam

Johnson

et al. 2004

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...

“…Fluorescence Microscopy-Expression and localization of GFP, GST-GFP, and GFP-␤-galactosidase fusion proteins in yeast was done essentially as described before (21). Specifically, the wild-type W303 yeast strain, strains disrupted for ASR1 or certain importins, strains encoding conditionally temperaturesensitive importin mutants, or the exportin mutant xpo1-1 were transformed with plasmids encoding GFP, GST-GFP, or GFP-␤-galactosidase fusion proteins.…”

Section: Cells Growth Microbiological Techniques and Plasmids-mentioning

confidence: 99%

A Novel Conserved Nuclear Localization Signal Is Recognized by a Group of Yeast Importins

Fries

Betz

Sohn

et al. 2007

Self Cite

Nucleo-cytoplasmic transport of proteins is mostly mediated by specific interaction between transport receptors of the importin ␤ family and signal sequences present in their cargo. While several signal sequences, in particular the classical nuclear localization signal (NLS) recognized by the heterodimeric importin ␣/␤ complex are well known, the signals recognized by other importin ␤-like transport receptors remain to be characterized in detail. Here we present the systematic analysis of the nuclear import of Saccharomyces cerevisiae Asr1p, a nonessential alcohol-responsive Ring/PHD finger protein that shuttles between nucleus and cytoplasm but accumulates in the nucleus upon alcohol stress. Nuclear import of Asr1p is constitutive and mediated by its C-terminal domain. A short sequence comprising residues 243-280 is sufficient and necessary for active targeting to the nucleus. Moreover, the nuclear import signal is conserved from yeast to mammals. In vitro, the nuclear localization signal of Asr1p directly interacts with the importins Kap114p, Kap95p, Pse1p, Kap123p, or Kap104p, interactions that are sensitive to the presence of RanGTP. In vivo, these importins cooperate in nuclear import. Interestingly, the same importins mediate nuclear transport of histone H2A. Based on mutational analysis and sequence comparison with a region mediating nuclear import of histone H2A, we identified a novel type of NLS with the consensus sequence R/KxxL(x) n V/ YxxV/IxK/RxxxK/R that is recognized by five yeast importins and connects them into a highly efficient network for nuclear import of proteins.Trafficking of proteins and RNAs between cytoplasm and nucleus is essential for the maintenance of cell function and crucially involved in adaptation to altered cellular conditions. Nucleo-cytoplasmic transport of most proteins and some RNAs is mediated by soluble transport receptors of the importin ␤ family (1-3). Directionality of nucleo-cytoplasmic transport is brought about by the small GTPase Ran (Gsp1p in Saccharomyces cerevisiae). The high concentration of RanGTP in the nucleus and of RanGDP in the cytoplasm, called RanGTP gradient, regulates the interaction of importins/exportins with their cargoes. This gradient is generated by the exclusive nuclear localization of the guanine nucleotide exchange factor RCC1/RanGEF (Prp20p in S. cerevisiae) and the nuclear exclusion of the RanGTPase-activating protein RanGAP1 (Rna1p in S. cerevisiae). To initiate nuclear import, an importin forms a dimeric complex with its cytoplasmic cargo. Following docking and translocation through the nuclear pore complex, the importin-cargo complex is dissociated by binding of RanGTP to the importin. Nuclear export starts with formation of a trimeric cargo-exportin-RanGTP complex, which is dissociated in the cytoplasm upon GTP hydrolysis.The complete genome of S. cerevisiae encodes 14 members of the importin ␤-like proteins, 10 of which function as importins, while three function as exportins. Msn5p is an exception since it mediates both import and ex...