Phosphorylation of the Cdc42 exchange factor Cdc24 by the PAK-like kinase Cla4 may regulate polarized growth in yeast.

Rho-type GTPases control many cytoskeletal rearrangements, but their regulation remains poorly understood. Here, we show that in S. cerevisiae, activation of the CDK Cdc28-Cln2 at bud emergence triggers relocalization of Cdc24, the GEF for Cdc42, from the nucleus to the polarization site, where it is stably maintained by binding to the adaptor Bem1. Locally activated Cdc42 then polarizes the cytoskeleton in a manner dependent on its effectors Bni1 and the PAK-like kinase Cla4. In addition, Cla4 induces phosphorylation of Cdc24, leading to its dissociation from Bem1 at bud tips, thereby ending polarized bud growth in vivo. Our results thus suggest a dynamic temporal and spatial regulation of the Cdc42 module: Cdc28-Cln triggers actin polarization by activating Cdc42, which in turn restricts its own activation via a negative feedback loop acting on its GEF Cdc24.

Nuclear sequestration of the exchange factor Cdc24 by Far1 regulates cell polarity during yeast mating.

Cytoskeletal rearrangements during the cell cycle and in response to signals are regulated by small Rho-type GTPases, but it is not known how these GTPases are activated in a spatial and temporal manner. Here we show that Cdc24, the guanine-nucleotide exchange factor for the yeast GTPase Cdc42, is sequestered in the cell nucleus by Far1. Export of Cdc24 to a site of cell polarization is mediated by two mechanisms. At bud emergence, activation of the G1 cyclin-dependent kinase Cdc28-Cln triggers degradation of Far1 and, as a result, relocation of Cdc24 to the cytoplasm. Cells overexpressing a non-degradable Far1 were unable to polarize their actin cytoskeleton because they failed to relocate Cdc24 to the incipient bud site. In contrast, in response to mating pheromones, the Far1-Cdc24 complex is exported from the nucleus by Msn5. This mechanism ensures that Cdc24 is targeted to the site of receptor-associated heterotrimeric G-protein activation at the plasma membrane, thereby allowing polarization of the actin cytoskeleton along the morphogenetic gradient of pheromone. Either degradation of Far1 or its nuclear export by Msn5 was sufficient for cell growth, suggesting that the two mechanisms are redundant for cell viability. Taken together, our results indicate that Far1 functions as a nuclear anchor for Cdc24. This sequestration regulates cell polarity in response to pheromones by restricting activation of Cdc42 to the site of pheromone receptor activation.

The Cdc42p effector Gic2p is targeted for ubiquitin-dependent degradation by the SCFGrr1 complex.

Cdc42p, a Rho-related GTP-binding protein, regulates cytoskeletal polarization and rearrangements in eukaryotic cells. In yeast, Gic1p and Gic2p are effectors of Cdc42p involved in actin polarization at bud emergence. Gic2p is expressed in a cell cycle-dependent manner and rapidly disappears shortly after bud emergence concomitant with the activation of the G1 cyclin-dependent kinase Cdc28p-Clnp. Here we have shown that the rapid disappearance of Gic2p results from ubiquitin-dependent proteolysis. Biochemical and genetic evidence demonstrates that degradation of Gic2p required the Skp1-cullin-F-box protein complex (SCF) components Cdc34p, Cdc53p, Skp1p and Grr1p, but not Cdc4p. Phosphorylation of several C-terminal sites of Gic2p served as part of the recognition signal for ubiquitination. In addition, binding of Gic2p to Cdc42p was a prerequisite for degradation, suggesting that specifically the active form of Gic2p is targeted for destruction. Finally, our data indicate that degradation of Gic2p may be part of a mechanism which restricts cytoskeletal polarization in the G1 phase of the cell cycle.

Novel Cdc42-binding proteins Gic1 and Gic2 control cell polarity in yeast.

Cdc42p, a Rho-related GTP-binding protein, regulates cytoskeletal polarization and rearrangements in eukaryotic cells, but the effectors mediating this control remain unknown. Through the use of the complete yeast genomic sequence, we have identified two novel Cdc42p targets, Gic1p and Gic2p, which contain consensus Cdc42/Rac interactive-binding (CRIB) domains and bind specifically to Cdc42p-GTP. Gic1p and Gic2p colocalize with Cdc42p as cell polarity is established during the cell cycle and during mating in response to pheromones. Cells deleted for both GIC genes exhibit defects in actin and microtubule polarization similar to those observed in cdc42 mutants. Finally, the interaction of the Gic proteins and Cdc42p is essential, as mutations in the CRIB domain of Gic2p that eliminate Cdc42p binding disrupt Gic2p localization and function. Thus, Gic1p and Gic2p define a novel class of Cdc42p targets that are specifically required for cytoskeletal polarization in vivo.

p25rum1 promotes proteolysis of the mitotic B-cyclin p56cdc13 during G1 of the fission yeast cell cycle.

The fission yeast Schizosaccharomyces pombe CDK inhibitor p25rum1 plays a major role in regulating cell cycle progression during G1. Here we show that p25rum1 associates with the CDK p34cdc2/p56cdc13 during G1 in normally cycling cells and is required for the rapid proteolysis of p56cdc13. In vitro binding data indicate that p25rum1 has specificity for the B-cyclin p56cdc13 component of the CDK and can bind the cyclin even in the absence of the cyclin destruction box. At the G1-S-phase transition, p25rum1 levels decrease and p56cd13 levels increase. We also show that on release from a G1 block, the rapid disappearance of p25rum1 requires the activity of the CDK p34cdc2/cig1p and that this same CDK phosphorylates p25rum1 in vitro. We propose that the binding of p25rum1 to p56cdc13 promotes cyclin proteolysis during G1, with p25rum1 possibly acting as an adaptor protein, promoting transfer of p56cdc13 to the proteolytic machinery. At the G1-S-phase transition, p25rum1 becomes targeted for proteolysis by a mechanism which may involve p34cdc2/cig1p phosphorylation. As a consequence, at this point in the cell cycle p56cdc13 proteolysis is inhibited, leading to a rise of p56cdc13 levels in preparation for mitosis.

Ultrastructural changes in the Schizosaccharomyces pombe nucleolus following the disruption of the gar2+ gene, which encodes a nucleolar protein structurally related to nucleolin.

The nucleolar protein gar2, from the fission yeast Schizosaccharomyces pombe, is the functional homolog of NSR1 from Saccharomyces cerevisiae, and is structurally related to nucleolin from vertebrates. By immunocytochemistry at the electron microscope level, we show that gar2 co-localizes with RNA polymerase I and the gar1 protein along the dense fibrillar component of the nucleolus in a wild-type strain of S. pombe, suggesting that gar2 is involved in the transcription and/or in the early steps of maturation of the ribosomal RNAs. Since the effects of disruption of the gar2+ gene might also shed light on the role of the gar2 protein, we analyzed the ultrastructure of the nucleolus of a gar2-disruption mutant. The nucleolus of the gar2- mutant is dramatically reorganized when compared with that of the wild-type gar2+ strain: a truncated protein containing the NH2-terminus of the gar2 protein is accumulated in an unusual nucleolar "dense body". Our results also suggest that the NH2-terminus might be sufficient for nucleolar localization via interaction with specific nucleolar components and support the hypothesis that gar2 in wild-type S. pombe interacts with nascent pre-rRNA via its two RNA-binding domains in combination with the glycine/arginine-rich domain. We also report that disruption of the gar2+ gene results in a mutant that is defective in cytokinesis and nuclear division.

Mitosis-specific phosphorylation of gar2, a fission yeast nucleolar protein structurally related to nucleolin.

The nucleolar protein gar2 of fission yeast is structurally related to the multifunctional nucleolar protein nucleolin from vertebrates and has been shown to be implicated in production of 18S rRNA. gar2 contains several potential casein kinase 2 (CK2) phosphorylation sites and a single putative p34(cdc2 )phosphorylation site in the consensus S50PKK. Here, we show that, like nucleolin, gar2 is phosphorylated in vitro by both highly purified CK2 from CHO cells and p34(cdc2 )from starfish oocytes. Moreover, the substitution of alanine for the N-terminal serine 50 abolishes phosphorylation by p34(cdc2 )in vitro. We also provide evidence that gar2 is phosphorylated in vitro by a p13(suc1)-Sepharose-bound kinase from Schizosaccharomyces pombe extracts that displays cell cycle-regulated activity similar to that of the p34(cdc2(kinase. In vivo 32P labeling of cells indicates that gar2 is a phosphoprotein and that incorporation of phosphate on residue 50 occurs specifically at mitosis. Taken together, these results lead us to propose that gar2 is likely to be an in vivo substrate for the mitotic p34(cdc2 )kinase. However, this posttranslational modification of the gar2 protein does not appear to be essential for normal production of 18S rRNA.

gar2 is a nucleolar protein from Schizosaccharomyces pombe required for 18S rRNA and 40S ribosomal subunit accumulation.

Several nucleolar proteins, such as nucleolin, NOP1/fibrillarin, SSB1, NSR1 and GAR1 share a common glycine and arginine rich structural motif called the GAR domain. To identify novel nucleolar proteins from fission yeast we screened Schizosaccharomyces pombe genomic DNA libraries with a probe encompassing the GAR structural motif. Here we report the identification and characterization of a S.pombe gene coding for a novel nucleolar protein, designated gar2. The structure of the fission yeast gar2 is reminiscent of that of nucleolin from vertebrates and NSR1 from Saccharomyces cerevisiae. In addition, like these proteins, gar2 has a nucleolar localisation. The disruption of the gar2+ gene affects normal cell growth, leads to an accumulation of 35S pre-rRNA and a decrease of mature 18S rRNA steady state levels. Moreover, ribosomal profiles of the mutant show an increase of free 60S ribosomal subunits and an absence of free 40S ribosomal subunits. gar2 is able to rescue a S.cerevisiae mutant lacking NSR1, thus establishing gar2 as a functional homolog of NSR1. We propose that gar2 helps the assembly of pre-ribosomal particles containing 18S rRNA.

HLA class II associations with idiopathic nephrotic syndrome in children.

The occasional familial occurrence of idiopathic nephrotic syndrome (NS) points to a genetic predisposition. Reports on associations with certain HLA class II antigens support this hypothesis. In order to define the immunogenetic background of NS more precisely, HLA class II allele frequencies in 161 children with NS were studied by restriction fragment length polymorphism (RFLP) typing. The patient cohorts consisted of 87 children from Southwest-France and 74 from Southwest-Germany. The control group consisted of 118 French and 101 German unrelated individuals from the same geographical areas. HLA alleles were defined in patients with steroid-sensitive (SS) and steroid-resistant (SR) NS and in controls. RFLP typing revealed that the previously reported association between SSNS and HLA-DR7 is confined to the RFLP split 7.1 (DRB1*07) with a combined relative risk (RRcomb) of 6.2. HLA-DQB typing showed an increased frequency of the allele DQB2b (DQB1*0201) (RRcomb = 7.8). HLA-DQA typing showed an association of SSNS with DQA3 (DQA1*0201,0301,0302) (RRcomb = 4.1). The highest RR (16.5) for SSNS was found in German patients who carried the two DRB1 specificities 17.1 (DRB1*0301) and 7.1 (DRB1*07). All associations were stronger in SS patients with frequent relapses or steroid dependency than in non- or infrequent relapsers. SR patients exhibited no significant associations with HLA class II alleles.

Structure of the differentially expressed mouse U3A gene.

Two markedly different forms of U3 RNA are present in mouse, the relative abundance of which largely depends upon the tissues. In all cases studied so far, the more abundant form is U3B, encoded by four previously characterized genes. We report here the isolation and analysis of the unique gene encoding the U3A variant, which completes the characterization of the mouse U3 multigene family. Comparisons with rat U3 genes indicate that the diversification of the A and B forms has predated the mouse/rat separation. The two forms of U3 RNA are submitted to similar, but not identical, primary and secondary structure constraints. As for the sequences flanking the RNA coding region, similar observations emerge for both types of genes: for each type, the 5' flanks are strongly conserved between mouse and rat, over at least the proximal 500 bp, whereas only about 30 bp of proximal 3' flanks are preserved, which include a signal for the formation of vertebrate U small nRNA 3' end. By contrast the 5' flanks of the two types of genes diverge extensively from each other, either in mouse or in rat, and could be involved in the differential expression of the two forms. Even over the few conserved motifs thought to be involved in the basic transcriptional control of vertebrate U small nRNA genes, the A and B forms of U3 genes exhibit specific differences maintained in the two rodent species.