Shelley Sazer

Phosphorylation at Thr167 is required for Schizosaccharomyces pombe p34cdc2 function.

Eukaryotic cell cycle progression requires the periodic activation and inactivation of a protein‐serine/threonine kinase which in fission yeast is encoded by the cdc2+ gene. The activity of this gene product, p34cdc2, is controlled by numerous interactions with other proteins and by its phosphorylation state. In fission yeast, p34cdc2 is phosphorylated on two sites, one of which has been identified as Tyr15. Dephosphorylation of Tyr15 regulates the initiation of mitosis. To understand more completely the regulation of p34cdc2 kinase activity, we have identified the second site of phosphorylation as Thr167, a residue conserved amongst all p34cdc2 homologues. By analysing the phenotypes of cells expressing various position 167 mutations and performing in vitro experiments, we establish that Thr167 phosphorylation is required for p34cdc2 kinase activity at mitosis and is involved in the association of p34cdc2 with cyclin B. Dephosphorylation of Thr167 might also play a role in the exit from mitosis.

The search for the primary function of the Ran GTPase continues

It has been nearly 20 years since the discovery of the first component of the Ran-GTPase pathway. Since then, nearly 100 articles, more than half of which have been published in the past three years, have reported the identification of additional components of the system and the existence of their structural and functional homologues in organisms ranging from yeast to man. The Ran system affects a vast array of nuclear processes including RNA metabolism, DNA replication, chromosome condensation and decondensation, and nucleocytoplasmic transport of protein and RNA. The current challenge is to identify the molecular targets that link the Ran-GTPase system to this collection o f nuclear processes.

The Fission Yeast Nup107-120 Complex Functionally Interacts with the Small GTPase Ran/Spi1 and Is Required for mRNA Export, Nuclear Pore Distribution, and Proper Cell Division

ABSTRACT We have characterized Schizosaccharomyces pombe open reading frames encoding potential orthologues of constituents of the evolutionarily conserved Saccharomyces cerevisiae Nup84 vertebrate Nup107-160 nuclear pore subcomplex, namely Nup133a, Nup133b, Nup120, Nup107, Nup85, and Seh1. In spite of rather weak sequence conservation, in vivo analyses demonstrated that these S. pombe proteins are localized at the nuclear envelope. Biochemical data confirmed the organization of these nucleoporins within conserved complexes. Although examination of the S. cerevisiae and S. pombe deletion mutants revealed different viability phenotypes, functional studies indicated that the involvement of this complex in nuclear pore distribution and mRNA export has been conserved between these highly divergent yeasts. Unexpectedly, microscopic analyses of some of the S. pombe mutants revealed cell division defects at the restrictive temperature (abnormal septa and mitotic spindles and chromosome missegregation) that were reminiscent of defects occurring in several S. pombe GTPase Ran (RanSp)/Spi1 cycle mutants. Furthermore, deletion of nup120 moderately altered the nuclear location of RanSp/Spi1, whereas overexpression of a nonfunctional RanSp/Spi1-GFP allele was specifically toxic in the Δnup120 and Δnup133b mutant strains, indicating a functional and genetic link between constituents of the S. pombe Nup107-120 complex and of the RanSp/Spi1 pathway.

Redundant control of rereplication in fission yeast

The initiation of DNA replication at replication origins in eukaryotic cells is tightly controlled to ensure that the genome is duplicated only once each cell cycle. We present evidence that in fission yeast, independent regulation of two essential components of the initiation complex, Cdc18 and Cdt1, contributes to the prevention of reinitiation of DNA replication. Cdc18 is negatively controlled by cyclin-dependent kinase (CDK) phosphorylation, but low level expression of a mutant form of Cdc18 lacking CDK phosphorylation sites (Cdc18CDK) is not sufficient to induce rereplication. Similar to Cdc18, Cdt1 is expressed periodically in the cell cycle, accumulating in the nucleus in G1 and declining in G2. When Cdt1 is expressed constitutively from an ectopic promoter, it accumulates in the nucleus throughout the cell cycle but does not promote reinitiation. However, constitutive expression of Cdt1, together with Cdc18CDK, is sufficient to induce extra rounds of DNA replication in the absence of mitosis. Significantly greater levels of rereplication can be induced by coexpression of Cdc18CDK and a Cdt1 mutant lacking a conserved C-terminal motif. In contrast, uncontrolled DNA replication does not occur when either mutant protein is expressed in the absence of the other. Constitutive expression of wild-type or mutant Cdt1 also leads to an increase in the levels of Cdc18CDK, possibly as a result of increased protein stability. Our data are consistent with the hypothesis that control of rereplication depends on a redundant mechanism in which negative regulation of Cdt1 functions in parallel with the negative regulation of Cdc18.

Fission yeast Lem2 and Man1 perform fundamental functions of the animal cell nuclear lamina

In animal cells the nuclear lamina, which consists of lamins and lamin-associated proteins, serves several functions: it provides a structural scaffold for the nuclear envelope and tethers proteins and heterochromatin to the nuclear periphery. In yeast, proteins and large heterochromatic domains including telomeres are also peripherally localized, but there is no evidence that yeast have lamins or a fibrous nuclear envelope scaffold. Nonetheless, we found that the Lem2 and Man1 proteins of the fission yeast Schizosaccharomyces pombe, evolutionarily distant relatives of the Lap2/Emerin/Man1 (LEM) sub-family of animal cell lamin-associated proteins, perform fundamental functions of the animal cell lamina. These integral inner nuclear membrane localized proteins, with nuclear localized DNA binding Helix-Extension-Helix (HEH) domains, impact nuclear envelope structure and integrity, are essential for the enrichment of telomeres at the nuclear periphery and by means of their HEH domains anchor chromatin, most likely transcriptionally repressed heterochromatin, to the nuclear periphery. These data indicate that the core functions of the nuclear lamina are conserved between fungi and animal cells and can be performed in fission yeast, without lamins or other intermediate filament proteins.

Stage-specific activity of the Leishmania major CRK3 kinase and functional rescue of a Schizosaccharomyces pombe cdc2 mutant

Cell cycle control by cdc2-related kinases (CRKs) is essential to the regulation of cell proliferation and developmental processes in many organisms. Alternating phases of growth, arrest, and differentiation are characteristics of the infectious cycle of many trypanosomatid parasites, raising the possibility that members of the trypanosomatid CRK gene family participate in the regulation of these essential processes. Here we describe properties of the CRK3 gene from Leishmania major, which encodes a 36 kDa protein kinase showing 60% amino acid sequence identity with human CDK2, including several conserved sites implicated in regulation of kinase activity. CRK3 mRNA was constitutively expressed throughout the parasite life cycle, but histone H1 kinase activity of an epitope tagged CRK3 protein was greater in log-phase than in stationary-phase promastigotes. When integrated into the genome and expressed at the optimal level, CRK3 was able to rescue the growth defect of a Schizosaccharomyces pombe cdc2 mutant (cdc2-33(ts)), indicating that CRK3 is a functional homolog of cdc2. Mutants of CRK3 at several key regulatory residues showed the expected dominant negative effects on the S. pombe mutant. This is the first example of functional expression of a trypanosomatid CRK in yeast, opening the way for further genetic studies within this amenable organism.

Evolutionary cell biology: Two origins, one objective

All aspects of biological diversification ultimately trace to evolutionary modifications at the cellular level. This central role of cells frames the basic questions as to how cells work and how cells come to be the way they are. Although these two lines of inquiry lie respectively within the traditional provenance of cell biology and evolutionary biology, a comprehensive synthesis of evolutionary and cell-biological thinking is lacking. We define evolutionary cell biology as the fusion of these two eponymous fields with the theoretical and quantitative branches of biochemistry, biophysics, and population genetics. The key goals are to develop a mechanistic understanding of general evolutionary processes, while specifically infusing cell biology with an evolutionary perspective. The full development of this interdisciplinary field has the potential to solve numerous problems in diverse areas of biology, including the degree to which selection, effectively neutral processes, historical contingencies, and/or constraints at the chemical and biophysical levels dictate patterns of variation for intracellular features. These problems can now be examined at both the within- and among-species levels, with single-cell methodologies even allowing quantification of variation within genotypes. Some results from this emerging field have already had a substantial impact on cell biology, and future findings will significantly influence applications in agriculture, medicine, environmental science, and synthetic biology.

The Ran GTPase System in Fission Yeast Affects Microtubules and Cytokinesis in Cells That Are Competent for Nucleocytoplasmic Protein Transport

ABSTRACT Misregulation of the evolutionarily conserved GTPase Ran in fission yeast results in defects in several cellular processes in cells that are competent for nucleocytoplasmic protein transport. These results suggest that transport is neither the only nor the primary Ran-dependent process in living cells. The ability of Ran to independently regulate multiple cellular processes in vivo is demonstrated by showing that (i) eight different transport-competent RanGEF (guanine nucleotide exchange factor) mutants have defects in mitotic spindle formation; (ii) the RanGEF temperature-sensitive mutant pim1-d1 has abnormal actin ring structures at the septum. Overexpression of Imp2p, which specifically destabilizes these structures, restores viability. (iii) Ran-dependent processes differ in their requirements for active Ran in vivo. Microtubule function, cytokinesis, and nuclear envelope structure are the Ran-dependent processes most sensitive to the amount of Ran protein in the cell, whereas nucleocytoplasmic protein transport is the most robust. Therefore, the ability of Ran from Schizosaccharomyces pombe to independently regulate multiple cellular processes may reflect differences in its interactions with the binding proteins that mediate these functions and explain the complex phenotypic consequences of its misregulation in vivo.

Nuclear envelope: Nuclear pore complexity

A new study shows that the filamentous fungus, Aspergillus nidulans, which has a closed mitosis, does not maintain a continuous permeability barrier during mitosis. This work challenges current views of the differences between closed and open mitosis and has implications for understanding mitotic specific changes in the nuclear pore complex and Ran GTPase system in lower eukaryotes.

Characterization of a Nuclear Protein Conferring Brefeldin A Resistance in Schizosaccharomyces pombe

The fungal metabolite brefeldin A disrupts protein secretion and causes the redistribution of the Golgi complex to the endoplasmic reticulum. Previously we isolated six genes that, when present in multiple copies, confer brefeldin A resistance to wild type Schizosaccharomyces pombe. Here we describe the characterization of one of these genes, hba1. This gene encodes an essential protein that shares homology with the mammalian protein RanBP1 and the protein encoded by the Saccharomyces cerevisiae gene YRB1 and contains a peptide motif present in several proteins found within the nuclear pore complex. The protein encoded by hba1 is localized to the nucleus, and it was determined that this protein is phosphorylated in vivo. The characterization of hba1 thus demonstrates a novel mechanism of drug resistance in S. pombe.