Fission yeast pak1+ encodes a protein kinase that interacts with Cdc42p and is involved in the control of cell polarity and mating

A STE20/p65pak homolog was isolated from fission yeast by PCR. The pak1+ gene encodes a 72 kDa protein containing a putative p21-binding domain near its amino-terminus and a serine/threonine kinase domain near its carboxyl-terminus. The Pak1 protein autophosphorylates on serine residues and preferentially binds to activated Cdc42p both in vitro and in vivo. This binding is mediated through the p21 binding domain on Pak1p and the effector domain on Cdc42p. Overexpression of an inactive mutant form of pak1 gives rise to cells with markedly abnormal shape with mislocalized actin staining. Pak1 overexpression does not, however, suppress lethality associated with cdc42-null cells or the morphologic defeat caused by overexpression of mutant cdc42 alleles. Gene disruption of pak1+ establishes that, like cdc42+, pak1+ function is required for cell viability. In budding yeast, pak1+ expression restores mating function to STE20-null cells and, in fission yeast, overexpression of an inactive form of Pak inhibits mating. These results indicate that the Pak1 protein is likely to be an effector for Cdc42p or a related GTPase, and suggest that Pak1p is involved in the maintenance of cell polarity and in mating.     

Evidence for an age-related dysfunction in the antiproliferative response to transforming growth factor-beta in vascular smooth muscle cells

The p34cdc2 protein kinase plays a key role in the control of the mitotic cell cycle of fission yeast, being required for both entry into S-phase and for entry into mitosis in the mitotic cell cycle, as well as for the initiation of the second meiotic nuclear division. In recent years, structural and functional homologues of p34cdc2, as well as several of the proteins that interact with and regulate p34cdc2 function in fission yeast, have been identified in a wide range of higher eukaryotic cell types, suggesting that the control mechanisms uncovered in this simple eukaryote are likely to be well conserved across evolution. Here we describe the construction and characterisation of a fission yeast strain in which the endogenous p34cdc2 protein is entirely absent and is replaced by its human functional homologue p34CDC2. We have used this strain to analyse aspects of the function of the human p34CDC2 protein genetically. We show that the function of the human p34CDC2 protein in fission yeast cells is dependent upon the action of the protein tyrosine phosphatase p80cdc25, that it responds to altered levels of both the mitotic inhibitor p107wee1 and the p34cdc2-binding protein p13suc1, and is lethal in combination with the mutant B-type cyclin p56cdc13-117. In addition, we demonstrate that the human p34CDC2 protein is proficient for fission yeast meiosis, and examine the behaviour of two mutant p34CDC2 proteins in fission yeast.     

Conjugation in S. pombe: identification of a microtubule-organising centre, a requirement for microtubules and a role for Mad2

During the G1 phase of the cell cycle, cells of the fission yeast Schizosaccharomyces pombe can be induced to mate by nitrogen starvation and the presence of mating pheromones. Polarised growth towards cells of the opposite mating type (P or M) leads to the formation of a projection tip and, upon contact, localised cell wall degradation results in conjugation and cell fusion [1]. Here, we have investigated the role of microtubules in this process. We describe a previously unidentified microtubule-organising centre (MTOC) that forms at projection tips upon cell-to-cell contact, before cells fuse. Treatment of mating cells with the microtubule-destabilising drug thiabendazole (TBZ) showed that microtubule integrity was required for mating at two distinct stages: during projection tip formation and cell fusion. Projection tip formation requires filamentous (F) actin function [2] and microtubules are required for the localisation of F actin to the projection tip. We also identify a role during mating for Mad2--a mitotic checkpoint protein that is required in all eukaryotes to maintain the mitotic state in response to microtubule depolymerisation [3]. S. pombe mad2 mutant cells were compromised in their ability to mate upon removal of TBZ, indicating that in fission yeast, in the absence of microtubules, Mad2 is also required to maintain mating competence.     

A homologue of Saccharomyces cerevisiae Dpm1p is not sufficient for synthesis of dolichol-phosphate-mannose in mammalian cells

Dolichol-phosphate-mannose (Dol-P-Man) serves as a donor of mannosyl residues in major eukaryotic glycoconjugates. It donates four mannosyl residues in the N-linked oligosaccharide precursor and all three mannosyl residues in the core of the glycosylphosphatidylinositol anchor. In yeasts it also donates one mannose to the O-linked oligosaccharide. The yeast DPM1 gene encodes a Dol-P-Man synthase that is a transmembrane protein expressed in the endoplasmic reticulum. We cloned human and mouse homologues of DPM1, termed hDPM1 and mDPM1, respectively, both of which encode proteins of 260 amino acids, having 30% amino acid identity with yeast Dpm1 protein but lacking a hydrophobic transmembrane domain, which exists in the yeast synthase. Human and mouse DPM1 cDNA restored Dol-P-Man synthesis in mouse Thy-1-deficient mutant class E cells. Mouse class E mutant cells had an inactivating mutation in the mDPM1 gene, indicating that mDPM1 is the gene for class E mutant. In contrast, hDPM1 and mDPM1 cDNA did not complement another Dol-P-Man synthesis mutant, hamster Lec15 cells, whereas yeast DPM1 restored both mutants. Therefore, in contrast to yeast, mammalian cells require hDPM1/mDPM1 protein and a product of another gene that is defective in Lec15 mutant cells for synthesis of Dol-P-Man.     

Structure-function comparisons of the proapoptotic protein Bax in yeast and mammalian cells

Expression of the proapoptotic protein Bax under the control of a GAL10 promoter in Saccharomyces cerevisiae resulted in galactose-inducible cell death. Immunofluorescence studies suggested that Bax is principally associated with mitochondria in yeast cells. Removal of the carboxyl-terminal transmembrane (TM) domain from Bax [creating Bax (deltaTM)] prevented targeting to mitochondrial and completely abolished cytotoxic function in yeast cells, suggesting that membrane targeting is crucial for Bax-mediated lethality. Fusing a TM domain from Mas70p, a yeast mitochondrial outer membrane protein, to Bax (deltaTM) restored targeting to mitochondria and cytotoxic function in yeast cells. Deletion of four well-conserved amino acids (IGDE) from the BH3 domain of Bax ablated its ability to homodimerize and completely abrogated lethality in yeast cells. In contrast, several Bax mutants which retained ability to homodimerize (deltaBH1, deltaBH2, and delta1-58) also retained at least partial lethal function in yeast cells. In coimmunoprecipitation experiments, expression of the wild-type Bax protein in Rat-1 fibroblasts and 293 epithelial cells induced apoptosis, whereas the Bax (deltaIGDE) mutant failed to induce apoptosis and did not associate with endogenous wild-type Bax protein. In contrast to yeast cells, Bax (deltaTM) protein retained cytotoxic function in Rat-1 and 293 cells, was targeted largely to mitochondria, and dimerized with endogenous Bax in mammalian cells. Thus, the dimerization-mediating BH3 domain and targeting to mitochondrial membranes appear to be essential for the cytotoxic function of Bax in both yeast and mammalian cells.     

The fission yeast chromo domain encoding gene chp1(+) is required for chromosome segregation and shows a genetic interaction with alpha-tubulin

In eukaryotes, the segregation of chromosomes is co-ordinated by the centromere and must proceed accurately if aneuploidy and cell death are to be avoided. The fission yeast centromere is complex, containing highly repetitive regions of DNA showing the characteristics of heterochromatin. Two proteins, Swi6p and Clr4p, that are associated with the fission yeast centromere also contain a chromo (chromatin organisation modifier) domain and are required for centromere function. We have analysed a novel fission yeast gene encoding a putative chromo domain called chp 1(+) (chromo domain protein in Schizosaccharomyces p ombe ). In the absence of Chp1p protein, cells are viable but show chromosome segregation defects such as lagging chromosomes on the spindle during anaphase and high rates of minichromosome loss, phenotypes which are also displayed by swi 6 and clr 4. A fusion protein between green fluorescent protein (GFP) and Chp1p, like Swi6p, is localized to discrete sites within the nucleus. In contrast to Swi6p and Clr4p, Chp1p is not required to repress silent mating-type genes. We demonstrate a genetic interaction between chp 1(+) and alpha-tubulin ( nda 2(+)) and between swi 6(+) and beta-tubulin ( nda 3(+)). Chp1p and Swi6p proteins may be components of the kinetochore which captures and stabilizes the microtubules of the spindle.     

Protein kinase mutants of human ATR increase sensitivity to UV and ionizing radiation and abrogate cell cycle checkpoint control

In fission yeast, the rad3 gene product plays a critical role in sensing DNA structure defects and activating damage response pathways. A structural homologue of rad3 in humans (ATR) has been identified based on sequence similarity in the protein kinase domain. General information regarding ATR expression, protein kinase activity, and cellular localization is known, but its function in human cells remains undetermined. In the current study, the ATR protein was examined by gel filtration of protein extracts and was found to exist predominantly as part of a large protein complex. A kinase-inactivated form of the ATR gene was prepared by site-directed mutagenesis and was used in transfection experiments to probe the function of this complex. Introduction of this kinase-dead ATR into a normal fibroblast cell line, an ATM-deficient fibroblast line derived from a patient with ataxia-telangiectasia, or a p53 mutant cell line all resulted in significant losses in cell viability. Clones expressing the kinase-dead ATR displayed increased sensitivity to x-rays and UV and a loss of checkpoint control. We conclude that ATR functions as a critical part of a protein complex that mediates responses to ionizing and UV radiation in human cells. These responses include effects on cell viability and cell cycle checkpoint control.     

Fission yeast mitotic regulator Dsk1 is an SR protein-specific kinase

Intricate interplay may exist between pre-mRNA splicing and the cell division cycle, and fission yeast Dsk1 appears to play a role in such a connection. Previous genetic analyses have implicated Dsk1 in the regulation of chromosome segregation at the metaphase/anaphase transition. Yet, its protein sequence suggests that Dsk1 may function as a kinase specific for SR proteins, a family of pre-mRNA splicing factors containing arginine-serine repeats. Using an in vitro system with purified components, we showed that Dsk1 phosphorylated human and yeast SR proteins with high specificity. The Dsk1-phosphorylated SF2/ASF protein was recognized strongly by a monoclonal antibody (mAb104) known to bind the in vivo phosphoepitope shared by SR proteins, indicating that the phosphorylation sites resided in the RS domain. Moreover, the fission yeast U2AF65 homolog, Prp2/Mis11 protein, was phosphorylated more efficiently by Dsk1 than by a human SR protein-specific kinase, SRPK1. Thus, these in vitro results suggest that Dsk1 is a fission yeast SR protein-specific kinase, and Prp2/Mis11 is likely an in vivo target for Dsk1. Together with previous genetic data, the studies support the notion that Dsk1 may play a role in coordinating pre-mRNA splicing and the cell division cycle.     

The dynamin-related GTPase, Dnm1p, controls mitochondrial morphology in yeast

The Saccharomyces cerevisiae Dnm1 protein is structurally related to dynamin, a GTPase required for membrane scission during endocytosis. Here we show that Dnm1p is essential for the maintenance of mitochondrial morphology. Disruption of the DNM1 gene causes the wild-type network of tubular mitochondrial membranes to collapse to one side of the cell but does not affect the morphology or distribution of other cytoplasmic organelles. Dnm1 proteins containing point mutations in the predicted GTP-binding domain or completely lacking the GTP-binding domain fail to rescue mitochondrial morphology defects in a dnm1 mutant and induce dominant mitochondrial morphology defects in wild-type cells. Indirect immunofluorescence reveals that Dnm1p is distributed in punctate structures at the cell cortex that colocalize with the mitochondrial compartment. These Dnm1p-containing structures remain associated with the spherical mitochondria found in an mdm10 mutant strain. In addition, a portion of Dnm1p cofractionates with mitochondrial membranes during differential sedimentation and sucrose gradient fractionation of wild-type cells. Our results demonstrate that Dnm1p is required for the cortical distribution of the mitochondrial network in yeast, a novel function for a dynamin-related protein.     

Complementation of the DNA repair-deficient swi10 mutant of fission yeast by the human ERCC1 gene

In human cells DNA damage caused by UV light is mainly repaired by the nucleotide excision repair pathway. This mechanism involves dual incisions on both sides of the damage catalyzed by two nucleases. In mammalian cells XPG cleaves 3' of the DNA lesion while the ERCC1-XPF complex makes the 5' incision. The amino acid sequence of the human excision repair protein ERCC1 is homologous with the fission yeast Swi10 protein. In order to test whether these proteins are functional homologues, we overexpressed the human gene in a Schizosaccharomyces pombe swi10 mutant. A swi10 mutation has a pleiotropic effect: it reduces the frequency of mating type switching (a mitotic transposition event from a silent cassette into the expression site) and causes increased UV sensitivity. We found that the full-length ERCC1 gene only complements the transposition defect of the fission yeast mutant, while a C-terminal truncated ERCC1 protein also restores the DNA repair capacity of the yeast cells. Using the two-hybrid system of Saccharomyces cerevisiae we show that only the truncated human ERCC1 protein is able to interact with the S . pombe Rad16 protein, which is the fission yeast homologue of human XPF. This is the first example yet known that a human gene can correct a yeast mutation in nucleotide excision repair.     

The small GTP-binding protein Rho1 is a multifunctional protein that regulates actin localization, cell polarity, and septum formation in the fission yeast Schizosaccharomyces pombe

BACKGROUND: The small GTP-binding protein Rho has been shown to regulate the formation of the actin cytoskeleton in animal cells. We have previously isolated two rho genes, rho1+ and rho2+, from the fission yeast Schizosaccharomyces pombe in order to investigate the function of Rho using genetic techniques. In this paper, we report the cellular function of Rho1. RESULTS: We found that Rho1 is essential for cell viability and cell polarity using gene disruption and by exogenous expression of botulinum C3 ADP-ribosyltransferase. In cells expressing either a constitutively active Rho1 or a dominant-negative Rho1, actin patches were delocalized. Both the cell wall and secondary septum were thick and stratified in cells expressing the constitutively active Rho1, while the cell wall of cells expressing the dominant-negative Rho1 seemed to be loosely organized. Furthermore, inactivation of Rho1 is apparently required for the separation of daughter cells. Cell fractionation studies suggested that Rho1 is predominantly membrane-bound. Moreover, we observed that Rho1 is localized to the cell periphery and to the septum. CONCLUSIONS: Rho1 is involved in actin patch localization, the control of cell polarity, the regulation of septation, and cell wall synthesis.     

Expression of rat cathepsin D cDNA in Saccharomyces cerevisiae: implications for intracellular targeting of cathepsin D to vacuoles

To investigate the intracellular transport mechanisms of lysosomal cathepsin D in yeast cells, we produced cathepsin D in Saccharomyces cerevisiae by placing the coding region under the control of the promoter of the yeast glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene. Immunoblotting analysis by the use of an antibody specific for rat cathepsin D coding sequence produced an intermediate species which had a slightly higher molecular weight than that of the mature cathepsin D. Cell fractionation experiments demonstrated that the cathepsin D polypeptide was colocalized to the yeast vacuoles with the marker enzyme carboxypeptidase Y in a Ficoll step gradient. A biosynthesis study with pulse-chase kinetic analysis revealed that the precursor polypeptide was accurately sorted to the yeast vacuoles as determined by cell fractionation, and that N-linked carbohydrate modifications were not required for vacuolar sorting of this protein. To elucidate the role of the propeptide region of cathepsin D, which might function in the intracellular targeting to the vacuole, a deletion mutant of cathepsin D lacking the propeptide was prepared and its intracellular targeting was examined after transfection into yeast cells. Immunoblotting analysis demonstrated that the propeptide-deleted mutant protein was recovered in a low quantity as compared with that in the case of yeast cells expressing the wild-type protein in the isolated vacuolar fraction. Immunofluorescence analysis revealed that the deletion mutant protein appeared to be accumulated within the intracellular small vesicles but not in the carboxypeptidase Y-positive vacuoles. Overall, these results indicate that the rat cathepsin D precursor polypeptide is recognized by mechanisms similar to those involved in the intracellular sorting of vacuolar proteins through the ER/Golgi/vacuolar sorting pathway in yeast cells, and that the propeptide has an important function in translocation of the cathepsin D polypeptide to the vacuole.     

Isolation and molecular characterization of the Arabidopsis TPS1 gene, encoding trehalose-6-phosphate synthase

An Arabidopsis thaliana cDNA clone, AtTPS1, that encodes a trehalose-6-phosphate synthase was isolated. The identity of this protein is supported by both structural and functional evidence. On one hand, the predicted sequence of the protein encoded by AtTPS1 showed a high degree of similarity with trehalose-6-phosphate synthases of different organisms. On the other hand, expression of the AtTPS1 cDNA in the yeast tps1 mutant restored its ability to synthesize trehalose and suppressed its growth defect related to the lack of trehalose-6-phosphate. Genomic organization and expression analyses suggest that AtTPS1 is a single-copy gene and is expressed constitutively at very low levels.     

A human peptidyl-prolyl isomerase essential for regulation of mitosis

The NIMA kinase is essential for progression through mitosis in Aspergillus nidulans, and there is evidence for a similar pathway in other eukaryotic cells. Here we describe the human protein Pin1, a peptidyl-prolyl cis/trans isomerase (PPIase) that interacts with NIMA. PPIases are important in protein folding, assembly and/or transport, but none has so far been shown to be required for cell viability. Pin1 is nuclear PPIase containing a WW protein interaction domain, and is structurally and functionally related to Ess1/Ptf1, an essential protein in budding yeast. PPIase activity is necessary for Ess1/Pin1 function in yeast. Depletion of Pin1/Ess1 from yeast or HeLa cells induces mitotic arrest, whereas HeLa cells overexpressing Pin1 arrest in the G2 phase of the cell cycle. Pin1 is thus an essential PPIase that regulates mitosis presumably by interacting with NIMA and attenuating its mitosis-promoting activity.     

Parents'' sung performances for infants

We have cloned an unique gene encoding the heavy chain of a type II myosin in the fission yeast, Schizosaccharomyces pombe. The myo2+ gene encodes a protein of 1526 amino acids with a predicted molecular weight of 177 kDa and containing consensus binding motifs for both essential and regulatory light chains. The S. pombe myo2+ head domain is 45% identical to myosin IIs from Saccharomyces cerevisiae and Homo sapiens and 40% identical to Drosophila melanogaster Structurally, myo2+ most closely resembles budding yeast MYO1, the tails of both myosin IIs containing a number of proline residues that are predicted to substantially disrupt the ability of these myosins to form coiled coils. The myo2+ gene is located on chromosome III, 8.3 map units from ade6+. Deletion of approximately 70% of the coding sequence of myo2+ is lethal but myo2delta spores can acquire a suppressor mutation that allows them to form viable microcolonies consisting of filaments of branched cells with aberrant septa. Overexpression of myo2+ results in the inhibition of cytokinesis; cells become elongated and multinucleate and fail to assemble a functional cytokinetic actin ring and are either aseptate or form aberrant septa. These results suggest that a contractile actin-myosin based cytokinetic mechanism appeared early in the evolution of eukaryotic cells and further emphasise the utility of fission yeast as a model organism in which to study the molecular and cellular basis of cytokinesis.     

Requirements for efficient production and transduction of human immunodeficiency virus type 1-based vectors

Phosphatidylglycerophosphate (PGP) synthase catalyzes the first step in the cardiolipin (CL) branch of phospholipid biosynthesis in mammalian cells. In this study, we isolated a Chinese hamster ovary (CHO) cDNA encoding a putative protein similar in sequence to the yeast PGS1 gene product, PGP synthase. The gene for the isolated CHO cDNA was named PGS1. Expression of the CHO PGS1 cDNA in CHO-K1 cells and production of a recombinant CHO PGS1 protein with a N-terminal extension in Escherichia coli resulted in 15-fold and 90-fold increases of PGP synthase specific activity, respectively, establishing that CHO PGS1 encodes PGP synthase. A PGP synthase-defective CHO mutant, PGS-S, isolated previously (Ohtsuka, T., Nishijima, M., and Akamatsu, Y. (1993) J. Biol. Chem. 268, 22908-22913) exhibits striking reductions in biosynthetic rate and cellular content of phosphatidylglycerol (PG) and CL and shows mitochondrial morphological and functional abnormalities. The CHO PGS-S mutant transfected with the CHO PGS1 cDNA exhibited 620-fold and 7-fold higher PGP synthase activity than mutant PGS-S and wild type CHO-K1 cells, respectively, and had a normal cellular content and rate of biosynthesis of PG and CL. In contrast to mutant PGS-S, the transfectant had morphologically normal mitochondria. When the transfectant and mutant PGS-S cells were cultivated in a glucose-depleted medium, in which cellular energy production mainly depends on mitochondrial function, the transformant but not mutant PGS-S was capable of growth. These results demonstrated that the morphological and functional defects displayed by the PGS-S mutant are due directly to the reduced ability to make normal levels of PG and/or CL.     

The fission yeast Cdc1 protein, a homologue of the small subunit of DNA polymerase delta, binds to Pol3 and Cdc27

cdc1+ is required for cell cycle progression in Schizosaccharomyces pombe. Cells carrying temperature-sensitive cdc1 mutants undergo cell cycle arrest when shifted to the restrictive temperature, becoming highly elongated. Here we describe the cloning and sequencing of cdc1+, which is shown to encode a 462 residue protein that displays significant sequence similarity to the small subunit of mammalian DNA polymerase delta. cdc1+ interacts genetically with pol3+, which encodes the large subunit of DNA polymerase delta in fission yeast, and the Cdc1 protein binds to Pol3 in vitro, strongly suggesting that Cdc1 is likely to be the small subunit of Pol delta. In addition, we show that cdc1+ overexpression is sufficient to rescue cells carrying temperature-sensitive cdc27 alleles and that the Cdc1 and Cdc27 proteins interact in vivo and in vitro. Deletion of either cdc1+ or cdc27+ results in cell cycle arrest with the arrested cells having a single nucleus with 2C DNA content. No evidence was obtained for a cut phenotype, indicating that neither cdc1+ nor cdc27+ is required for checkpoint function. cdc1 mutant cells are supersensitive to the DNA synthesis inhibitor hydroxyurea and to the DNA damaging agent MMS, display increased frequency of mini-chromosome loss and have an extended S phase.     

The yeast halotolerance determinant Hal3p is an inhibitory subunit of the Ppz1p Ser/Thr protein phosphatase

Components of cellular stress responses can be identified by correlating changes in stress tolerance with gain or loss of function of defined genes. Previous work has shown that yeast cells deficient in Ppz1 protein phosphatase or overexpressing Hal3p, a novel regulatory protein of unknown function, exhibit increased resistance to sodium and lithium, whereas cells lacking Hal3p display increased sensitivity. These effects are largely a result of changes in expression of ENA1, encoding the major cation extrusion pump of yeast cells. Disruption or overexpression of HAL3 (also known as SIS2) has no effect on salt tolerance in the absence of PPZ1, suggesting that Hal3p might function upstream of Ppz1p in a novel signal transduction pathway. Hal3p is recovered from crude yeast homogenates by using immobilized, bacterially expressed Ppz1p fused to glutathione S-transferase, and it also copurifies with affinity-purified glutathione S-transferase-Ppz1p from yeast extracts. In both cases, the interaction is stronger when only the carboxyl-terminal catalytic phosphatase domain of Ppz1p is expressed. In vitro experiments reveal that the protein phosphatase activity of Ppz1p is inhibited by Hal3p. Overexpression of Hal3p suppresses the reduced growth rate because of the overexpression of Ppz1p and aggravates the lytic phenotype of a slt2/mpk1 mitogen-activated protein kinase mutant (thus mimicking the deletion of PPZ1). Therefore, Hal3p might modulate diverse physiological functions of the Ppz1 phosphatase, such as salt stress tolerance and cell cycle progression, by acting as a inhibitory subunit.     

Purification of Hsk1, a minichromosome maintenance protein kinase from fission yeast

Members of the Cdc7 family of protein kinases are essential for the initiation of DNA replication in all eukaryotes, but their precise biochemical function is unclear. We have purified the fission yeast Cdc7 homologue Hsk1 approximately 30,000-fold, to near homogeneity. Purified Hsk1 has protein kinase activity on several substrates and is capable of autophosphorylation. Point mutations in highly conserved regions of Hsk1 inactivate the kinase in vitro and in vivo. Overproduction of two of the mutant hsk1 alleles blocks initiation of DNA replication and deranges the mitotic checkpoint, a phenotype consistent with a role for Hsk1 in the early stages of initiation. The purified Hsk1 kinase can be separated into two active forms, a Hsk1 monomer and a heterodimer consisting of Hsk1 complexed with a co-purifying polypeptide, Dfp1. Association with Dfp1 stimulates phosphorylation of exogenous substrates but has little effect on autokinase activity. We have identified Dfp1 as the fission yeast homologue of budding yeast Dbf4. Purified Hsk1 phosphorylates the Cdc19 (Mcm2) subunit of the six-member minichromosome maintenance protein complex purified from fission yeast. Since minichromosome maintenance proteins have been implicated in the initiation of DNA replication, the essential function of Hsk1 at the G1/S transition may be mediated by phosphorylation of Cdc19. Furthermore, the phosphorylation of critical substrates by Hsk1 kinase is likely regulated by association with a Dbf4-like co-factor.