Peter A. Fantes

Since its humble start as a model organism in two European laboratories in the 1940s and 1950s, the fission yeast Schizosaccharomyces pombe has grown to become one of the best-studied eukaryotes today. This article outlines the way in which interest in S. pombe developed and spread from Europe to Japan, North America, and elsewhere from its beginnings up to the first International Meeting devoted to this yeast in 1999. We describe the expansion of S. pombe research during this period with an emphasis on many of the individual researchers involved and their interactions that resulted in the development of today’s vibrant community.

Cdc37 is an essential molecular chaperone found in fungi and metazoa whose main specificity is for certain protein kinases. Cdc37 can act as an Hsp90 cochaperone or alone; in yeasts, the interaction with Hsp90 is weak and appears not to be essential for Cdc37 function. Numerous genetic interactions between Cdc37 and likely client proteins have been observed in yeasts, but biochemical confirmation has been reported in only a few cases. We and others have generated and characterized temperature-sensitive cdc37 alleles in S. pombe and have used them to investigate the cellular roles of Cdc37: previous work has shown that mitotic Cdc2 is a major client. In this paper, we describe a screen for mutations synthetically lethal with a cdc37ts mutant with the aim of identifying genes encoding further client proteins of Cdc37. Ten such strains were isolated, and genomic libraries were screened for rescuing plasmids. In one case, a truncated cdc7 gene was identified. Further experiments showed that the mutation in this strain was indeed in cdc7 . Cdc7 is a protein kinase required for septum initiation, and we show that its kinase activity is greatly reduced when Cdc37 function is impaired. Cdc7 normally locates to the spindle pole body during mitosis, and this appears to be unaffected in the cdc37ts mutant. Other evidence suggests that, in addition to mitosis and septum initiation, Cdc37 may also be required for septum cleavage.

Cdc37 is a molecular chaperone whose clients are predominantly protein kinases, many of which are important in cell-cycle progression. Temperature-sensitive mutants of cdc37 in Schizosaccharomyces pombe are lethal at the restrictive temperature, arresting cell division within a single cell cycle. These mutant cells elongate during incubation at the restrictive temperature, consistent with a cell-cycle defect. The cell-cycle arrest arises from defective function of the mutant Cdc37 proteins rather than a reduction in Cdc37 protein levels. Around 80% of the arrested, elongated cells contain a single nucleus and replicated (2C) DNA content, indicating that these mutants arrest the cell cycle in G2 or mitosis (M). Cytological observations show that the majority of cells arrest in G2. In fission yeast, a G2 cell-cycle arrest can arise by inactivation of the cyclin-dependent kinase (Cdk) Cdc2 that regulates entry into mitosis. Studies of the cdc37 temperature-sensitive mutants show a genetic interaction with some cdc2 alleles and overexpression of cdc2 rescues the lethality of some cdc37 alleles at the restrictive temperature, suggesting that Cdc2 is a likely client for the Cdc37 molecular chaperone. In cdc37 temperature-sensitive mutants at the restrictive temperature, the level of Cdc2 protein remains constant but Cdc2 protein kinase activity is greatly reduced. Inactivation of Cdc2 appears to result from the inability to form complexes with its mitotic cyclin partner Cdc13. Further evidence for Cdc2 being a client of Cdc37 in S. pombe comes from the identification of genetic and biochemical interactions between these proteins.

Cdc37 is a molecular chaperone that interacts with a range of clients and co-chaperones, forming various high molecular mass complexes. Cdc37 sequence homology among species is low. High homology between yeast and metazoan proteins is restricted to the extreme N-terminal region, which is known to bind clients that are predominantly protein kinases. We show that despite the low homology, both Saccharomyces cerevisiae and human Cdc37 are able to substitute for the Schizosaccharomyces pombe protein in a strain deleted for the endogenous cdc37 gene. Expression of a construct consisting of only the N-terminal domain of S. pombe Cdc37, lacking the postulated heat-shock protein (Hsp) 90-binding and homodimerization domains, can also sustain cellular viability, indicating that Cdc37 dimerization and interactions with the cochaperone Hsp90 may not be essential for Cdc37 function in S. pombe. Biochemical investigations showed that a small proportion of total cellular Cdc37 occurs in a high molecular mass complex that also contains Hsp90. These data indicate that the N-terminal domain of Cdc37 carries out essential functions independently of the Hsp90-binding domain and dimerization of the chaperone itself.

The identification of a Schizosaccharomyces pombe homologue of the cdc37 gene is described. The gene product is most similar to the budding yeast homologue, but shows similarity to metazoan Cdc37 proteins, with a region of high similarity at the extreme N-terminus. Gene transplacement experiments in diploid cells followed by tetrad dissection show that the gene is essential. Depletion of the gene product after switching off expression of cdc37 from the regulatable nmt81 promoter results in cessation of growth and division. The cells arrest heterogeneously, with a significant proportion showing mitotic defects; paradoxically, a proportion of the cells show a short-cell phenotype consistent with an advanced cell cycle.

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⁺andwis4⁺, 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 win1mutations. 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 wee1-50mutant and its suppression by wis genes.