Nuclear mRNA Export Requires Complex Formation between Mex67p and Mtr2p at the Nuclear Pores

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The ubiquitin-related protein SUMO-1 is covalently attached to proteins by SUMO-1 ligases. We have performed a proteome-wide analysis of sumoylated substrate proteins in yeast. Employing the powerful affinity purification of Protein A-Smt3 (Smt3 is the yeast homologue of SUMO-1) from yeast lysates in combination with tandem liquid chromatography mass spectrometry, we have isolated potential Smt3-carrying substrate proteins involved in DNA replication and repair, chromatin remodeling, transcription activation, Pol-I, Pol-II, and Pol-III transcription, 5 pre-mRNA capping, 3 pre-mRNA processing, proteasome function, and tubulin folding. Employing tandem affinity purifications or a rapid biochemical assay referred to as "SUMO fingerprint," we showed that several subunits of RNA polymerases I, II, and III, members of the transcription repression and chromatin remodeling machineries previously not known to be sumoylated, are modified by SUMO-1. Thus, the identification of a broad range of SUMO-1 substrate proteins is expected to lead to further insight into the regulatory aspects of sumoylation.The ubiquitin-like protein SUMO-1 (Smt3 in yeast) is a 100-residue protein that is conjugated to substrate proteins by sequential thioester transfer reactions via specific E1 1 activating enzymes (Uba2/Aos1) and E2 (Ubc9) conjugating enzyme (1, 2). SUMO is conjugated to specific lysine residues on substrate proteins typically exhibiting consensus sites hKxE, where h is a hydrophobic amino acid (3, 4). Unlike ubiquitylation, sumoylation of target proteins does not lead to proteasomal degradation but can affect diverse functions of the protein, such as subcellular localization, protein/DNA interaction, or enzymatic activity (5, 6).Smt3 is highly conserved and essential in yeast. In addition, the conjugating and deconjugating machinery are conserved from yeast to humans and perform essential functions in yeast. The only yeast proteins known to be modified by Smt3 are the nonessential septins involved in cytokinesis and the essential Pol30, Top2, and Pds5 (7-9). A growing number of proteins that are modified by SUMO-1 in mammalian cells have been reported (6). Recent proteomic approaches in mammalian cells have identified new proteins that are subject to SUMO-1 and SUMO-2 modification (10 -12). The confirmation of sumoylation of target proteins in these approaches was limited by the need for protein specific antibodies.Previously, we have shown that the deconjugating enzyme Ulp1 is tethered to the nuclear pore channel via nuclear import receptors (13). The biological significance of such a tethering mechanism is poorly understood because of the lack of knowledge about its substrate proteins. Yeast has served as a powerful, rapid, genetic and in vivo biochemical model system to gain mechanistic insights into protein function in eukaryotes. Hence, we applied a proteomic approach in yeast to unravel the SUMO proteome. We have enriched sumoylated proteins from yeast cell lysates using the high affinity tag of Protein A (ProtA). In this...

Section: Methodsmentioning

confidence: 99%

A Proteome-wide Approach Identifies Sumoylated Substrate Proteins in Yeast

Panse

Hardeland

Werner³

et al. 2004

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“…These factors include Gle1p, Gle2p, Dbp5p, Mex67p, and Mtr2p. All of these factors except Gle2p are essential, and in wild type cells all localize predominantly at the NPC with some also showing low levels of cytoplasmic or nucleoplasmic staining (19,22,29,(45)(46)(47). Therefore, each was a candidate for perturbation by the overexpression of the Nup116-GLFG region.…”

Section: Mex67-gfp Accumulates In the Nucleus Of Cells Overexpressingmentioning

confidence: 99%

“…The essential factor Gle1p interacts with Dbp5p (20), and gle1 mutants are synthetically lethal with either rip1/nup42 or nup100 null mutants (19,21). The essential mRNA export factor Mex67p forms a heterodimeric complex with Mtr2p, and Mtr2p mediates association with Nup85p (22). The vertebrate Mex67p homologue, TAP, directly associates with vertebrate Nup214p, Nup98p, and hCG1 (23,24).…”

mentioning

confidence: 99%

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

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...

“…Mex67 forms a heterodimer with another export factor, Mtr2, binds the hnRNP-like factor Yra1, and interacts with FG (phenylalanine, glycine) repeats in several Nups (27)(28)(29). Func-tional associations have also been established between Mex67 and the TREX complex through Yra1 and the splicing factor Sub2 (30 -32).…”

mentioning

confidence: 99%

Nuclear Export of the Yeast mRNA-binding Protein Nab2 Is Linked to a Direct Interaction with Gfd1 and to Gle1 Function

Suntharalingam¹,

Alcázar-Román

Wente

2004

Nuclear export of mRNA is mediated by interactions between soluble factors and nuclear pore complex (NPC) proteins. In Saccharomyces cerevisiae, Nab2 is an essential RNA-binding protein that shuttles between the nucleus and cytoplasm. The mechanism for trafficking of Nab2-bound mRNA through the NPC has not been defined. Gle1 is also required for mRNA export, and Gle1 interactions with NPC proteins, the RNA helicase Dbp5, and Gfd1 have been reported. Here we report that Nab2, Gfd1, and Gle1 associate in a complex. By using immobilized recombinant Gfd1, Nab2 was isolated from total yeast lysate. A similar biochemical assay with immobilized recombinant Nab2 resulted in coisolation of Gfd1 and Gle1. A Nab2-Gfd1 complex was also identified by coimmunoprecipitation from yeast lysates. In vitro binding assays with recombinant proteins revealed a direct association between Nab2 and Gfd1, and two-hybrid assays delineated Gfd1 binding to the N-terminal Nab2 domain. This N-terminal Nab2 domain is distinct from its RNA binding domains suggesting Nab2 could bind Gfd1 and RNA simultaneously. As Nab2 export was blocked in a gle1 mutant at the restrictive temperature, we propose a model wherein Gfd1 serves as a bridging factor between Gle1 and Nab2-bound mRNA during export.