Saccharomyces cerevisiae GTPase complex: Gtr1p-Gtr2p regulates cell-proliferation through Saccharomyces cerevisiae Ran-binding protein, Yrb2p

A Gtr1p GTPase, the GDP mutant of which suppresses both temperature-sensitive mutants of Saccharomyces cerevisiae RanGEF/Prp20p and RanGAP/Rna1p, was presently found to interact with Yrb2p, the S. cerevisiae homologue of mammalian Ran-binding protein 3. Gtr1p bound the Ran-binding domain of Yrb2p. In contrast, Gtr2p, a partner of Gtr1p, did not bind Yrb2p, although it bound Gtr1p. A triple mutant: yrb2delta gtr1delta gtr2delta was lethal, while a double mutant: gtr1delta gtr2delta survived well, indicating that Yrb2p protected cells from the killing effect of gtr1delta gtr2delta. Recombinant Gtr1p and Gtr2p were purified as a complex from Escherichia coli. The resulting Gtr1p-Gtr2p complex was comprised of an equal amount of Gtr1p and Gtr2p, which inhibited the Rna1p/Yrb2 dependent RanGAP activity. Thus, the Gtr1p-Gtr2p cycle was suggested to regulate the Ran cycle through Yrb2p.     

The nucleoporin Nup60p functions as a Gsp1p-GTP-sensitive tether for Nup2p at the nuclear pore complex

The nucleoporins Nup60p, Nup2p, and Nup1p form part of the nuclear basket structure of the Saccharomyces cerevisiae nuclear pore complex (NPC). Here, we show that these necleoporins can be isolated from yeast extracts by affinity chromatography on karyopherin Kap95p-coated beads. To characterize Nup60p further, Nup60p-coated beads were used to capture its interacting proteins from extracts. We find that Nup60p binds to Nup2p and serves as a docking site for Kap95p-Kap60p heterodimers and Kap123p. Nup60p also binds Gsp1p-GTP and its guanine nucleotide exchange factor Prp20p, and functions as a Gsp1p guanine nucleotide dissociation inhibitor by reducing the activity of Prp20p. Yeast lacking Nup60p exhibit minor defects in nuclear export of Kap60p, nuclear import of Kap95p-Kap60p-dependent cargoes, and diffusion of small proteins across the NPC. Yeast lacking Nup60p also fail to anchor Nup2p at the NPC, resulting in the mislocalization of Nup2p to the nucleoplasm and cytoplasm. Purified Nup60p and Nup2p bind each other directly, but the stability of the complex is compromised when Kap60p binds Nup2p. Gsp1p-GTP enhances by 10-fold the affinity between Nup60p and Nup2p, and restores binding of Nup2p-Kap60p complexes to Nup60p. The results suggest a dynamic interaction, controlled by the nucleoplasmic concentration of Gsp1p-GTP, between Nup60p and Nup2p at the NPC.     

Nmd3p is a Crm1p-dependent adapter protein for nuclear export of the large ribosomal subunit

In eukaryotic cells, nuclear export of nascent ribosomal subunits through the nuclear pore complex depends on the small GTPase Ran. However, neither the nuclear export signals (NESs) for the ribosomal subunits nor the receptor proteins, which recognize the NESs and mediate export of the subunits, have been identified. We showed previously that Nmd3p is an essential protein from yeast that is required for a late step in biogenesis of the large (60S) ribosomal subunit. Here, we show that Nmd3p shuttles and that deletion of the NES from Nmd3p leads to nuclear accumulation of the mutant protein, inhibition of the 60S subunit biogenesis, and inhibition of the nuclear export of 60S subunits. Moreover, the 60S subunits that accumulate in the nucleus can be coimmunoprecipitated with the NES-deficient Nmd3p. 60S subunit biogenesis and export of truncated Nmd3p were restored by the addition of an exogenous NES. To identify the export receptor for Nmd3p we show that Nmd3p shuttling and 60S export is blocked by the Crm1p-specific inhibitor leptomycin B. These results identify Crm1p as the receptor for Nmd3p export. Thus, export of the 60S subunit is mediated by the adapter protein Nmd3p in a Crm1p-dependent pathway.     

SUMOylation mediates the disassembly of the Smad4 nuclear export complex via RanGAP1 in KELOIDS

Sentrin/small ubiquitin-like modifier (SUMO) has emerged as a powerful mediator regulating biological processes and participating in pathophysiological processes that cause human diseases, such as cancer, myocardial fibrosis and neurological disorders. Sumoylation has been shown to play a positive regulatory role in keloids. However, the sumoylation mechanism in keloids remains understudied. We proposed that sumoylation regulates keloids via a complex. RanGAP1 acted as a synergistic, functional partner of SUMOs in keloids. Nuclear accumulation of Smad4, a TGF-β/Smad pathway member, was associated with RanGAP1 after SUMO1 inhibition. RanGAP1*SUMO1 mediated the nuclear accumulation of Smad4 due to its impact on nuclear export and reduction in the dissociation of Smad4 and CRM1. We clarified a novel mechanism of positive regulation of sumoylation in keloids and demonstrated the function of sumoylation in Smad4 nuclear export. The NPC-associated RanGAP1*SUMO1 complex functions as a disassembly machine for the export receptor CRM1 and Smad4. Our research provides new perspectives for the mechanisms of keloids and nucleocytoplasmic transport.     

RAE1 is a shuttling mRNA export factor that binds to a GLEBS-like NUP98 motif at the nuclear pore complex through multiple domains

Gle2p is implicated in nuclear export of poly(A)+ RNA and nuclear pore complex (NPC) structure and distribution in Saccharomyces cerevisiae. Gle2p is anchored at the nuclear envelope (NE) via a short Gle2p-binding motif within Nup116p called GLEBS. The molecular mechanism by which Gle2p and the Gle2p-Nup116p interaction function in mRNA export is unknown. Here we show that RAE1, the mammalian homologue of Gle2p, binds to a GLEBS-like NUP98 motif at the NPC through multiple domains that include WD-repeats and a COOH-terminal non-WD-repeat extension. This interaction is direct, as evidenced by in vitro binding studies and chemical cross-linking. Microinjection experiments performed in Xenopus laevis oocytes demonstrate that RAE1 shuttles between the nucleus and the cytoplasm and is exported from the nucleus in a temperature-dependent and RanGTP-independent manner. Docking of RAE1 to the NE is highly dependent on new mRNA synthesis. Overexpression of the GLEBS-like motif also inhibits NE binding of RAE1 and induces nuclear accumulation of poly(A)+ RNA. Both effects are abrogated either by the introduction of point mutations in the GLEBS-like motif or by overexpression of RAE1, indicating a direct role for RAE1 and the NUP98-RAE1 interaction in mRNA export. Together, our data suggest that RAE1 is a shuttling transport factor that directly contributes to nuclear export of mRNAs through its ability to anchor to a specific NUP98 motif at the NPC.     

Structure and assembly of the Nup84p complex

The Nup84p complex consists of five nucleoporins (Nup84p, Nup85p, Nup120p, Nup145p-C, and Seh1p) and Sec13p, a bona fide subunit of the COPII coat complex. We show that a pool of green fluorescent protein-tagged Sec13p localizes to the nuclear pores in vivo, and identify sec13 mutant alleles that are synthetically lethal with nup85Delta and affect the localization of a green fluorescent protein-Nup49p reporter protein. In the electron microscope, sec13 mutants exhibit structural defects in nuclear pore complex (NPC) and nuclear envelope organization. For the assembly of the complex, Nup85p, Nup120p, and Nup145p-C are essential. A highly purified Nup84p complex was isolated from yeast under native conditions and its molecular mass was determined to be 375 kD by quantitative scanning transmission electron microscopy and analytical ultracentrifugation, consistent with a monomeric complex. Furthermore, the Nup84p complex exhibits a Y-shaped, triskelion-like morphology 25 nm in diameter in the transmission electron microscope. Thus, the Nup84p complex constitutes a paradigm of an NPC structural module with distinct composition, structure, and a role in nuclear mRNA export and NPC bio- genesis.     

Chtop is a component of the dynamic TREX mRNA export complex

The TREX complex couples nuclear pre‐mRNA processing with mRNA export and contains multiple protein components, including Uap56, Alyref, Cip29 and the multi‐subunit THO complex. Here, we have identified Chtop as a novel TREX component. We show that both Chtop and Alyref activate the ATPase and RNA helicase activities of Uap56 and that Uap56 functions to recruit both Alyref and Chtop onto mRNA. As observed with the THO complex subunit Thoc5, Chtop binds to the NTF2‐like domain of Nxf1, and this interaction requires arginine methylation of Chtop. Using RNAi, we show that co‐knockdown of Alyref and Chtop results in a potent mRNA export block. Chtop binds to Uap56 in a mutually exclusive manner with Alyref, and Chtop binds to Nxf1 in a mutually exclusive manner with Thoc5. However, Chtop, Thoc5 and Nxf1 exist in a single complex in vivo. Together, our data indicate that TREX and Nxf1 undergo dynamic remodelling, driven by the ATPase cycle of Uap56 and post‐translational modifications of Chtop.     

A novel family of nuclear transport receptors mediates the export of messenger RNA to the cytoplasm

Fully processed mRNAs are exported to the cytoplasm where they direct protein synthesis. A general feature of mRNA export is that it is an active, receptor-mediated process. The mRNA export receptors are thought to recognize and bind to the mRNA-export cargoes either directly or indirectly (via adaptor proteins) and facilitate their translocation across the central channel of the nuclear pore complex (NPC). On the cytoplasmic side of the NPC, the exported mRNA is released and the receptor returns to the nucleoplasm, without the cargo, to initiate additional rounds of export. Recent, studies in yeast and in higher eukaryotes have led to the elucidation of an evolutionarily conserved pathway for the export of bulk mRNA to the cytoplasm.     

Domain topology of the p62 complex within the 3-D architecture of the nuclear pore complex

The nuclear pore complex (NPC) is the only known gateway for exchange of macromolecules between the cytoplasm and nucleus of eukaryotic cells. One key compound of the NPC is the p62 subcomplex, which consists of the nucleoporins p62, p54, and p58/p45 and is supposed to be involved in nuclear protein import and export. Here we show the localization of distinct domains of the p62 complex by immuno-electron microscopy using isolated nuclei from Xenopus oocytes. To determine the exact position of the p62 complex, we examined the localization of the C and N-terminal domains of p62 by immunogold-labeling using domain-specific antibodies against p62. In addition we expressed epitope-tagged versions of p62, p54, and p58 in Xenopus oocytes and localized the domains with antibodies against the tags. This first systematic analysis of the domain topology of the p62 complex within the NPC revealed that the p62 complex is anchored to the cytoplasmic face of the NPC most likely by the coiled-coil domains of the three nucleoporins. Furthermore, we found the phenylalanine-glycine (FG)-repeat domain of p62, but not of p58 and p54, to be of mobile and flexible nature.     

The Rix1 (Ipi1p-2p-3p) complex is a critical determinant of DNA replication licensing independent of their roles in ribosome biogenesis

Several replication-initiation proteins are assembled stepwise onto replicators to form pre-replicative complexes (pre-RCs) to license eukaryotic DNA replication. We performed a yeast functional proteomic screen and identified the Rix1 complex members (Ipi1p-Ipi2p/Rix1-Ipi3p) as pre-RC components and critical determinants of replication licensing and replication-initiation frequency. Ipi3p interacts with pre-RC proteins, binds chromatin predominantly at ARS sequences in a cell cycle-regulated and ORC- and Noc3p-dependent manner and is required for loading Cdc6p, Cdt1p and MCM onto chromatin to form pre-RC during the M-to-G₁ transition and for pre-RC maintenance in G₁ phase-independent of its role in ribosome biogenesis. Moreover, Ipi1p and Ipi2p, but not other ribosome biogenesis proteins Rea1p and Utp1p, are also required for pre-RC formation and maintenance, and Ipi1p, -2p and -3p are interdependent for their chromatin association and function in pre-RC formation. These results establish a new framework for the hierarchy of pre-RC proteins, where the Ipi1p-2p-3p complex provides a critical link between ORC-Noc3p and Cdc6p-Cdt1p-MCM in replication licensing.     

Phosphorylation regulates the interaction and complex formation between wt p53 protein and PARP-1

We recently characterized the interaction between poly(ADP-ribose) polymerase-1 (PARP-1) and the product of the tumor suppressor gene p53. We investigated which domains of human PARP-1 and of human wild-type (wt) p53 were involved in this protein-protein interaction. We generated baculoviral constructs encoding full length or distinct functional domains of both proteins. Full length PARP-1 was simultaneously coexpressed in insect cells with full length wt p53 protein or its distinct truncated fragments and vice versa. Reciprocal immunoprecipitation of Sf9 cell lysates revealed that the central and carboxy-terminal fragments of p53 were sufficient to confer binding to PARP-1, whereas the amino-terminal part harboring the transactivation functional domain was dispensable. On the other hand, the amino-terminal and central fragments of PARP-1 were necessary for complex formation with p53 protein. As the most important features of p53 protein are regulated by phosphorylation, we addressed the question of whether its phosphorylation is essential for binding between the two proteins. Baculovirally expressed wt p53 was post-translationally modified. At least six distinct p53 isomeres were resolved by immunoblotting following two-dimensional separation of baculovirally expressed wt p53 protein. Using specific phospho-serine antibodies, we identified phosphorylation of baculovirally expressed p53 protein at five distinct sites. To define the role of p53 phosphorylation, pull-down assays using untreated and dephosphorylated p53 protein were performed. Dephosphorylated p53 failed to bind PARP-1 indicating that complex formation between both proteins is regulated by phosphorylation of p53. The marked phosphorylation of p53 at Ser392 observed in unstressed cells suggests that the phosphorylated carboxy-terminal part of p53 undergoes complex formation with PARP-1 resulting in masking of the NES and thereby preventing its export. The functional significance of the interaction between both proteins was investigated at two different conditions: inactivation of PARP-1 and overexpression of PARP-1. Our results unequivocally show that the presence of PARP-1 regulates the basal expression of wt p53 in unstressed cells.     

Building arks for tRNA: Structure and function of the Arc1p family of non-catalytic tRNA-binding proteins

Following the intricate architecture of the eukaryotic cell, protein synthesis involves formation of many macromolecular assemblies, some of which are composed by tRNA-aminoacylation enzymes. Protein-protein and protein-tRNA interactions in these complexes can be facilitated by non-catalytic tRNA-binding proteins. This review focuses on the dissection of the molecular, structural and functional properties of a particular family of such proteins: yeast Arc1p and its homologues in prokaryotes and higher eukaryotes. They represent paradigms of the strategies employed for the organization of sophisticated and dynamic nanostructures supporting spatio-temporal cellular organization.     

Saccharomyces cerevisiae Ndc1p Is a Shared Component of Nuclear Pore Complexes and Spindle Pole Bodies

We report a novel connection between nuclear pore complexes (NPCs) and spindle pole bodies (SPBs) revealed by our studies of the Saccharomyces cerevisiae NDC1 gene. Although both NPCs and SPBs are embedded in the nuclear envelope (NE) in yeast, their known functions are quite distinct. Previous work demonstrated that NDC1 function is required for proper SPB duplication (Winey, M., M.A. Hoyt, C. Chan, L. Goetsch, D. Botstein, and B. Byers. 1993. J. Cell Biol. 122:743–751). Here, we show that Ndc1p is a membrane protein of the NE that localizes to both NPCs and SPBs. Indirect immunofluorescence microscopy shows that Ndc1p displays punctate, nuclear peripheral localization that colocalizes with a known NPC component, Nup49p. Additionally, distinct spots of Ndc1p localization colocalize with a known SPB component, Spc42p. Immunoelectron microscopy shows that Ndc1p localizes to the regions of NPCs and SPBs that interact with the NE. The NPCs in ndc1-1 mutant cells appear to function normally at the nonpermissive temperature. Finally, we have found that a deletion of POM152, which encodes an abundant but nonessential nucleoporin, suppresses the SPB duplication defect associated with a mutation in the NDC1 gene. We show that Ndc1p is a shared component of NPCs and SPBs and propose a shared function in the assembly of these organelles into the NE.     

Structure-based thermodynamic analysis of the dissociation of protein phosphatase-1 catalytic subunit and microcystin-LR docked complexes

The relationship between the structure of a free ligand in solution and the structure of its bound form in a complex is of great importance to the understanding of the energetics and mechanism of molecular recognition and complex formation. In this study, we use a structure-based thermodynamic approach to study the dissociation of the complex between the toxin microcystin-LR (MLR) and the catalytic domain of protein phosphatase-1 (PP-1c) for which the crystal structure of the complex is known. We have calculated the thermodynamic parameters (enthalpy, entropy, heat capacity, and free energy) for the dissociation of the complex from its X-ray structure and found the calculated dissociation constant (4.0 x 10(-11)) to be in excellent agreement with the reported inhibitory constant (3.9 x 10(-11)). We have also calculated the thermodynamic parameters for the dissociation of 47 PP-1c:MLR complexes generated by docking an ensemble of NMR solution structures of MLR onto the crystal structure of PP-1c. In general, we observe that the lower the root-mean-square deviation (RMSD) of the docked complex (compared to the X-ray complex) the closer its free energy of dissociation (deltaGd(o)) is to that calculated from the X-ray complex. On the other hand, we note a significant scatter between the deltaGd(o) and the RMSD of the docked complexes. We have identified a group of seven docked complexes with deltaGd(o) values very close to the one calculated from the X-ray complex but with significantly dissimilar structures. The analysis of the corresponding enthalpy and entropy of dissociation shows a compensation effect suggesting that MLR molecules with significant structural variability can bind PP-1c and that substantial conformational flexibility in the PP-1c:MLR complex may exist in solution.     

An ER membrane protein,Sop4, facilitates ER export of the yeast plasma membrane [H+]ATPase,Pma1

We have analyzed the mechanism by which Sop4, a novel ER membrane protein, regulates quality control and intracellular transport of Pma1–7, a mutant plasma membrane ATPase. At the restrictive temperature, newly synthesized Pma1–7 is targeted for vacuolar degradation instead of being correctly delivered to the cell surface. Loss of Sop4 at least partially corrects vacuolar mislocalization, allowing Pma1–7 routing to the plasma membrane. Ste2–3 is a mutant pheromone receptor which, like Pma1–7, is defective in targeting to the cell surface, resulting in a mating defect. sop4Δ suppresses the mating defect of ste2–3 cells as well as the growth defect of pma1–7 . Visualization of newly synthesized Pma1–7 in sop4Δ cells by indirect immunofluorescence reveals delayed export from the ER. Similarly, ER export of wild-type Pma1 is delayed in the absence of Sop4 although intracellular transport of Gas1 and CPY is unaffected. These observations suggest a model in which a selective increase in ER residence time for Pma1–7 may allow it to achieve a more favorable conformation for subsequent delivery to the plasma membrane. In support of this model, newly synthesized Pma1–7 is also routed to the plasma membrane upon release from a general block of ER-to-Golgi transport in sec13–1 cells.     

Genetic and biochemical interactions between the Arp2/3 complex, Cmd1p, casein kinase II, and Tub4p in yeast

Arc35p, a component of the Arp2/3 complex, plays at least two distinct roles, regulating the actin cytoskeleton, but also microtubule function during cell division. Both functions involve calmodulin (CMD1). To investigate the pathway affecting microtubule function, we identified genes that are able to suppress the temperature-sensitive growth defect of the arc35-1 strain. Genes encoding gamma-tubulin (TUB4) or any subunit of casein kinase II (CKII) suppressed this growth defect, but did not suppress the growth defect of a mutant in another subunit of the Arp2/3 complex, arp2-1. We could also show a physical association of Arc35p with subunits of CKII, Cmd1p, and Tub4p. Based on the exclusive localization of Arc35p to the cytosolic Arp2/3 complex and on mutant phenotypes, we propose that the role of the Arc35p/CKII interaction might be to activate a cytosolic pool of gamma-tubulin, likely via calmodulin, for its nuclear and/or cytoplasmic functions.     

Release of the export adapter, Nmd3p, from the 60S ribosomal subunit requires Rpl10p and the cytoplasmic GTPase Lsg1p

In eukaryotes, nuclear export of the large (60S) ribosomal subunit requires the adapter protein Nmd3p to provide the nuclear export signal. Here, we show that in yeast release of Nmd3p from 60S subunits in the cytoplasm requires the ribosomal protein Rpl10p and the G-protein, Lsg1p. Mutations in LSG1 or RPL10 blocked Nmd3-GFP shuttling into the nucleus and export of pre-60S subunits from the nucleus. Overexpression of NMD3 alleviated the export defect, indicating that the block in 60S export in lsg1 and rpl10 mutants results indirectly from failing to recycle Nmd3p. The defect in Nmd3p recycling and the block in 60S export in both lsg1 and rpl10 mutants was also suppressed by mutant Nmd3 proteins that showed reduced binding to 60S subunits in vitro. We propose that the correct loading of Rpl10p into 60S subunits is required for the release of Nmd3p from subunits by Lsg1p. These results suggest a coupling between recycling the 60S export adapter and activation of 60S subunits for translation.     

Binding of the Mex67p/Mtr2p heterodimer to FXFG, GLFG, and FG repeat nucleoporins is essential for nuclear mRNA export

It is not known how Mex67p and Mtr2p, which form a heterodimer essential for mRNA export, transport mRNPs through the nuclear pore. Here, we show that the Mex67p/Mtr2p complex binds to all of the repeat types (GLFG, FXFG, and FG) found in nucleoporins. For this interaction, complex formation between Mex67p and Mtr2p has to occur. MEX67 and MTR2 also genetically interact with different types of repeat nucleoporins, such as Nup116p, Nup159p, Nsp1p, and Rip1p/Nup40p. These data suggest a model in which nuclear mRNA export requires the Mex67p/Mtr2p heterodimeric complex to directly contact several repeat nucleoporins, organized in different nuclear pore complex subcomplexes, as it carries the mRNP cargo through the nuclear pore.