Random mutagenesis and functional analysis of the Ran-binding protein, RanBP1

Ran GTPase is required for nucleocytoplasmic transport of many types of cargo. Several proteins that recognize Ran in its GTP-bound state (Ran x GTP) possess a conserved Ran-binding domain (RanBD). Ran-binding protein-1 (RanBP1) has a single RanBD and is required for RanGAP-mediated GTP hydrolysis and release of Ran from nuclear transport receptors (karyopherins). In budding yeast (Saccharomyces cerevisiae), RanBP1 is encoded by the essential YRB1 gene; expression of mouse RanBP1 cDNA rescues the lethality of Yrb1-deficient cells. We generated libraries of mouse RanBP1 mutants and examined 11 mutants in vitro and for their ability to complement a temperature-sensitive yrb1 mutant (yrb1-51(ts)) in vivo. In 9 of the mutants, the alteration was a change in a residue (or 2 residues) that is conserved in all known RanBDs. However, 4 of these 9 mutants displayed biochemical properties indistinguishable from that of wild-type RanBP1. These mutants bound to Ran x GTP, stimulated RanGAP, inhibited the exchange activity of RCC1, and rescued growth of the yrb1-51(ts) yeast cells. Two of the 9 mutants altered in residues thought to be essential for interaction with Ran were unable to rescue growth of the yrb1(ts) mutant and did not bind detectably to Ran in vitro. However, one of these 2 mutants (and 2 others that were crippled in other RanBP1 functions) retained some ability to co-activate RanGAP. A truncated form of RanBP1 (lacking its nuclear export signal) was able to complement the yrb1(ts) mutation. When driven from the YRB1 promoter, 4 of the 5 mutants most impaired for Ran binding were unable to rescue growth of the yrb1(ts) cells; remarkably, these mutants could nevertheless form ternary complexes with importin-5 or importin-beta and Ran-GTP. The same mutants stimulated only inefficiently RanGAP-mediated GTP hydrolysis of the Ran x GTP x importin-5 complex. Thus, the essential biological activity of RanBP1 in budding yeast correlates not with Ran x GTP binding per se or with the ability to form ternary complexes with karyopherins, but with the capacity to potentiate RanGAP activity toward GTP-bound Ran in these complexes.     

Differential transmission of G1 cell cycle arrest and mating signals by Saccharomyces cerevisiae Ste5 mutants in the pheromone pathway.

Saccharomyces cerevisiae Ste5 is a scaffold protein that recruits many pheromone signaling molecules to sequester the pheromone pathway from other homologous mitogen-activated protein kinase pathways. G1 cell cycle arrest and mating are two different physiological consequences of pheromone signal transduction and Ste5 is required for both processes. However, the roles of Ste5 in G1 arrest and mating are not fully understood. To understand the roles of Ste5 better, we isolated 150 G1 cell cycle arrest defective STE5 mutants by chemical mutagenesis of the gene. Here, we found that two G1 cell cycle arrest defective STE5 mutants (ste5M(D248V) and ste5(delta-776)) retained mating capacity. When overproduced in a wild-type strain, several ste5 mutants also showed different dominant phenotypes for G1 arrest and mating. Isolation and characterization of the mutants suggested separable roles of Ste5 in G1 arrest and mating of S. cerevisiae. In addition, the roles of Asp-248 and Tyr-421, which are important for pheromone signal transduction were further characterized by site-directed mutagenesis studies.     

Mutants in a yeast Ran binding protein are defective in nuclear transport.

Ran, a Ras-like GTPase, has been implicated in controlling the movement of proteins and RNAs in and out of the nucleus. We have constructed strains of Saccharomyces cerevisiae which produce fusion proteins containing glutathione-S-transferase (GST) fused to Gsp1p, which encodes the essential yeast Ran homolog, and a mutant form of Gsp1p that mimics the GTP-bound state. A major protein with the apparent size of 34 kDa co-purifies with the GTP-bound form of Gsp1p. This protein was identified as Yrb1p (Yeast Ran Binding Protein) and stimulates GTP hydrolysis by Gsp1p in the presence of Rna1p, the Gsp1 GTPase activating protein. Yrb1p is located in the cytoplasm with some concentration at the nuclear periphery. Temperature-sensitive yrb1 mutants are defective in nuclear protein import and RNA export. A mutation in the highly conserved Ran binding region of Yrb1p reduces its ability to interact with Gsp1p. These data indicate that Yrb1p functions with Gsp1p and suggest that together they can control transport of macromolecules across the nuclear envelope.     

Genetic Relationships between the G Protein β_entitystart_#947_entit Complex, Ste5p, Ste20p and Cdc42p: Investigation of Effector Roles in the Yeast Pheromone Response Pathway

The Saccharomyces cerevisiae G protein βγ dimer, Ste4p/Ste18p, acts downstream of the α subunit, Gpa1p, to activate the pheromone response pathway and therefore must interact with a downstream effector. Synthetic sterile mutants that exacerbate the phenotype of ste4-ts mutations were isolated to identify proteins that functionally interact with Ste4p. The identification of a ste18 mutant indicated that this screen could identify proteins that interact directly with Ste4p. The other mutations were in STE5 and the STE20 kinase gene, which act near Ste4p in the pathway, and a new gene called STE21. ste20 null mutants showed residual mating, suggesting that another kinase may provide some function. Overexpression of Ste5p under galactose control activated the pheromone response pathway. This activation was dependent on Ste4p and Ste18p and partially dependent on Ste20p. These results cannot be explained by the linear pathway of Ste4p -> Ste20p -> Ste5p. Overexpression of Cdc42p resulted in a slight increase in pheromone induction of a reporter gene, and overexpression of activated forms of Cdc42p resulted in a further twofold increase. Mutations in pheromone response pathway components did not suppress the lethality associated with the activated CDC42 mutations, suggesting that this effect is independent of the pheromone response pathway.     

Nuclear protein import, but not mRNA export, is defective in all Saccharomyces cerevisiae mutants that produce temperature-sensitive forms of the Ran GTPase homologue Gsp1p

A series of ts mutations in the GSP1 gene of Saccharomyces cerevisiae was isolated by error-prone PCR. A total of 25 ts gsp1 strains was obtained. Each of these mutants showed between one and seven different amino acid alterations. In several of these ts gsp1 strains, the same amino acid residues in Gsp1p were repeatedly mutated, indicating that our screen for ts gsp1 mutations was saturating. All of the ts gsp1 strains isolated had a defect in nuclear protein import, but only 16 of the 25 ts gsp1 strains had a defect in mRNA export. Thus, Gsp1p is suggested to be directly involved in nuclear protein import, but not in mRNA export. Following release from α-factor arrest, 11 of the ts gsp1 mutants arrested in G1; the remainder did not show any specific cell-cycle arrest, at 37°?C, the nonpermissive temperature. While the mutants that are defective in both mRNA export and protein import have a tendency to arrest in G1, there was no clear correlation between the cell cycle phenotype and the defects in mRNA export and nuclear protein import. Based on this, we assume that Ran/Gsp1p GTPase regulates the cell cycle and the nucleus/cytosol exchange of macromolecules through interactions with effectors that were independent of each other, and are differentially affected by mutation.     

The yeast Apq12 protein affects nucleocytoplasmic mRNA transport

An important step in mRNA biogenesis is the export of mRNA from the nucleus to the cytoplasm. In this work, we provide evidence that the previously uncharacterized gene APQ12 functions in nucleocytoplasmic mRNA transport in Saccharomyces cerevisiae. First, apq12delta strains manifest 3' hyperadenylated mRNA similar to other previously characterized RNA export mutants. Second, bulk poly(A)+ RNA is retained in the nucleus in apq12delta cells. Third, an Apq12p-GFP chimeric protein is localized to the nuclear periphery. Fourth, mRNA in apq12delta cells is stabilized, consistent with a defect in the rate of nuclear export. Interestingly, apq12delta mutants are severely compromised for growth and display atypical cell morphology. Because this aberrant cell morphology is not seen with other viable export mutants, Apq12p must have either an additional cellular function, or preferentially impinge on the export of mRNAs regulating cell growth. Together, these findings support a role for APQ12 in nucleocytoplasmic transport of mRNA.     

The alpha-factor receptor C-terminus is important for mating projection formation and orientation in Saccharomyces cerevisiae

Successful mating of MATa Saccharomyces cerevisiae cells is dependent on Ste2p, the alpha-factor receptor. Besides receiving the pheromone signal and transducing it through the G-protein coupled MAP kinase pathway, Ste2p is active in the establishment and orientation of the mating projection. We investigated the role of the carboxyl terminus of the receptor in mating projection formation and orientation using a spatial gradient assay. Cells carrying the ste2-T326 mutation, truncating 105 of the 135 amino acids in the receptor tail including a motif necessary for its ligand-mediated internalization, display slow onset of projection formation, abnormal shmoo morphology, and reduced ability to orient the mating projection toward a pheromone source. This reduction was due to the increased loss of mating projection orientation in a pheromone gradient. Cells with a mutated endocytosis motif were defective in reorientation in a pheromone gradient. ste2-Delta296 cells, which carry a complete truncation of the Ste2p tail, exhibit a severe defect in projection formation, and those projections that do form are unable to orient in a pheromone gradient. These results suggest a complex role for the Ste2p carboxy-terminal tail in the formation, orientation, and directional adjustment of the mating projection, and that endocytosis of the receptor is important for this process. In addition, mutations in RSR1/BUD1 and SPA2, genes necessary for budding polarity, exhibited little or no defect in formation or orientation of mating projections. We conclude that mating projection orientation depends upon the carboxyl terminus of the pheromone receptor and not the directional machinery used in budding.     

Nuclear protein import, but not mRNA export, is defective in all Saccharomyces cerevisiae mutants that produce temperature-sensitive forms of the Ran GTPase homologue Gsp1p

A series of ts mutations in the GSP1 gene of Saccharomyces cerevisiae was isolated by error-prone PCR. A total of 25 ts gsp1 strains was obtained. Each of these mutants showed between one and seven different amino acid alterations. In several of these ts gsp1 strains, the same amino acid residues in Gsp1p were repeatedly mutated, indicating that our screen for ts gsp1 mutations was saturating. All of the ts gsp1 strains isolated had a defect in nuclear protein import, but only 16 of the 25 ts gsp1 strains had a defect in mRNA export. Thus, Gsp1p is suggested to be directly involved in nuclear protein import, but not in mRNA export. Following release from α-factor arrest, 11 of the ts gsp1 mutants arrested in G1; the remainder did not show any specific cell-cycle arrest, at 37° C, the nonpermissive temperature. While the mutants that are defective in both mRNA export and protein import have a tendency to arrest in G1, there was no clear correlation between the cell cycle phenotype and the defects in mRNA export and nuclear protein import. Based on this, we assume that Ran/Gsp1p GTPase regulates the cell cycle and the nucleus/cytosol exchange of macromolecules through interactions with effectors that were independent of each other, and are differentially affected by mutation. Received: 30 June 1997 / Accepted: 23 October 1997     

The yeast a-factor transporter Ste6p, a member of the ABC superfamily, couples ATP hydrolysis to pheromone export

ATP-binding cassette (ABC) proteins transport a diverse collection of substrates. It is presumed that these proteins couple ATP hydrolysis to substrate transport, yet ATPase activity has been demonstrated for only a few. To provide direct evidence for such activity in Ste6p, the yeast ABC protein required for the export of a-factor mating pheromone, we established conditions for purification of Ste6p in biochemical quantities from both yeast and Sf9 insect cells. The basal ATPase activity of purified and reconstituted Ste6p (V(max) = 18 nmol/mg/min; K(m) for MgATP = 0.2 mm) compares favorably with several other ABC proteins and was inhibited by orthovanadate in a profile diagnostic of ABC transporters (apparent K(I) = 12 microm). Modest stimulation (approximately 40%) was observed upon the addition of a-factor either synthetic or in native form. We also used an 8-azido-[alpha-(32)P]ATP binding and vanadate-trapping assay to examine the behavior of wild-type Ste6p and two different double mutants (G392V/G1087V and G509D/G1193D) shown previously to be mating-deficient in vivo. Both mutants displayed a diminished ability to hydrolyze ATP, with the latter uncoupled from pheromone transport. We conclude that Ste6p catalyzes ATP hydrolysis coupled to a-factor transport, which in turn promotes mating.     

Yrb2p, a Nup2p-related yeast protein, has a functional overlap with Rna1p, a yeast Ran-GTPase-activating protein.

The Ran-GTPase cycle is important for nucleus-cytosol exchange of macromolecules and other nuclear processes. We employed the two-hybrid method to identify proteins interacting with Ran and the Ran GTP/GDP exchange factor. Using PRP20, encoding the Ran GTP/GDP exchange factor, we identified YRB1, previously identified as a protein able to interact with human Ran GTP/GDP exchange factor RCC1 in the two-hybrid system. Using GSP1, encoding the yeast Ran, as bait, we isolated YRB2. YRB2 encodes a protein containing a Ran-binding motif similar to that found in Yrb1p and Nup2p. Yrb1p is located in the cytosol whereas Nup2p is nuclear. Similar to Yrb1p, Yrb2p bound to GTP-Gsp1p but not to GDP-Gsp1p and enhanced the GTPase-activating activity of Rna1p. However, unlike Yrb1p, Yrb2p did not inhibit the nucleotide-releasing activity of Prp20p. While overproduction of Yrb1p inhibited the growth of a mutant possessing a PRP20 mutation (srm1-1) and suppressed the rna1-1 mutation, overproduction of Yrb2p showed no effect on the growth of these mutants. Disruption of YRB2 made yeast cold sensitive and was synthetically lethal with rna1-1 but not with nup2delta. Nuclear protein import and the mRNA export were normal in strains possessing mutations of YRB2. We propose that Yrb2p is involved in the nuclear processes of the Ran-GTPase cycle which are not related to nucleus-cytosol exchange of macromolecules.     

Saccharomyces cerevisiae a-factor mutants reveal residues critical for processing, activity, and export

The Saccharomyces cerevisiae mating pheromone a-factor provides a paradigm for understanding the biogenesis of prenylated fungal pheromones. The biogenesis of a-factor involves multiple steps: (i) C-terminal CAAX modification (where C is cysteine, A is aliphatic, and X is any residue) which includes prenylation, proteolysis, and carboxymethylation (by Ram1p/Ram2p, Ste24p or Rce1p, and Ste14p, respectively); (ii) N-terminal processing, involving two sequential proteolytic cleavages (by Ste24p and Axl1p); and (iii) nonclassical export (by Ste6p). Once exported, mature a-factor interacts with the Ste3p receptor on MATalpha cells to stimulate mating. The a-factor biogenesis machinery is well defined, as is the CAAX motif that directs C-terminal modification; however, very little is known about the sequence determinants within a-factor required for N-terminal processing, activity, and export. Here we generated a large collection of a-factor mutants and identified residues critical for the N-terminal processing steps mediated by Ste24p and Axl1p. We also identified mutants that fail to support mating but do not affect biogenesis or export, suggesting a defective interaction with the Ste3p receptor. Mutants significantly impaired in export were also found, providing evidence that the Ste6p transporter recognizes sequence determinants as well as CAAX modifications. We also performed a phenotypic analysis of the entire set of isogenic a-factor biogenesis machinery mutants, which revealed information about the dependency of biogenesis steps upon one another, and demonstrated that export by Ste6p requires the completion of all processing events. Overall, this comprehensive analysis will provide a useful framework for the study of other fungal pheromones, as well as prenylated metazoan proteins involved in development and aging.     

Mating-defective ste mutations are suppressed by cell division cycle start mutations in Saccharomyces cerevisiae.

Temperature-sensitive mutants which arrest in the G1 phase of the cell cycle have been described for the yeast Saccharomyces cerevisiae. One class of these mutants (carrying cdc28, cdc36, cdc37, or cdc39) forms a shmoo morphology at restrictive temperature, characteristic of mating pheromone-arrested wild-type cells. Therefore, one hypothesis to explain the control of cell division by mating factors states that mating pheromones arrest wild-type cells by inactivating one or more of these CDC gene products. A class of mutants (carrying ste4, ste5, ste7, ste11, or ste12) which is insensitive to mating pheromone and sterile has also been described. One possible function of the STE gene products is the inactivation of the CDC gene products in the presence of a mating pheromone. A model incorporating these two hypotheses predicts that such STE gene products will not be required for mating in strains carrying an appropriate cdc lesion. This prediction was tested by assaying the mating abilities of double mutants for all of the pairwise combinations of cdc and ste mutations. Lesions in either cdc36 or cdc39 suppressed the mating defect due to ste4 and ste5. Allele specificity was observed in the suppression of both ste4 and ste5. The results indicate that the CDC36, CDC39, STE4, and STE5 gene products interact functionally or physically or both in the regulation of cell division mediated by the presence or absence of mating pheromones. The cdc36 and cdc39 mutations did not suppress ste7, ste11, or ste12. Lesions in cdc28 or cdc37 did not suppress any of the ste mutations. Other models of CDC and STE gene action which predicted that some of the cdc and ste mutations would be alleles of the same locus were tested. None of the cdc mutations was allelic to the ste mutations and, therefore, these models were eliminated.     

Mapping of a yeast G protein betagamma signaling interaction.

The mating pathway of Saccharomyces cerevisiae is widely used as a model system for G protein-coupled receptor-mediated signal transduction. Following receptor activation by the binding of mating pheromones, G protein betagamma subunits transmit the signal to a MAP kinase cascade, which involves interaction of Gbeta (Ste4p) with the MAP kinase scaffold protein Ste5p. Here, we identify residues in Ste4p required for the interaction with Ste5p. These residues define a new signaling interface close to the Ste20p binding site within the Gbetagamma coiled-coil. Ste4p mutants defective in the Ste5p interaction interact efficiently with Gpa1p (Galpha) and Ste18p (Ggamma) but cannot function in signal transduction because cells expressing these mutants are sterile. Ste4 L65S is temperature-sensitive for its interaction with Ste5p, and also for signaling. We have identified a Ste5p mutant (L196A) that displays a synthetic interaction defect with Ste4 L65S, providing strong evidence that Ste4p and Ste5p interact directly in vivo through an interface that involves hydrophobic residues. The correlation between disruption of the Ste4p-Ste5p interaction and sterility confirms the importance of this interaction in signal transduction. Identification of the Gbetagamma coiled-coil in Ste5p binding may set a precedent for Gbetagamma-effector interactions in more complex organisms.     

Rat8p/Dbp5p is a shuttling transport factor that interacts with Rat7p/Nup159p and Gle1p and suppresses the mRNA export defect of xpo1-1 cells.

In a screen for temperature-sensitive mutants of Saccharomyces cerevisiae defective for mRNA export, we previously identified the essential DEAD-box protein Dbp5p/Rat8p and the nucleoporin Rat7p/Nup159p. Both are essential for mRNA export. Here we report that Dbp5p and Rat7p interact through their Nterminal domains. Deletion of this portion of Rat7p (Rat7pDeltaN) results in strong defects in mRNA export and eliminates association of Dbp5p with nuclear pores. Overexpression of Dbp5p completely suppressed the growth and mRNA export defects of rat7DeltaN cells and resulted in weaker suppression in cells carrying rat7-1 or the rss1-37 allele of GLE1. Dbp5p interacts with Gle1p independently of the N-terminus of Dbp5p. Dbp5p shuttles between nucleus and cytoplasm in an Xpo1p-dependent manner. It accumulates in nuclei of xpo1-1 cells and in cells with mutations affecting Mex67p (mex67-5), Gsp1p (Ran) or Ran effectors. Overexpression of Dbp5p prevents nuclear accumulation of mRNA in xpo1-1 cells, but does not restore growth, suggesting that the RNA export defect of xpo1-1 cells may be indirect. In a screen for high-copy suppressors of the rat8-2 allele of DBP5, we identified YMR255w, now called GFD1. Gfd1p is not essential, interacts with Gle1p and Rip1p/Nup42p, and is found in the cytoplasm and at the nuclear rim.