The wis1 protein kinase is a dosage-dependent regulator of mitosis in Schizosaccharomyces pombe.

The wis1+ gene encodes a newly identified mitotic control element in Schizosaccharomyces pombe. It was isolated by virtue of its interaction with the mitotic control genes cdc25, wee1 and win1. The wis1+ gene potentially encodes a 66 kDa protein with homology to the serine/threonine family of protein kinases. wis1+ plays an important role in the regulation of entry into mitosis, as it shares with cdc25+ and nim1+/cdr1+ the property of inducing mitosis in a dosage-dependent manner. Increased levels of wis1+ expression cause mitotic initiation to occur at a reduced cell size. Loss of wis1+ function does not prevent vegetative growth and division, though wis1- cells show an elongated morphology, indicating that their entry into mitosis and cell division is delayed relative to wild type cells. wis1- cells undergo a rapid reduction of viability upon entry into stationary phase, suggesting a role for wis1+ in the integration of nutritional sensing with the control over entry into mitosis.     

Two cytoplasmic candidates for immunophilin action are revealed by affinity for a new cyclophilin: one in the presence and one in the absence of CsA.

We report the cloning and characterization of a new binding protein for the immunosuppressive drug cyclosporin A (CsA). This new cyclophilin, cyclophilin C (cyp C), shows extensive homology with all previously identified cyclophilins. Cyp C mRNA is expressed in a restricted subset of tissues relative to cyclophilins A and B, but is present in those tissues reported to be most affected by CsA therapy. A cyp C fusion protein has peptidyl-prolyl isomerase activity, and CsA inhibits this activity. Using the cyp C fusion protein as an affinity ligand to probe cellular extracts, we find that the cyp C fusion protein binds specifically to a 77 kd protein in the absence of CsA, while in the presence of CsA it instead binds specifically to a 55 kd protein. We propose that the p77 is involved in cyp C native function and that the p55 is involved in signal transduction events blocked by treatment with immunosuppressive levels of CsA.     

CNS1 Encodes an Essential p60/Sti1 Homolog in Saccharomyces cerevisiae That Suppresses Cyclophilin 40 Mutations and Interacts with Hsp90

Cyclophilins are cis-trans-peptidyl-prolyl isomerases that bind to and are inhibited by the immunosuppressant cyclosporin A (CsA). The toxic effects of CsA are mediated by the 18-kDa cyclophilin A protein. A larger cyclophilin of 40 kDa, cyclophilin 40, is a component of Hsp90-steroid receptor complexes and contains two domains, an amino-terminal prolyl isomerase domain and a carboxy-terminal tetratricopeptide repeat (TPR) domain. There are two cyclophilin 40 homologs in the yeast Saccharomyces cerevisiae, encoded by the CPR6 and CPR7 genes. Yeast strains lacking the Cpr7 enzyme are viable but exhibit a slow-growth phenotype. In addition, we show here that cpr7 mutant strains are hypersensitive to the Hsp90 inhibitor geldanamycin. When overexpressed, the TPR domain of Cpr7 alone complements both cpr7 mutant phenotypes, while overexpression of the cyclophilin domain of Cpr7, full-length Cpr6, or human cyclophilin 40 does not. The open reading frame YBR155w, which has moderate identity to the yeast p60 homolog STI1, was isolated as a high-copy-number suppressor of the cpr7 slow-growth phenotype. We show that this Sti1 homolog Cns1 (cyclophilin seven suppressor) is constitutively expressed, essential, and found in protein complexes with both yeast Hsp90 and Cpr7 but not with Cpr6. Cyclosporin A inhibited Cpr7 interactions with Cns1 but not with Hsp90. In summary, our findings identify a novel component of the Hsp90 chaperone complex that shares function with cyclophilin 40 and provide evidence that there are functional differences between two conserved sets of Hsp90 binding proteins in yeast.     

Mcs4, a Two-Component System Response Regulator Homologue, Regulates the Schizosaccharomyces Pombe Cell Cycle Control

The Schizosaccharomyces pombe cdc2-3w wee1-50 double mutant displays a temperature-sensitive lethal phenotype termed mitotic catastrophe. Six mitotic catastrophe suppressor (mcs1-6) genes were identified in a genetic screen designed to identify regulators of cdc2. Mutations in mcs1-6 suppress the cdc2-3w wee1-50 temperature-sensitive growth defect. Here, the cloning of mcs4 is described. The mcs4 gene product displays significant sequence homology to members of the two-component system response regulator protein family. Strains carrying the mcs4 and cdc25 mutations display a synthetic osmotic lethal phenotype along with an inability to grow on minimal synthetic medium. These phenotypes are suppressed by a mutation in wee1. In addition, the wis1 gene, encoding a stress-activated mitogen-activated protein kinase kinase, was identified as a dosage suppressor in this screen. These findings link the two-component signal transduction system to stress response and cell cycle control in S. pombe.     

Isolation and characterization of a second protein tyrosine phosphatase gene, PTP2, from Saccharomyces cerevisiae.

A putative protein tyrosine phosphatase (PTPase) gene, PTP2, was cloned from Saccharomyces cerevisiae. The complete yeast PTP2 gene encodes a 750-amino acid residue protein with a predicted mass of 86 kDa. The conserved PTPase domain was localized in the C-terminal half of the protein. Amino acid sequence alignment of the yeast PTPase domain with other phosphatases indicated approximately 20-25% sequence identity with the mammalian PTPase and a similar degree of identity with the PTPase encoded by the yeast PTP1 gene. The PTP2 gene is closely linked to the yeast RET1 and STE4 genes and is localized on the right arm of chromosome 15. Gene disruption experiments demonstrated that neither PTP2 alone nor PTP2 in combination with PTP1 was essential for growth under the conditions tested. The ability of PTP2 to complement the cdc25-22 mutant of Schizosaccharomyces pombe was also examined, and unlike the human T-cell PTPase, which was able to complement the cdc25-22 mutant, the S. cerevisiae PTP2 was unable to complement the cdc25-22 mutant of S. pombe.     

The Fission Yeast Mitotic Regulator win1+ Encodes an MAP Kinase Kinase Kinase That Phosphorylates and Activates Wis1 MAP Kinase Kinase in Response to High Osmolarity

The Schizosaccharomyces pombe win1-1 mutant has a defect in the G2-M transition of the cell cycle. Although the defect is suppressed by wis1⁺ and wis4⁺, which are components of a stress-activated MAP kinase pathway that links stress response and cell cycle control, the molecular identity of Win1 has not been known. We show here that win1⁺ encodes a polypeptide of 1436 residues with an apparent molecular size of 180 kDa and demonstrate that Win1 is a MAP kinase kinase kinase that phosphorylates and activates Wis1. Despite extensive similarities between Win1 and Wis4, the two MAP kinase kinase kinases have distinct functions. Wis4 is able to compensate for loss of Win1 only under unstressed conditions to maintain basal Wis1 activity, but it fails to suppress the osmosignaling defect conferred by win1 mutations. The win1-1 mutation is a spontaneous duplication of 16 nucleotides, which leads to a frameshift and production of a truncated protein lacking the kinase domain. We discuss the cell cycle phenotype of the win1-1 cdc25-22 wee1-50 mutant and its suppression by wis genes.     

Counteractive roles of protein phosphatase 2C (PP2C) and a MAP kinase kinase homolog in the osmoregulation of fission yeast.

With the goal of discovering the cellular functions of type 2C protein phosphatases, we have cloned and analyzed two ptc (phosphatase two C) genes, ptc2+ and ptc3+, from the fission yeast Schizosaccharomyces pombe. Together with the previously identified ptc1+ gene, the enzymes encoded by these genes account for approximately 90% of the measurable PP2C activity in fission yeast cells. No obvious growth defects result from individual disruptions of ptc genes, but a delta ptc1 delta ptc3 double mutant displays aberrant cell morphology and temperature-sensitive cell lysis that is further accentuated in a delta ptc1 delta ptc2 delta ptc3 triple mutant. These phenotypes are almost completely suppressed by the presence of osmotic stabilizers, strongly indicating that PP2C has an important role in osmoregulation. Genetic suppression of delta ptc1 delta ptc3 lethality identified two loci, mutations of which render cells hypersensitive to high-osmolarity media. One locus is identical to wis1+, encoding a MAP kinase kinase (MEK) homolog. The Wis1 sequence is most closely related to the Saccharomyces cerevisiae MEK encoded by PBS2, which is required for osmoregulation. These data indicate that divergent yeasts have functionally conserved MAP kinase pathways, which are required to increase intracellular osmotic concentrations in response to osmotic stress. Moreover, our observations implicate PP2C enzymes as also having an important role in signal transduction processes involved in osmoregulation, probably acting to negatively regulate the osmosensing signal that is transmitted through Wis1 MAP kinase kinase.     

Severe growth defect in a Schizosaccharomyces pombe mutant defective in intron lariat degradation.

The cDNAs and genes encoding the intron lariat-debranching enzyme were isolated from the nematode Caenorhabditis elegans and the fission yeast Schizosaccharomyces pombe based on their homology with the Saccharomyces cerevisiae gene. The cDNAs were shown to be functional in an interspecific complementation experiment; they can complement an S. cerevisiae dbr1 null mutant. About 2.5% of budding yeast S. cerevisiae genes have introns, and the accumulation of excised introns in a dbr1 null mutant has little effect on cell growth. In contrast, many S. pombe genes contain introns, and often multiple introns per gene, so that S. pombe is estimated to contain approximately 40 times as many introns as S. cerevisiae. The S. pombe dbr1 gene was disrupted and shown to be nonessential. Like the S. cerevisiae mutant, the S. pombe null mutant accumulated introns to high levels, indicating that intron lariat debranching represents a rate-limiting step in intron degradation in both species. Unlike the S. cerevisiae mutant, the S. pombe dbr1::leu1+ mutant had a severe growth defect and exhibited an aberrant elongated cell shape in addition to an intron accumulation phenotype. The growth defect of the S. pombe dbr1::leu1+ strain suggests that debranching activity is critical for efficient intron RNA degradation and that blocking this pathway interferes with cell growth.     

Cyclophilin sensitivity to sanglifehrin A can be correlated to the same specific tryptophan residue as cyclosporin A

Sanglifehrin A (SFA) is a recently discovered immunosuppressant drug that shares its intracellular target with the major immunosuppressant drug cyclosporin A (CsA). Both bind to and inhibit the cyclophilins, a diverse family of proteins found throughout nature that share a conserved catalytic domain. Although they share this common protein target, the mechanism of action of the cyclophilin-SFA complex has been reported as distinct from that of the well-studied cyclophilin-CsA complex. The X-ray structure of a macrolide analogue of SFA's cyclic region complexed with cyclophilin A has recently been resolved, but this left the placement of the linear region of SFA unresolved. Using five cyclophilins from the fission yeast Schizosaccharomyces pombe, and a mutant of one of these proteins, SpCyp3-F128W, we have shown that the sensitivity of cyclophilins to SFA can be correlated to the same specific tryptophan residue that has previously been identified to correlate to CsA sensitivity, and that the tail of SFA may be responsible for mediating this sensitivity.     

High level expression and characterization of the cyclophilin B gene from the anaerobic fungus Orpinomyces sp. strain PC-2

Cyclophilins are an evolutionarily conserved family of peptidyl-prolyl cis-trans isomerases (PPIases). A cyclophilin B (cypB) gene from the anaerobic fungus Orpinomyces sp. strain PC-2 was cloned and overexpressed in Escherichia coli. It was expressed as an amino-terminal 6 x His-tagged recombinant protein to facilitate purification. Highly purified protein (26.5 kDa) was isolated by two chromatographic steps involving affinity and gel filtration for biochemical studies of the enzyme. The recombinant CypB displayed PPIase activity with a k(cat)/K(m) of 8.9 x 10(6) M(-1) s(-1) at 10 degrees C and pH 7.8. It was inhibited by cyclosporin A (CsA) with an IC(50) of 23.5 nM, similar to those of the native protein and other cyclophilin B enzymes from animals. Genomic DNA analysis of cypB revealed that it was present as a single copy in Orpinomyces PC-2 and contained two introns, indicating it has a eukaryotic origin. It is one of the most heavily interrupted genes with intron sequences found in anaerobic fungi. The three-dimensional model of Orpinomyces PC-2 CypB was predicted with a homology modeling approach using the Swiss-Model Protein Modeling Server and three dimensional structure of human CypB as a template. The overall architecture of the CypB molecule is very similar to that of human CypB.     

FK-506, a potent novel immunosuppressive agent, binds to a cytosolic protein which is distinct from the cyclosporin A-binding protein, cyclophilin

A novel macrolide antibiotic, FK-506, isolated from Streptomyces tsukubaensis, has been shown to be a potent immunosuppressive agent in vivo and in vitro. FK-506 shares a number of immunosuppressive properties with the cyclic peptide, cyclosporin A (CsA), although 10 to 100 times more potent in this regard. These similarities suggest that both agents may share a similar mechanism(s) of action at the biochemical level. We have identified a cytoplasmic binding protein for FK-506 in the human T cell line, JURKAT, using [3H]FK-506. The FK-506 binding protein has a mr of 10 to 12 kDa (as determined by gel filtration), is heat stable and does not bind CsA. This contrasts with the CsA binding protein, cyclophilin, in that cyclophilin is heat labile and has a mr of 15 to 17 kDa. Our data suggest that FK-506 binds to a low m.w. protein(s) in JURKAT cells, which is distinct from cyclophilin. This protein may mediate the immunosuppressive effects of FK-506 in T cells. In addition, our results suggest that the immunosuppressive activity of FK-506, as with CsA, is mediated by an intracellular mechanism.     

Vacuolar protein sorting in fission yeast: cloning, biosynthesis, transport, and processing of carboxypeptidase Y from Schizosaccharomyces pombe.

PCR was used to isolate a carboxypeptidase Y (CPY) homolog gene from the fission yeast Schizosaccharomyces pombe. The cloned S. pombe cpy1+ gene has a single open reading frame, which encodes 950 amino acids with one potential N-glycosylation site. It appears to be synthesized as an inactive pre-pro protein that likely undergoes processing following translocation into appropriate intracellular organelles. The C-terminal mature region is highly conserved in other serine carboxypeptidases. In contrast, the N-terminal pro region containing the vacuolar sorting signal in CPY from Saccharomyces cerevisiae shows fewer identical residues. The pro region contains two unusual repeating sequences; repeating sequence I consists of seven contiguous repeating segments of 13 amino acids each, and repeating sequence II consists of seven contiguous repeating segments of 9 amino acids each. Pulse-chase radiolabeling analysis revealed that Cpy1p was initially synthesized in a 110-kDa pro-precursor form and via the 51-kDa single-polypeptide-chain intermediate form which has had its pro segment removed is finally converted to a heterodimer, the mature form, which is detected as a 32-kDa protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. Like S. cerevisiae CPY, S. pombe Cpy1p does not require the N-linked oligosaccharide moiety for vacuolar delivery. To investigate the vacuolar sorting signal of S. pombe Cpy1p, we have constructed cpy1+-SUC2 gene fusions that direct the synthesis of hybrid proteins consisting of N-terminal segments of various lengths of S. pombe Cpy1p fused to the secreted enzyme S. cerevisiae invertase. The N-terminal 478 amino acids of Cpy1 are sufficient to direct delivery of a Cpy1-Inv hybrid protein to the vacuole. These results showed that the pro peptide of Cpy1 contains the putative vacuolar sorting signal.     

Identification and characterization of Moca-cyp. A Drosophila melanogaster nuclear cyclophilin

Cyclophilins are enzymes catalyzing the cis-trans isomerization of peptidyl-prolyl bonds and belong to the enzyme class of peptidyl-prolyl cis-trans isomerases (PPIases), which includes two more families (FK506 binding proteins and parvulins). We report the characterization of a novel cyclophilin (Moca-cyp) isolated from Drosophila melanogaster. The single-copy Moca-cyp gene, which is localized on chromosome 3R, was cloned and sequenced. The sequence alignment of the gene against Moca-cyp cDNA allowed us to define its intron/exon structure and to identify a variant cDNA corresponding to an alternatively spliced mRNA. By embryo in situ RNA hybridization and immunostaining, we show that the expression of Moca-cyp is regulated during embryogenesis of Drosophila. The 120-kDa nuclear Moca-cyp protein belongs to a subfamily of large cyclophilins sharing structural and enzymatic features: their highly conserved N-terminal PPIase domain is extended by a positively charged and divergent C-terminal tail. Compared with cyclophilin 18, the enzymatic activity carried by the PPIase domain of Moca-cyp is low, exhibits characteristic substrate specificity, and shows a reduced sensitivity to the drug cyclosporin A (CsA). The reduced affinity for CsA is one of the typical features linking members of this subfamily and is probably the consequence of two amino acid substitutions within their active site. Another structural feature shared by members of this subfamily is a conserved polypeptidic segment ("moca" domain) that we report for the first time. The moca domain is located within the C-terminal tail and is the exclusive hallmark of a group of large cyclophilins found in multicellular organisms of the animal kingdom.     

Mutation of a conserved glycine residue modifies the vanadate sensitivity of the plasma membrane H+-ATPase from Schizosaccharomyces pombe

The structural gene pma+1 for the H+-ATPase from the fission yeast Schizosaccharomyces pombe has been isolated and sequenced. The intron-less gene encodes for a protein of Mr = 99,769 which is 75% homologous to those of Saccharomyces cerevisiae and Neurospora crassa. The S. pombe pma+1 gene complements not only S. pombe pma-1-1 but also S. cerevisiae pma-1-4 mutants selected for in vitro vanadate-resistant ATPase activity. The sequence of the S. pombe mutant pma-1-1 allele reveals that the glycine residue 268, which is perfectly conserved in the transduction domain of all animal and fungal transport ATPases sequenced so far, is modified into an aspartate residue by the mutation. Replacement of glycine 268 by aspartate has been monitored by the appearance of a new PvuI restriction site in the mutant DNA. Mitotic cosegregation has been observed between the PvuI site and vanadate-resistant ATPase activity in a growing population of S. pombe transformants.     

A Magnaporthe grisea cyclophilin acts as a virulence determinant during plant infection

Cyclophilins are peptidyl prolyl cis-trans isomerases that are highly conserved throughout eukaryotes and that are best known for being the cellular target of the immunosuppressive drug cyclosporin A (CsA). The activity of CsA is caused by the drug forming a complex with cyclophilin A and inhibiting the calmodulin-dependent phosphoprotein phosphatase calcineurin. We have investigated the role of CYP1, a cyclophilin-encoding gene in the phytopathogenic fungus Magnaporthe grisea, which is the causal agent of rice blast disease. CYP1 putatively encodes a mitochondrial and cytosolic form of cyclophilin, and targeted gene replacement has shown that CYP1 acts as a virulence determinant in rice blast. Cyp1 mutants show reduced virulence and are impaired in associated functions, such as penetration peg formation and appressorium turgor generation. CYP1 cyclophilin also is the cellular target for CsA in Magnaporthe, and CsA was found to inhibit appressorium development and hyphal growth in a CYP1-dependent manner. These data implicate cyclophilins as virulence factors in phytopathogenic fungi and also provide evidence that calcineurin signaling is required for infection structure formation by Magnaporthe.     

Inhibition of G2/M progression in Schizosaccharomyces pombe by a mutant calmodulin kinase II with constitutive activity.

Intracellular signaling by the second messenger Ca2+ through its receptor calmodulin (CaM) regulates cell function via the activation of CaM-dependent enzymes. Previous studies have shown that cell cycle progression at G1/S and G2/M is sensitive to intracellular CaM levels. However, little is known about the CaM-regulated enzymes involved. Protein phosphorylation has been shown to be important for cell-cycle regulation. Because CaM regulates several protein kinases, and at least one protein phosphatase, our studies are focusing on the roles of these enzymes within the cell cycle. As an initial approach to this problem, cDNAs encoding either normal or mutant calcium/calmodulin kinase II (CaMKII) have been expressed in Schizosaccharomyces pombe. The results show that overexpression of a constitutively active mutant CaMKII caused cell-cycle arrest in G2. Arrest was associated with a failure to activate the p34/cdc2 protein kinase. Expression of the mutant CaMKII in strains of S. pombe with altered timing of mitosis revealed that this effect is not mediated either by cdc25+ or wee1+, suggesting that CaMKII may regulate G2/M progression by another mechanism.     

Assembly of the Rieske iron-sulphur protein into the cytochrome bf complex in thylakoid membranes of isolated pea chloroplasts.

The assembly of the Rieske iron-sulphur protein into the cytochrome bf complex was examined following import of 35S-labeled precursor protein by isolated pea chloroplasts. Rieske protein assembled into the cytochrome bf complex was resolved from unassembled Rieske protein and from other membrane complexes by nondenaturing gel electrophoresis of dodecyl maltoside-solubilized thylakoid membranes. Four mutant forms of the Rieske protein were able to assemble into the cytochrome bf complex in isolated chloroplasts. These were a triple substitution mutant, C107S/H109R/C112S, replacing conserved residues involved in the ligation of the [2Fe-2S] centre; the mutant Delta45-52 which removed a glycine-rich region predicted to form a flexible hinge between the hydrophobic membrane-associated region and the hydrophilic lumenal domain; and mutants Delta168-173 and Delta177-179 which removed two C-terminal regions, which are highly conserved in chloroplast and cyanobacterial Rieske proteins. This indicates that the [2Fe-2S] cluster, the glycine-rich region and the C-terminal region are not essential for stable assembly of the Rieske protein into the cytochrome bf complex in isolated chloroplasts.