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In Saccharomyces cerevisiae , Mrc1 (homolog of human Claspin and mediator of replication checkpoint) is not only a part of the replication machine, but also participates in the replication stress response when DNA replication is blocked by hydroxyurea. Since Mrc1 is expressed in a small amount in cells and has many proteins interacting with it as a mediator, it is difficult to obtain Mrc1 with high concentration and purity. This article reports the purification of a stable truncation of Mrc1 and the full length Mrc1. High concentration and high purity of Mrc1 was obtained and the three-dimensional structure of Mrc1 was analyzed, which is a ring with a hole in the center. At the same time, we found that Mrc1 has an interaction with Rad24-RFC a clamp loader in the replication checkpoint, and can form a complex with it, implying that we can assemble large replication checkpoint complexes in vitro . These results initially reveal the ring structure of Mrc1 and its interaction with Rad24-RFC in replication checkpoints in S. cerevisiae .

Budding yeast Dmc1 is a member of the RecA family of strand exchange proteins essential for homologous recombination (HR) during meiosis. Dmc1 mediates the steps of homology search and DNA strand exchange reactions that are central to HR. To achieve optimum activity, Dmc1 requires a number of accessory factors. Although methods for purification of Dmc1 and many of its associated factors have been described (Binz, Dickson, Haring, & Wold, 2006; Busygina et al., 2013; Chan, Brown, Qin, Handa, & Bishop, 2014; Chi et al., 2006; Cloud, Chan, Grubb, Budke, & Bishop, 2012; Nimonkar, Amitani, Baskin, & Kowalczykowski, 2007; Van Komen, Macris, Sehorn, & Sung, 2006), Dmc1 has been particularly difficult to purify because of its tendency to aggregate. Here, we provide an alternative and simple high-yield purification method for recombinant Dmc1 that is active and responsive to stimulation by accessory factors. The same method may be used for purification of recombinant Rdh54 (a.k.a. Tid1) and other HR proteins with minor adjustments. We also describe an economical and sensitive D-loop assay for strand exchange proteins that uses fluorescent dye-tagged, rather than radioactive, ssDNA substrates., (© 2018 Elsevier Inc. All rights reserved.)

Tem1 is a small GTPase that controls the mitotic progression of Saccharomyces cerevisiae through the Mitotic Exit Network. Tem1 activity is tightly controlled in mitosis by Bub2 and Bfa1 and is also regulated by the spindle orientation checkpoint that monitors the correct alignment of the mitotic spindle with the mother-daughter axis. In this chapter we describe the purification of Tem1, Bfa1, and Bub2 and a detailed radioactive filter-binding assay to study the nucleotide binding properties of Tem1 and the role of its regulators Bfa1 and Bub2.

Virtually all eukaryotic cell functions and signaling pathways are regulated by protein phosphorylation. The Rad53 kinase plays crucial roles in the DNA damage response in Saccharomyces cerevisiae and is widely used as a surrogate marker for DNA damage checkpoint activation by diverse genotoxic agents. Most currently available assays for Rad53 activation are based on either electrophoretic mobility shifts or semiquantitative in situ autophosphorylation activity on protein blots. Here, we describe direct quantitative measures to assess Rad53 activity using immunoprecipitation kinase assays and quantitative mass spectrometric analysis of Rad53 activation loop autophosphorylation states. Both assays employ a highly specific Rad53 antibody, and thus enable the analysis of the untagged endogenous protein under physiological conditions. The principles of these assays are readily transferable to other protein kinases for which immunoprecipitation-grade antibodies are available, and thus potentially applicable to a wide range of eukaryotic signaling pathways beyond yeast., (© 2017 Elsevier Inc. All rights reserved.)

In budding yeast, the monopolin complex mediates sister kinetochore cross-linking and co-orientation in meiosis I. The CK1δ kinase Hrr25 is critical for sister kinetochore co-orientation, but its roles are not well understood. Here, we present the structures of Hrr25 and its complex with the monopolin subunit Mam1. Hrr25 possesses a "central domain" that packs tightly against the kinase C-lobe, adjacent to the binding site for Mam1. Together, the Hrr25 central domain and Mam1 form a novel, contiguous embellishment to the Hrr25 kinase domain that affects Hrr25 conformational dynamics and enzyme kinetics. Mam1 binds a hydrophobic surface on the Hrr25 N-lobe that is conserved in CK1δ-family kinases, suggesting a role for this surface in recruitment and/or regulation of these enzymes throughout eukaryotes. Finally, using purified proteins, we find that Hrr25 phosphorylates the kinetochore receptor for monopolin, Dsn1. Together with our new structural insights into the fully assembled monopolin complex, this finding suggests that tightly localized Hrr25 activity modulates monopolin complex-kinetochore interactions through phosphorylation of both kinetochore and monopolin complex components., (© 2016 The Authors.)

Loading of the ring-shaped Mcm2-7 replicative helicase around DNA licenses eukaryotic origins of replication. During loading, Cdc6, Cdt1, and the origin-recognition complex (ORC) assemble two heterohexameric Mcm2-7 complexes into a head-to-head double hexamer that facilitates bidirectional replication initiation. Using multi-wavelength single-molecule fluorescence to monitor the events of helicase loading, we demonstrate that double-hexamer formation is the result of sequential loading of individual Mcm2-7 complexes. Loading of each Mcm2-7 molecule involves the ordered association and dissociation of distinct Cdc6 and Cdt1 proteins. In contrast, one ORC molecule directs loading of both helicases in each double hexamer. Based on single-molecule FRET, arrival of the second Mcm2-7 results in rapid double-hexamer formation that anticipates Cdc6 and Cdt1 release, suggesting that Mcm-Mcm interactions recruit the second helicase. Our findings reveal the complex protein dynamics that coordinate helicase loading and indicate that distinct mechanisms load the oppositely oriented helicases that are central to bidirectional replication initiation., (Copyright © 2015 Elsevier Inc. All rights reserved.)

The role of Dmc1 as a meiosis-specific general recombinase was first demonstrated in Saccharomyces cerevisiae. Progress in understanding the biochemical mechanism of ScDmc1 has been hampered by its tendency to form inactive aggregates. We have found that the inclusion of ATP during protein purification prevents Dmc1 aggregation. ScDmc1 so prepared is capable of forming D-loops and responsive to its accessory factors Rad54 and Rdh54. Negative staining electron microscopy and iterative helical real-space reconstruction revealed that the ScDmc1-ssDNA nucleoprotein filament harbors 6.5 protomers per turn with a pitch of ∼106Å. The ScDmc1 purification procedure and companion molecular analyses should facilitate future studies on this recombinase., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Licensing of origins of eukaryotic DNA replication involves the loading of six minichromosome maintenance proteins (Mcm2-7) into pre-replicative complexes (pre-RCs). The assembly of the pre-RC is restricted to G1 phase of the cell cycle, which is crucial to ensure once per cell cycle DNA replication. Mcm2-7 is loaded by the action of the origin recognition complex (ORC), Cdc6 and Cdt1 and requires ATP. In vitro reconstitution of this reaction has shown that Mcm2-7 is loaded onto DNA as a symmetrical head-to-head double hexamer. We describe in detail how pre-RC proteins are purified and used to reconstitute pre-RC formation in vitro. This method is useful for studying the biochemical mechanisms of Mcm2-7 loading as well as subsequent steps in DNA replication., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The assembly and disassembly of chromatin impacts all DNA-dependent processes in eukaryotes. These processes are intricately regulated through stepwise mechanisms, requiring multiple proteins, posttranslational modifications, and remodeling enzymes, as well as specific proteins to chaperone the highly basic and aggregation-prone histone proteins. The histone chaperones are acidic proteins that perform the latter function by maintaining the stability of the histones when they are not associated with DNA and guiding the deposition and removal of histones from DNA. Understanding the thermodynamics of these processes provides deeper insights into the mechanisms of chromatin assembly and disassembly. Here we describe complementary thermodynamic and biochemical approaches for analysis of the interactions of a major chaperone of the H3/H4 dimer, anti-silencing function 1 (Asf1) with histones H3/H4, and DNA. Fluorescence quenching approaches are useful for measuring the binding affinity of Asf1 for histones H3/H4 under equilibrium conditions. Electrophoretic mobility shift analyses are useful for examining Asf1-mediated tetrasome (H3/H4-DNA) assembly and disassembly processes. These approaches potentially can be used more generally for the study of other histone chaperone-histone interactions and provide a means to dissect the role of posttranslational modifications and other factors that participate in chromatin dynamics., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Members of the structural maintenance of chromosome (SMC) family of proteins are essential regulators of genomic stability. In particular, the conserved Smc5-6 complex is required for efficient DNA repair, checkpoint signaling, and DNA replication in all eukaryotes. Despite these important functions, the actual nature of the DNA substrates recognized by the Smc5-6 complex in chromosomes is currently unknown. Furthermore, how the core SMC components of the Smc5-6 complex use their ATPase-driven mechanochemical activities to act on chromosomes is not understood. Here, we address these issues by purifying and defining the DNA-binding activity of Smc5. We show that Smc5 binds strongly and specifically to single-stranded DNA (ssDNA). Remarkably, this DNA-binding activity is independent of Smc6 and is observed with the monomeric form of Smc5. We further show that Smc5 ATPase activity is essential for its functions in vivo and that ATP regulates the association of Smc5 with its substrates in vitro. Finally, we demonstrate that Smc5 is able to bind efficiently to oligonucleotides consistent in size with ssDNA intermediates produced during DNA replication and repair. Collectively, our data on the DNA-binding activities of Smc5 provide a compelling molecular basis for the role of the Smc5-6 complex in the DNA damage response.

The licensing of eukaryotic DNA replication origins, which ensures once-per-cell-cycle replication, involves the loading of six related minichromosome maintenance proteins (Mcm2-7) into prereplicative complexes (pre-RCs). Mcm2-7 forms the core of the replicative DNA helicase, which is inactive in the pre-RC. The loading of Mcm2-7 onto DNA requires the origin recognition complex (ORC), Cdc6, and Cdt1, and depends on ATP. We have reconstituted Mcm2-7 loading with purified budding yeast proteins. Using biochemical approaches and electron microscopy, we show that single heptamers of Cdt1*Mcm2-7 are loaded cooperatively and result in association of stable, head-to-head Mcm2-7 double hexamers connected via their N-terminal rings. DNA runs through a central channel in the double hexamer, and, once loaded, Mcm2-7 can slide passively along double-stranded DNA. Our work has significant implications for understanding how eukaryotic DNA replication origins are chosen and licensed, how replisomes assemble during initiation, and how unwinding occurs during DNA replication.

The Cdc7-Dbf4 complex plays an instrumental role in the initiation of DNA replication and is a target of replication-checkpoint responses in Saccharomyces cerevisiae. Cdc7 is a conserved serine/threonine kinase whose activity depends on association with its regulatory subunit, Dbf4. A conserved sequence near the N-terminus of Dbf4 (motif N) is necessary for the interaction of Cdc7-Dbf4 with the checkpoint kinase Rad53. To understand the role of the Cdc7-Dbf4 complex in checkpoint responses, a fragment of Saccharomyces cerevisiae Dbf4 encompassing motif N was isolated, overproduced and crystallized. A complete native data set was collected at 100 K from crystals that diffracted X-rays to 2.75 A resolution and structure determination is currently under way.

The Mcm2-7 (minichromosome maintenance) complex is a toroidal AAA(+) ATPase and the putative eukaryotic replicative helicase. Unlike a typical homohexameric helicase, Mcm2-7 contains six distinct, essential, and evolutionarily conserved subunits. Precedence to other AAA(+) proteins suggests that Mcm ATPase active sites are formed combinatorially, with Walker A and B motifs contributed by one subunit and a catalytically essential arginine (arginine finger) contributed by the adjacent subunit. To test this prediction, we used copurification experiments to identify five distinct and stable Mcm dimer combinations as potential active sites; these subunit associations predict the architecture of the Mcm2-7 complex. Through the use of mutant subunits, we establish that at least three sites are active for ATP hydrolysis and have a canonical AAA(+) configuration. In isolation, these five active-site dimers have a wide range of ATPase activities. Using Walker B and arginine finger mutations in defined Mcm subunits, we demonstrate that these sites similarly make differential contributions toward viability and ATP hydrolysis within the intact hexamer. Our conclusions predict a structural discontinuity between Mcm2 and Mcm5 and demonstrate that in contrast to other hexameric helicases, the six Mcm2-7 active sites are functionally distinct.

Origin recognition complex (ORC), a six-protein complex, is the most likely initiator of chromosomal DNA replication in eukaryotes. Throughout the cell cycle, ORC binds to chromatin at origins of DNA replication and functions as a 'landing pad' for the binding of other proteins, including Cdt1p, to form a prereplicative complex. In this study, we used yeast two-hybrid analysis to examine the interaction between Cdt1p and every ORC subunit. We observed potent interaction with Orc6p, and weaker interaction with Orc2p and Orc5p. Coimmunoprecipitation assay confirmed that Cdt1p interacted with Orc6p, as well as with Orc1p and Orc2p. We mapped the C-terminal region, and a middle region of Orc6p (amino acids residues 394-435, and 121-175, respectively), as important for interaction with Cdt1p. Cdt1p was purified to examine its direct interaction with ORC, and its effect on the activity of ORC. Glutathione-S-transferase pull-down analysis revealed that Cdt1p binds directly to ORC. Cdt1p neither bound to origin DNA and ATP nor affected ORC-binding to origin DNA and ATP. These results suggest that interaction of Cdt1p with ORC is involved in the formation of the prereplicative complex, rather than in regulation of the activity of ORC.

The spindle assembly checkpoint (SAC) coordinates mitotic progression with sister chromatid alignment. In mitosis, the checkpoint machinery accumulates at kinetochores, which are scaffolds devoted to microtubule capture. The checkpoint protein Mad2 (mitotic arrest deficient 2) adopts two conformations: open (O-Mad2) and closed (C-Mad2). C-Mad2 forms when Mad2 binds its checkpoint target Cdc20 or its kinetochore receptor Mad1. When unbound to these ligands, Mad2 folds as O-Mad2. In HeLa cells, an essential interaction between C- and O-Mad2 conformers allows Mad1-bound C-Mad2 to recruit cytosolic O-Mad2 to kinetochores. In this study, we show that the interaction of the O and C conformers of Mad2 is conserved in Saccharomyces cerevisiae. MAD2 mutant alleles impaired in this interaction fail to restore the SAC in a mad2 deletion strain. The corresponding mutant proteins bind Mad1 normally, but their ability to bind Cdc20 is dramatically impaired in vivo. Our biochemical and genetic evidence shows that the interaction of O- and C-Mad2 is essential for the SAC and is conserved in evolution.

Ime2 is the founding member of a family of protein kinases that are required for effective progression through meiotic development. Ime2 is essential for the induction of meiosis-specific genes and for the activation of meiotic DNA replication in the budding yeast Saccharomyces cerevisiae. Aside from the fact that Ime2 is a protein kinase and shares several amino acid motifs with cyclin dependent kinases, virtually nothing is known about its enzymatic properties or substrates. Biochemical characterization of Ime2 has been hindered by its low abundance and short half-life. We have created baculovirus expression vectors to produce recombinant Ime2 in insect cells. In this report, we describe the overproduction of Ime2 and its purification using affinity chromatography. Using this procedure, we have been able to purify up to 2mg Ime2 from 1L of infected insect cells. The Ime2 isolated by this method displays properties similar to those of the native enzyme that has been immunoprecipitated from yeast. The high level expression of Ime2 in this system and its ease of purification will be beneficial for more extensive biochemical analysis of Ime2 and related meiosis-specific kinases.

Schizosaccharomyces pombe Cdc5p and its Saccharomyces cerevisiae ortholog, Cef1p, are essential Myb-related proteins implicated in pre-mRNA splicing and contained within large multiprotein complexes. Here we describe the tandem affinity purification (TAP) of Cdc5p- and Cef1p-associated complexes. Using transmission electron microscopy, we show that the purified Cdc5p complex is a discrete structure. The components of the S. pombe Cdc5p/S. cerevisiae Cef1p complexes (termed Cwfs or Cwcs, respectively) were identified using direct analysis of large protein complex (DALPC) mass spectrometry (A. J. Link et al., Nat. Biotechnol. 17:676-682, 1999). At least 26 proteins were detected in the Cdc5p/Cef1p complexes. Comparison of the polypeptides identified by S. pombe Cdc5p purification with those identified by S. cerevisiae Cef1p purification indicates that these two yeast complexes are nearly identical in composition. The majority of S. pombe Cwf proteins and S. cerevisiae Cwc proteins are known pre-mRNA splicing factors including core Sm and U2 and U5 snRNP components. In addition, the complex contains the U2, U5, and U6 snRNAs. Previously uncharacterized proteins were also identified, and we provide evidence that several of these novel factors are involved in pre-mRNA splicing. Our data represent the first comprehensive analysis of CDC5-associated proteins in yeasts, describe a discrete highly conserved complex containing novel pre-mRNA splicing factors, and demonstrate the power of DALPC for identification of components in multiprotein complexes.

The CCR4-NOT complex is an evolutionarily conserved, transcriptional regulatory complex that is involved in controlling mRNA initiation, elongation and degradation. The CCR4-NOT proteins from Saccharomyces cerevisiae exist in two complexes, 1.9x10(6) Da and 1.0x10(6) Da (1.0 MDa) in size, and individual components of these complexes display such disparate functions as binding to and restricting TFIID functions, contacting SAGA and contributing to mRNA deadenylation. As a first step in characterizing the functional roles of the 1.0 MDa complex, we have purified it to near homogeneity. Mass spectrometric analysis was subsequently used to identify all the components of the complex. The 1.0 MDa complex was found to contain CCR4, CAF1, NOT1-5 and two new proteins, CAF40 and CAF130. CAF130 and CAF40 are two unique yeast proteins, with CAF40 displaying extensive homology to proteins from other eukaryotes. Immunoprecipitation and gel filtration experiments confirmed that CAF130 and CAF40 are components of both of the 1.9 MDa and 1.0 MDa CCR4-NOT complexes. Biochemical analysis indicated that the CAF40 and CAF130 proteins bind to the NOT1 protein and exist in a location separate from the two other subsets of proteins in the complex: the CCR4 and CAF1 proteins, and the NOT2, NOT4 and NOT5 proteins. Moreover, CAF40 was able to interact with human NOT1, suggesting that human CAF40 would also be a component of the recently identified human CCR4-NOT complex. Analysis of caf40 and caf130 deletions indicated that they elicited phenotypes shared by defects in other CCR4-NOT genes. The distinct location of CAF40 and CAF130 and the evolutionary conservation of CAF40 implicate them in novel roles in the function of the CCR4-NOT complex., (Copyright 2001 Academic Press.)

We have undertaken an extensive screen to identify Saccharomyces cerevisiae genes whose products are involved in cell cycle progression. We report the identification of 113 genes, including 19 hypothetical ORFs, which confer arrest or delay in specific compartments of the cell cycle when overexpressed. The collection of genes identified by this screen overlaps with those identified in loss-of-function cdc screens but also includes genes whose products have not previously been implicated in cell cycle control. Through analysis of strains lacking these hypothetical ORFs, we have identified a variety of new CDC and checkpoint genes.

Skp1p-cullin-F-box (SCF) protein complexes are ubiquitin ligases required for degradation of many regulatory proteins involved in cell cycle progression, morphogenesis, and signal transduction. Using a genetic screen, we have isolated a novel allele of the HRT1/RBX1 gene in budding yeast (hrt1-C81Y). hrt1-C81Y mutant cells exhibited an aberrant morphology but were viable at all temperatures. The cells displayed multiple genetic interactions with mutations in known SCF components and were defective for the degradation of several SCF targets including Gic2p, Far1p, Sic1p, and Cln2p. In addition, they also failed to degrade the F-box proteins Grr1p, Cdc4p, and Met30p. Wild-type Hrt1p but not Hrt1p-C81Y was able to bind multiple F-box proteins in an F-box-dependent manner. Hrt1p-C81Y harbors a single mutation in its ring-finger domain, which is conserved in subunits of distinct E3 ligases. Finally, Hrt1p was localized in both nucleus and cytoplasm and despite a short half-life was expressed constitutively throughout the cell cycle. Taken together, these results suggest that Hrt1p is a core subunit of multiple SCF complexes.

In the budding yeast Saccharomyces cerevisiae, Cdc37 is required for the productive formation of Cdc28-cyclin complexes. The cdc37-1 mutant arrests at Start with low levels of Cdc28 protein, which is predominantly unphosphorylated at Thr169, fails to bind cyclin, and has little protein kinase activity. We show here that Cdc28 and not cyclin is specifically defective in the cdc37-1 mutant and that Cdc37 likely does not act as an assembly factor for Cdc28-cyclin complex formation. We have also found that the levels and activity of the protein kinase Cak1 are significantly reduced in the cdc37-1 mutant. Pulse-chase analysis indicates that Cdc28 and Cak1 proteins are both destabilized when Cdc37 function is absent during but not after translation. In addition, Cdc37 promotes the production of Cak1, but not that of Cdc28, when coexpressed in insect cells. We conclude that budding yeast Cdc37, like its higher eukaryotic homologs, promotes the physical integrity of multiple protein kinases, perhaps by virtue of a cotranslational role in protein folding.

Rad24 functions in the DNA damage checkpoint pathway of Saccharomyces cerevisiae. Here, analysis of Rad24 in whole cell extracts demonstrated that its mass was considerably greater than its predicted molecular weight, suggesting that Rad24 is a component of a protein complex. The Rad24 complex was purified to homogeneity. In addition to Rad24, the complex included polypeptides of 40 kDa and 35 kDa. The 40 kDa species was found by mass spectrometry to contain Rfc2 and Rfc3, subunits of replication factor C (RFC), a five subunit protein that is required for the loading of polymerases onto DNA during replication and repair [3]. We hypothesised that other RFC subunits, all of which share sequence homologles with Rad24, might also be components of the Rad24 complex. Reciprocal co-immunoprecipitation studies were performed using extracts prepared from strains containing epitope-tagged RFC proteins. These experiments showed that the small RFC proteins, Rfc2, Rfc3, Rfc4 and Rfc5, interacted with Rad24, whereas the Rfc1 subunit did not. We suggest that this RFC-like Rad24 complex may function as a structure-specific sensor in the DNA damage checkpoint pathway.

A multisubunit cohesin complex holds sister chromatids together after DNA replication. Using chromatin immunoprecipitation, we detected cohesin association with centromeres and with discrete sites along chromosome arms from S phase until metaphase in S. cerevisiae. Short DNA sequences (130-280 bp) are sufficient to confer cohesin association. Cohesin association with a centromere depends on Mif2p, the centromere binding factor CBF3, and a centromere-specific histone variant, Cse4p. Because only active centromeres confer cohesin association with centromeric DNA, we suggest that cohesin is recruited by the same chromatin structure that confers the attachment of microtubules. Propagation of this structure might be partly epigenetic. Finally, cohesion associated with "minimal" centromeres is insufficient to resist the splitting force exerted by microtubules and appears to be reinforced by cohesion provided by their flanking DNA sequences.

Previously we showed that the yeast proteins Spt16 (Cdc68) and Pob3 are physically associated, and interact physically and genetically with the catalytic subunit of DNA polymerase alpha, Pol1 [Wittmeyer and Formosa (1997) Mol. Cell. Biol. 17, 4178-4190]. Here we show that purified Spt16 and Pob3 form a stable, abundant, elongated heterodimer and provide evidence that this is the functional form of these proteins. Genetic interactions between mutations in SPT16 and POB3 support the importance of the Spt16-Pob3 interaction in vivo. Spt16, Pob3, and Pol1 proteins were all found to localize to the nucleus in S. cerevisiae. A portion of the total cellular Spt16-Pob3 was found to be chromatin-associated, consistent with the proposed roles in modulating chromatin function. Some of the Spt16-Pob3 complex was found to copurify with the yeast DNA polymerase alpha/primase complex, further supporting a connection between Spt16-Pob3 and DNA replication.

The Cdc42p GTPase is involved in the signal transduction cascades controlling bud emergence and polarized cell growth in S. cerevisiae. Cells expressing the cdc42(V44A) effector domain mutant allele displayed morphological defects of highly elongated and multielongated budded cells indicative of a defect in the apical-isotropic switch in bud growth. In addition, these cells contained one, two, or multiple nuclei indicative of a G2/M delay in nuclear division and also a defect in cytokinesis and/or cell separation. Actin and chitin were delocalized, and septin ring structure was aberrant and partially delocalized to the tips of elongated cdc42(V44A) cells; however, Cdc42(V44A)p localization was normal. Two-hybrid protein analyses showed that the V44A mutation interfered with Cdc42p's interactions with Cla4p, a p21(Cdc42/Rac)-activated kinase (PAK)-like kinase, and the novel effectors Gic1p and Gic2p, but not with the Ste20p or Skm1p PAK-like kinases, the Bni1p formin, or the Iqg1p IQGAP homolog. Furthermore, the cdc42(V44A) morphological defects were suppressed by deletion of the Swe1p cyclin-dependent kinase inhibitory kinase and by overexpression of Cla4p, Ste20p, the Cdc12 septin protein, or the guanine nucleotide exchange factor Cdc24p. In sum, these results suggest that proper Cdc42p function is essential for timely progression through the apical-isotropic switch and G2/M transition and that Cdc42(V44A)p differentially interacts with a number of effectors and regulators.

In eukaryotes, protein function can be modulated by ligation to ubiquitin or to ubiquitin-like proteins (Ubl proteins). The vertebrate Ubl protein SUMO-1 is only 18% identical to ubiquitin but is 48% identical to the yeast protein Smt3. Both SUMO-1 and Smt3 are ligated to cellular proteins, and protein conjugation to SUMO-1/Smt3 is involved in many physiological processes. It remained unknown, however, whether deconjugation of SUMO-1/Smt3 from proteins is also essential. Here we describe a yeast Ubl-specific protease, Ulp1, which cleaves proteins from Smt3 and SUMO-1 but not from ubiquitin. Ulp1 is unrelated to any known deubiquitinating enzyme but shows distant similarity to certain viral proteases, indicating the existence of a widely conserved protease fold. Proteins related to Ulp1 are present in many organisms, including several human pathogens. The pattern of Smt3-coupled proteins in yeast changes markedly throughout the cell cycle, and specific conjugates accumulate in ulp1 mutants. Ulp1 has several functions, including an essential role in the G2/M phase of the cell cycle.

We have found that the guanine nucleotide exchange factor for ras, Cdc25p, interacts with Ssa1p in Saccharomyces cerevisiae. This interaction was observed with GST-fused Cdc25p polypeptides and confirmed by coimmunoprecipitation with the endogenous Cdc25p. Hsp82 appeared also to be co-immunoprecipitated with Cdc25p, albeit to a lower level than Hsp70. In a strain deleted for SSA1 and SSA2, we observed a reduced cellular content of Cdc25p. Consistent with a reduced activity of the cAMP-dependent PKA pathway, the rate of accumulation of both trehalose and glycogen was stimulated in the ssa-deleted strain. Expression of SSA1 reversed these effects, whereas co-expression of SSA1 and PDE2 restored high accumulation. The expression of genes repressed by cAMP, GAC1 and TPS1, fused to beta-galactosidase, was also stimulated by deletion of SSA genes. The effect of ssa deletion on glycogen accumulation was lost in a strain deleted for CDC25 rescued by the RAS2ile152 allele. Altogether, these results lead to the conclusion that Ssa1p positively controls the cAMP pathway through Cdc25p. We propose that this connection plays a critical role in the adaptation of cells to stress conditions.

We previously demonstrated that a combination of both positive and negative cis -acting upstream elements control the transcription of the gene encoding TBP ( SPT15 ) in Saccharomyces cerevisiae . One of these elements found in that study, resident between 5' flanking sequences -147 and -128 , and termed PED (for positive element distal), was found to play an essential positive role in driving transcription of the gene encoding TBP. In this report, we map at nucleotide-level resolution, the critical residues which comprise PED, purify and sequence the protein that binds to it and determine that this PED binding factor is Abf1p, an abundant yeast protein previously broadly implicated in both gene regulation and DNA replication. In the case of the TBP-encoding gene, however, Abf1p works through the PED element which is a non-consensus binding site. Based upon the work of others, the PED-variant ABF1 site would be predicted to be a very poor binding site for this factor yet Abf1p binds PED and a consensus ABF1 site with comparable affinity. These results are discussed in light of the broader context of Abf1p-mediated gene regulation.

The CDC6 gene product from Saccharomyces cerevisiae is required for transition from late G1 to S phase of the cell cycle. We have investigated the subcellular localization of the CDC6 protein in yeast to explore where Cdc6p exerts its gene function (s). Using affinity-purified sera we localized Cdc6p to the cytoplasm and the nuclear matrix by both subcellular fractionation and indirect immunofluorescence microscopy. The nuclear localization was confirmed to be in the nuclear scaffold by the low-salt extraction method. The Cdc6p cannot be detected in the mitochondrial or plasma membrane fractions. Using indirect immunofluorescence, we found that a subpopulation of Cdc6p migrated into the nucleus after G1/S transition and diminished after M phase, suggesting its temporal role in nuclear DNA replication. The predicted Cdc6p polypeptide contains a conserved nuclear localization, 27PLKRKKL33, similar to that of the SV40 large T antigen and other nuclear proteins. To test whether this peptide segment plays a role in mediating nuclear transport, we have carried out site-directed mutagenesis to alter the conserved 29Lys to Thr and Arg. The wild-type nuclear localization signal of Cdc6p was found to mediate the LacZ reporter gene fused to CDC6 efficiently to the nucleus, but not the mutated versions of the nuclear localization motif. The results suggested that 29Lys is important in mediating nuclear localization, the 29Thr and 29Arg mutant versions of the CDC6 gene were also unable to complement the cdc6 temperature-sensitive mutant. However, when these mutants were expressed from a multicopy plasmid, the mutated genes could complement the mutation. Similar results were obtained in the cdc6-disrupted cells. Taken together, we suggest that (i) Cdc6p is predominantly located in the cytoplasm, (ii) the nuclear entry of Cdc6p is cell cycle dependent, and (iii) nuclear entry of Cdc6p is mediated by its nuclear localization signal. The presence of Cdc6p in both the nucleus and the cytoplasm suggests a model that Cdc6p exerts its gene function in DNA replication and mitotic restraint in the cell cycle.

Mcm2, Mcm3, and Mcm5/Cdc46 are conserved proteins essential for the initiation of DNA synthesis at replication origins in Saccharomyces cerevisiae. The accumulation of these proteins in the nucleus before the onset of DNA synthesis suggests that they play a role in restricting DNA synthesis to once per cell cycle. In this work, we show that Mcm2, Mcm3, and Mcm5 self-interact and interact with one another to form complexes. Mcm2 and Mcm3 are abundant proteins, present in approximately 4 X 10(4) and 2 X 10(5) copies per cell, respectively. Reducing the dosage of Mcm2 by half results in diminished usage of specific replication origins. These results together suggest that a significant molar excess of Mcm proteins relative to replication origins is required for the proper initiation of all replication origins.

The Rho family small G proteins are implicated in various cell functions, such as cell morphological change, cell motility, and cytokinesis. However, their modes of action in regulating these cell functions remain to be clarified. In the present study, we have isolated a cDNA encoding a protein which interacts with the GTP-bound form, but not with the GDP-bound form, of the Rho family members, including RhoA, Racl, and Cdc42, by the yeast two-hybrid method. This protein is kinectin, known to be a vesicle membrane anchoring protein of kinesin, which is an ATPase motor transporting vesicles along microtubules.

The interaction of Saccharomyces cerevisiae Ras2p with the catalytic domain of the GDP/GTP exchange factors (GEFs) mouse CDC25(Mm), yeast Cdc25p, and Sdc25p was analyzed by introducing the substitution R80D/N81D into Ras2p S24N, a mutant that is shown to interfere with the Ras2p wild type (wt)-GEF interaction by forming a stable complex. The triple mutant, like Ras2p R80D/N81D, did not interfere with the action of GEF on Ras2p wt (or H-Ras p21) and was unable to form a stable complex with GEF. The GEF stimulation of the nucleotide dissociation of the triple mutant was virtually abolished and strongly decreased with the double mutant. The affinity of Ras2p S24N/R80D/N81D for GDP and GTP was decreased 3 and 4 orders of magnitude, respectively, like that of Ras2p S24N, whereas the double mutant behaved as Ras2p wt. Like Ras2p S24N and unlike Ras2p R80D/N81D, the GTP-bound triple mutant did not activate adenylyl cyclase. Thus, the triple mutant and Ras2p S24N have opposite properties toward the binding to GEF but similarly modified behaviors toward GDP, GTP, and adenylyl cyclase. This work emphasizes the determinant role of the distal switch II region of Ras2p for the interaction with GEF and the different structural background of the interaction with adenylyl cyclase.

We have investigated the role of the essential Rho1 GTPase in cell integrity signaling in budding yeast. Conditional rho1 mutants display a cell lysis defect that is similar to that of mutants in the cell integrity signaling pathway mediated by protein kinase C (Pkc1), which is suppressed by overexpression of Pkc1.rho1 mutants are also impaired in pathway activation in response to growth at elevated temperature. Pkc1 co-immunoprecipitates with Rho1 in yeast extracts, and recombinant Rho1 associates with Pkc1 in vitro in a GTP-dependent manner. Recombinant Rho1 confers upon Pkc1 the ability to be stimulated by phosphatidylserine, indicating that Rho1 controls signal transmission through Pkc1.

The Saccharomyces cerevisiae CDC3, CDC10, CDC11, and CDC12 genes encode a family of related proteins, the septins, which are involved in cell division and the organization of the cell surface during vegetative growth. A search for additional S. cerevisiae septin genes using the polymerase chain reaction identified SPR3, a gene that had been identified previously on the basis of its sporulation-specific expression. The predicted SPR3 product shows 25-40% identity in amino acid sequence to the previously known septins from S. cerevisiae and other organisms. Immunoblots confirmed the sporulation-specific expression of Spr3p and showed that other septins are also present at substantial levels in sporulating cells. Consistent with the expression data, deletion of SPR3 in either of two genetic backgrounds had no detectable effect on exponentially growing cells. In one genetic background, deletion of SPR3 produced a threefold reduction in sporulation efficiency, although meiosis appeared to be completed normally. In this background, deletion of CDC10 had no detectable effect on sporulation. In the other genetic background tested, the consequences of the two deletions were reversed. Immunofluorescence observations suggest that Spr3p, Cdc3p, and Cdc11p are localized to the leading edges of the membrane sacs that form near the spindle-pole bodies and gradually extend to engulf the nuclear lobes that contain the haploid chromosome sets, thus forming the spores. Deletion of SPR3 does not prevent the localization of Cdc3p and Cdc11p, but these proteins appear to be less well organized, and the intensity of their staining is reduced. Taken together, the results suggest that the septins play important but partially redundant roles during the process of spore formation.