Fine structure analysis of the yeast centrin, Cdc31p, identifies residues specific for cell morphology and spindle pole body duplication

Centrin/Cdc31p is a Ca2+-binding protein related to calmodulin found in the MTOC of diverse organisms. In yeast, Cdc31p localizes to the SPB where it interacts with Kar1p and is required for SPB duplication. Recent findings suggest that centrin also functions elsewhere in the cell. To dissect the functions of Cdc31p, we generated cdc31 mutations chosen only for temperature sensitivity, but otherwise unbiased as to phenotype. Three phenotypes of the cdc31 mutants, temperature sensitivity, G2/M arrest, and cell lysis, were not well correlated, indicating that the mutations may differentially affect Cdc31p's interactions with other proteins. Alleles near the C-terminal region exhibited high G2/M arrest and genetic interactions with kar1-Delta17, suggesting that this region modulates an SPB-related function. Alleles causing high lysis and reduced Kic1p kinase activity mapped to the middle of the gene, suggesting disruption of a KIC1-like function and defects in activating Kic1p. A third region conferred temperature sensitivity without affecting cell lysis or G2/M arrest, suggesting that it defines a third function. Mutations in the C-terminal region were also defective for interaction with Kic1p. Mapping the alleles onto a predicted structure of Cdc31p, we have identified surfaces likely to be important for interacting with both Kar1p and Kic1p.     

Sfi1p has conserved centrin-binding sites and an essential function in budding yeast spindle pole body duplication

Centrins are calmodulin-like proteins present in microtubule-organizing centers. The Saccharomyces cerevisiae centrin, Cdc31p, was functionally tagged with a single Z domain of protein A, and used in pull-down experiments to isolate Cdc31p-binding proteins. One of these, Sfi1p, localizes to the half-bridge of the spindle pole body (SPB), where Cdc31p is also localized. Temperature-sensitive mutants in SFI1 show a defect in SPB duplication and genetic interactions with cdc31-1. Sfi1p contains multiple internal repeats that are also present in a Schizosaccharomyces pombe protein, which also localizes to the SPB, and in several human proteins, one of which localizes close to the centriole region. Cdc31p binds directly to individual Sfi1 repeats in a 1:1 ratio, so a single molecule of Sfi1p binds multiple molecules of Cdc31p. The centrosomal human protein containing Sfi1 repeats also binds centrin in the repeat region, showing that this centrin-binding motif is conserved.     

Telomere-binding and Stn1p-interacting activities are required for the essential function of Saccharomyces cerevisiae Cdc13p

Yeast Saccharomyces cerevisiae Cdc13p is the telomere-binding protein that protects telomeres and regulates telomere length. It is documented that Cdc13p binds specifically to single-stranded TG_1–3 telomeric DNA sequences and interacts with Stn1p. To localize the region for single-stranded TG_1–3 DNA binding, Cdc13p mutants were constructed by deletion mutagenesis and assayed for their binding activity. Based on in vitro electrophoretic mobility shift assay, a 243-amino-acid fragment of Cdc13p (amino acids 451–693) was sufficient to bind single-stranded TG_1–3 with specificity similar to that of the native protein. Consistent with the in vitro observation, in vivo one-hybrid analysis also indicated that this region of Cdc13p was sufficient to localize itself to telomeres. However, the telomere-binding region of Cdc13p (amino acids 451–693) was not capable of complementing the growth defects of cdc13 mutants. Instead, a region comprising the Stn1p-interacting and telomere-binding region of Cdc13p (amino acids 252–924) complemented the growth defects of cdc13 mutants. These results suggest that binding to telomeres by Cdc13p is not sufficient to account for the cell viability, interaction with Stn1p is also required. Taken together, we have defined the telomere-binding domain of Cdc13p and showed that both binding to telomeres and Stn1p by Cdc13p are required to maintain cell growth.     

Kar1 binding to Sfi1 C-terminal regions anchors the SPB bridge to the nuclear envelope

The yeast spindle pole body (SPB) is the functional equivalent of the mammalian centrosome. The half bridge is a SPB substructure on the nuclear envelope (NE), playing a key role in SPB duplication. Its cytoplasmic components are the membrane-anchored Kar1, the yeast centrin Cdc31, and the Cdc31-binding protein Sfi1. In G1, the half bridge expands into the bridge through Sfi1 C-terminal (Sfi1-CT) dimerization, the licensing step for SPB duplication. We exploited photo-activated localization microscopy (PALM) to show that Kar1 localizes in the bridge center. Binding assays revealed direct interaction between Kar1 and C-terminal Sfi1 fragments. kar1Δ cells whose viability was maintained by the dominant CDC31-16 showed an arched bridge, indicating Kar1’s function in tethering Sfi1 to the NE. Cdc31-16 enhanced Cdc31–Cdc31 interactions between Sfi1–Cdc31 layers, as suggested by binding free energy calculations. In our model, Kar1 binding is restricted to Sfi1-CT and Sfi1 C-terminal centrin-binding repeats, and centrin and Kar1 provide cross-links, while Sfi1-CT stabilizes the bridge and ensures timely SPB separation.     

Interaction of Saccharomyces Cdc13p with Pol1p, Imp4p, Sir4p and Zds2p is involved in telomere replication, telomere maintenance and cell growth control

Telomeres are the physical ends of eukaryotic chromosomes. They are important for maintaining the integrity of chromosomes and this function is mediated through a number of protein factors. In Saccharomyces cerevisiae, Cdc13p binds to telomeres and affects telomere maintenance, telomere position effects and cell cycle progression through G₂/M phase. We identified four genes encoding Pol1p, Sir4p, Zds2p and Imp4p that interact with amino acids 1–252 of Cdc13p using a yeast two-hybrid screening system. Interactions of these four proteins with Cdc13p were through direct protein–protein interactions as judged by in vitro pull-down assays. Direct protein–protein interactions were also observed between Pol1p–Imp4p, Pol1p–Sir4p and Sir4p–Zds2p, whereas no interaction was detected between Imp4p–Sir4p and Zds2p–Imp4p, suggesting that protein interactions were specific in the complex. Pol1p was shown to interact with Cdc13p. Here we show that Zds2p and Imp4p also form a stable complex with Cdc13p in yeast cells, because Zds2p and Imp4p co-immunoprecipitate with Cdc13p, whereas Sir4p does not. The function of the N-terminal 1–252 region of Cdc13p was also analyzed. Expressing Cdc13(252–924)p, which lacks amino acids 1–252 of Cdc13p, causes defects in progressive cell growth and eventually arrested in the G₂/M phase of the cell cycle. These growth defects were not caused by progressive shortening of telomeres because telomeres in these cells were long. Point mutants in the amino acids 1–252 region of Cdc13p that reduced the interaction between Cdc13p and its binding proteins resulted in varying level of defects in cell growth and telomeres. These results indicate that the interactions between Cdc13(1–252)p and its binding proteins are important for the function of Cdc13p in telomere regulation and cell growth. Together, our results provide evidence for the formation of a Cdc13p-mediated telosome complex through its N-terminal region that is involved in telomere maintenance, telomere length regulation and cell growth control.     

Mps3p is a novel component of the yeast spindle pole body that interacts with the yeast centrin homologue Cdc31p

Accurate duplication of the Saccharomyces cerevisiae spindle pole body (SPB) is required for formation of a bipolar mitotic spindle. We identified mutants in SPB assembly by screening a temperature-sensitive collection of yeast for defects in SPB incorporation of a fluorescently marked integral SPB component, Spc42p. One SPB assembly mutant contained a mutation in a previously uncharacterized open reading frame that we call MPS3 (for monopolar spindle). mps3-1 mutants arrest in mitosis with monopolar spindles at the nonpermissive temperature, suggesting a defect in SPB duplication. Execution point experiments revealed that MPS3 function is required for the first step of SPB duplication in G1. Like cells containing mutations in two other genes required for this step of SPB duplication (CDC31 and KAR1), mps3-1 mutants arrest with a single unduplicated SPB that lacks an associated half-bridge. MPS3 encodes an essential integral membrane protein that localizes to the SPB half-bridge. Genetic interactions between MPS3 and CDC31 and binding of Cdc31p to Mps3p in vitro, as well as the fact that Cdc31p localization to the SPB is partially dependent on Mps3p function, suggest that one function for Mps3p during SPB duplication is to recruit Cdc31p, the yeast centrin homologue, to the half-bridge.     

Centrin/Cdc31 is a novel regulator of protein degradation

Rad23 is required for efficient protein degradation and performs an important role in nucleotide excision repair. Saccharomyces cerevisiae Rad23, and its human counterpart (hHR23), are present in a complex containing the DNA repair factor Rad4 (termed XPC, for xeroderma pigmentosum group C, in humans). XPC/hHR23 was also reported to bind centrin-2, a member of the superfamily of calcium-binding EF-hand proteins. We report here that yeast centrin, which is encoded by CDC31, is similarly present in a complex with Rad4/Rad23 (called NEF2). The interaction between Cdc31 and Rad23/Rad4 varied by growth phase and reflected oscillations in Cdc31 levels. Strikingly, a cdc31 mutant that formed a weaker interaction with Rad4 showed sensitivity to UV light. Based on the dual function of Rad23, in both DNA repair and protein degradation, we questioned if Cdc31 also participated in protein degradation. We report here that Cdc31 binds the proteasome and multiubiquitinated proteins through its carboxy-terminal EF-hand motifs. Moreover, cdc31 mutants were highly sensitive to drugs that cause protein damage, failed to efficiently degrade proteolytic substrates, and formed altered interactions with the proteasome. These findings reveal for the first time a new role for centrin/Cdc31 in protein degradation.     

The Rho1p Exchange Factor Rgf1p Signals Upstream from the Pmk1 Mitogen-activated Protein Kinase Pathway in Fission Yeast

The Schizosaccharomyces pombe exchange factor Rgf1p specifically regulates Rho1p during polarized growth. Rgf1p activates the β-glucan synthase (GS) complex containing the catalytic subunit Bgs4p and is involved in the activation of growth at the second end, a transition that requires actin reorganization. In this work, we investigated Rgf1p signaling and observed that Rgf1p acted upstream from the Pck2p-Pmk1p MAPK signaling pathway. We noted that Rgf1p and calcineurin play antagonistic roles in Cl⁻ homeostasis; rgf1Δ cells showed the vic phenotype (viable in the presence of immunosuppressant and chlorine ion) and were unable to grow in the presence of high salt concentrations, both phenotypes being characteristic of knockouts of the MAPK components. In addition, mutations that perturb signaling through the MAPK pathway resulted in defective cell integrity (hypersensitivity to caspofungin and β-glucanase). Rgf1p acts by positively regulating a subset of stimuli toward the Pmk1p-cell integrity pathway. After osmotic shock and cell wall damage HA-tagged Pmk1p was phosphorylated in wild-type cells but not in rgf1Δ cells. Finally, we provide evidence to show that Rgf1p regulates Pmk1p activation in a process that involves the activation of Rho1p and Pck2p, and we demonstrate that Rgf1p is unique in this signaling process, because Pmk1p activation was largely independent of the other two Rho1p-specific GEFs, Rgf2p and Rgf3p.     

Differential Interaction of Tomosyn with Syntaxin and SNAP25 Depends on Domains in the WD40 β-Propeller Core and Determines Its Inhibitory Activity

Neuronal exocytosis depends on efficient formation of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes and is regulated by tomosyn, a SNARE-binding protein. To gain new information about tomosyn''s activity, we characterized its mobility and organization on the plasma membrane (PM) in relation to other SNARE proteins and inhibition of exocytosis. By using direct stochastic optical reconstruction microscopy (dSTORM), we found tomosyn to be organized in small clusters adjacent to syntaxin clusters. In addition, we show that tomosyn is present in both syntaxin-tomosyn complexes and syntaxin-SNAP25-tomosyn complexes. Tomosyn mutants that lack residues 537–578 or 897–917 from its β-propeller core diffused faster on the PM and exhibited reduced binding to SNAP25, suggesting that these mutants shift the equilibrium between tomosyn-syntaxin-SNAP25 complexes on the PM to tomosyn-syntaxin complexes. As these deletion mutants impose less inhibition on exocytosis, we suggest that tomosyn inhibition is mediated via tomosyn-syntaxin-SNAP25 complexes and not tomosyn-syntaxin complexes. These findings characterize, for the first time, tomosyn''s dynamics at the PM and its relation to its inhibition of exocytosis.     

Interaction between Mitochondria and the Actin Cytoskeleton in Budding Yeast Requires Two Integral Mitochondrial Outer Membrane Proteins,Mmm1p and Mdm10p

Transfer of mitochondria to daughter cells during yeast cell division is essential for viable progeny. The actin cytoskeleton is required for this process, potentially as a track to direct mitochondrial movement into the bud. Sedimentation assays reveal two different components required for mitochondria–actin interactions: (1) mitochondrial actin binding protein(s) (mABP), a peripheral mitochondrial outer membrane protein(s) with ATP-sensitive actin binding activity, and (2) a salt-inextractable, presumably integral, membrane protein(s) required for docking of mABP on the organelle. mABP activity is abolished by treatment of mitochondria with high salt. Addition of either the salt-extracted mitochondrial peripheral membrane proteins (SE), or a protein fraction with ATP-sensitive actin-binding activity isolated from SE, to salt-washed mitochondria restores this activity. mABP docking activity is saturable, resistant to high salt, and inhibited by pre-treatment of salt-washed mitochondria with papain. Two integral mitochondrial outer membrane proteins, Mmm1p (Burgess, S.M., M. Delannoy, and R.E. Jensen. 1994. J.Cell Biol. 126:1375–1391) and Mdm10p, (Sogo, L.F., and M.P. Yaffe. 1994. J.Cell Biol. 126:1361– 1373) are required for these actin–mitochondria interactions. Mitochondria isolated from an mmm1-1 temperature-sensitive mutant or from an mdm10 deletion mutant show no mABP activity and no mABP docking activity. Consistent with this, mitochondrial motility in vivo in mmm1-1 and mdm10Δ mutants appears to be actin independent. Depolymerization of F-actin using latrunculin-A results in loss of long-distance, linear movement and a fivefold decrease in the velocity of mitochondrial movement. Mitochondrial motility in mmm1-1 and mdm10Δ mutants is indistinguishable from that in latrunculin-A–treated wild-type cells. We propose that Mmm1p and Mdm10p are required for docking of mABP on the surface of yeast mitochondria and coupling the organelle to the actin cytoskeleton.Mitochondria are indispensable organelles for normal eukaryotic cell function. Since mitochondria cannot be synthesized de novo, these organelles are inherited, i.e., transferred from mother to daughter during cell division. In the yeast Saccharomyces cerevisiae, vegetative cell division occurs by budding, a form of proliferation in which growth is directed toward the developing bud. Previous studies indicate that mitochondria undergo a series of cell cycle–linked motility events during normal inheritance in yeast (Simon et al., 1997). These are: (a) polarization of mitochondria towards the site of bud emergence in G₁ phase; (b) linear, polarized movement of mitochondria from mother cells to developing buds in S phase; (c) immobilization of newly inherited mitochondria in the bud tip during S and G₂ phases; and (d) release of immobilized mitochondria from the bud tip during M phase.There is mounting evidence that the actin cytoskeleton controls mitochondrial morphology and inheritance during vegetative yeast cell growth. The two major actin structures of yeast observed by light microscopy are patches and cables. Actin cables are bundles of actin filaments that extend from the mother into the bud. Mitochondria colocalize with these actin cables (Drubin et al., 1993; Lazzarino et al., 1994). Moreover, mutations such as deletion of the tropomyosin I gene, TPM1, or the mitochondrial distribution and morphology gene, MDM20, which selectively destabilize actin cables, result in the loss of polarized mitochondrial movement and reduce transfer of mitochondria into buds (Herman et al., 1997; Simon et al., 1997). Together, these studies indicate that normal mitochondrial inheritance in yeast requires association of mitochondria with actin cables.Cell-free studies reveal a possible mechanism underlying actin control of mitochondrial inheritance. Sedimentation assays document binding of mitochondria to the lateral surface of F-actin. This mitochondrial actin-binding activity is ATP-sensitive, saturable, reversible, and mediated by protein(s) on the mitochondrial surface (Lazzarino et al., 1994). In addition, ATP-driven, actin-dependent motor activity has been identified on the surface of mitochondria (Simon et al., 1995). These observations support a model of mitochondrial inheritance whereby mitochondria use an actin-dependent motor to drive their movement from mother to daughter cells along actin cable tracks.Yeast genetic screens have revealed several genes, collectively referred to as mdm (mitochondrial distribution and morphology) and mmm (maintenance of mitochondrial morphology), which are required for mitochondrial inheritance (McConnell et al., 1990; Burgess et al., 1994; Sogo and Yaffe, 1994). We have focused on two of these genes: MDM10 and MMM1. Deletion of MDM10 leads to the development of giant spherical mitochondria, presumably by the collapse of elongated mitochondria into a spherical mass (Sogo and Yaffe, 1994). Deletion of MMM1 (Burgess et al., 1994) produces a similar phenotype. In both mutants, the fraction of buds without mitochondria is high, indicating defective mitochondrial inheritance. The proteins encoded by these genes, Mdm10p and Mmm1p, appear to be integral membrane proteins in the mitochondrial outer membrane. Here, we report tests of the hypothesis that Mmm1p and Mdm10p are required to link mitochondria to the cytoskeleton.     

ATP-dependent Pre-replicative Complex Assembly Is Facilitated by Adk1p in Budding Yeast

Pre-replicative complex (pre-RC) assembly is a critical part of the mechanism that controls the initiation of DNA replication, and ATP binding and hydrolysis by multiple pre-RC proteins are essential for pre-RC assembly and activation. Here, we demonstrate that Adk1p (adenylate kinase 1 protein) plays an important role in pre-RC assembly in Saccharomyces cerevisiae. Isolated from a genetic screen, adk1^G20S cells with a mutation within the nucleotide-binding site were defective in replication initiation. adk1Δ cells were viable at 25 °C but not at 37°C. Flow cytometry indicated that both the adk1-td (temperature-inducible degron) and adk1^G20S mutants were defective in S phase entry. Furthermore, Adk1p bound to chromatin throughout the cell cycle and physically interacted with Orc3p, whereas the Adk1^G20S protein had a reduced ability to bind chromatin and Orc3p without affecting the cellular ATP level. In addition, Adk1p associated with replication origins by ChIP assay. Finally, Adk1-td protein depletion prevented pre-RC assembly during the M-to-G₁ transition. We suggest that Adk1p regulates ATP metabolism on pre-RC proteins to promote pre-RC assembly and activation.     

Cdc14 Early Anaphase Release,FEAR, Is Limited to the Nucleus and Dispensable for Efficient Mitotic Exit

Cdc14 phosphatase is a key regulator of exit from mitosis, acting primarily through antagonism of cyclin-dependent kinase, and is also thought to be important for meiosis. Cdc14 is released from its sequestration site in the nucleolus in two stages, first by the non-essential Cdc Fourteen Early Anaphase Release (FEAR) pathway and later by the essential Mitotic Exit Network (MEN), which drives efficient export of Cdc14 to the cytoplasm. We find that Cdc14 is confined to the nucleus during early mitotic anaphase release, and during its meiosis I release. Proteins whose degradation is directed by Cdc14 as a requirement for mitotic exit (e.g. the B-type cyclin, Clb2), remain stable during mitotic FEAR, a result consistent with Cdc14 being restricted to the nucleus and not participating directly in mitotic exit. Cdc14 released by the FEAR pathway has been proposed to have a wide variety of activities, all of which are thought to promote passage through anaphase. Proposed functions of FEAR include stabilization of anaphase spindles, resolution of the rDNA to allow its segregation, and priming of the MEN so that mitotic exit can occur promptly and efficiently. We tested the model for FEAR functions using the FEAR-deficient mutation net1-6cdk. Our cytological observations indicate that, contrary to the current model, FEAR is fully dispensable for timely progression through a series of anaphase landmarks and mitotic exit, although it is required for timely rDNA segregation. The net1-6cdk mutation suppresses temperature-sensitive mutations in MEN genes, suggesting that rather than activating mitotic exit, FEAR either inhibits the MEN or has no direct effect upon it. One interpretation of this result is that FEAR delays MEN activation to ensure that rDNA segregation occurs before mitotic exit. Our findings clarify the distinction between FEAR and MEN-dependent Cdc14 activities and will help guide emerging quantitative models of this cell cycle transition.     

Loss of CDC5 function in Saccharomyces cerevisiae leads to defects in Swe1p regulation and Bfa1p/Bub2p-independent cytokinesis

In many organisms, polo kinases appear to play multiple roles during M-phase progression. To provide new insights into the function of budding yeast polo kinase Cdc5p, we generated novel temperature-sensitive cdc5 mutants by mutagenizing the C-terminal domain. Here we show that, at a semipermissive temperature, the cdc5-3 mutant exhibited a synergistic bud elongation and growth defect with loss of HSL1, a component important for normal G(2)/M transition. Loss of SWE1, which phosphorylates and inactivates the budding yeast Cdk1 homolog Cdc28p, suppressed the cdc5-3 hsl1Delta defect, suggesting that Cdc5p functions at a point upstream of Swe1p. In addition, the cdc5-4 and cdc5-7 mutants exhibited chained cell morphologies with shared cytoplasms between the connected cell bodies, indicating a cytokinetic defect. Close examination of these mutants revealed delayed septin assembly at the incipient bud site and loosely organized septin rings at the mother-bud neck. Components in the mitotic exit network (MEN) play important roles in normal cytokinesis. However, loss of BFA1 or BUB2, negative regulators of the MEN, failed to remedy the cytokinetic defect of these mutants, indicating that Cdc5p promotes cytokinesis independently of Bfa1p and Bub2p. Thus, Cdc5p contributes to the activation of the Swe1p-dependent Cdc28p/Clb pathway, normal septin function, and cytokinesis.     

Substitution as a mechanism for genetic robustness: the duplicated deacetylases Hst1p and Sir2p in Saccharomyces cerevisiae

How duplicate genes provide genetic robustness remains an unresolved question. We have examined the duplicated histone deacetylases Sir2p and Hst1p in Saccharomyces cerevisiae and find that these paralogs with non-overlapping functions can provide genetic robustness against null mutations through a substitution mechanism. Hst1p is an NAD⁺-dependent histone deacetylase that acts with Sum1p to repress a subset of midsporulation genes. However, hst1Δ mutants show much weaker derepression of target loci than sum1Δ mutants. We show that this modest derepression of target loci in hst1Δ strains occurs in part because Sir2p substitutes for Hst1p. Sir2p contributes to repression of the midsporulation genes only in the absence of Hst1p and is recruited to target promoters by a physical interaction with the Sum1 complex. Furthermore, when Sir2p associates with the Sum1 complex, the complex continues to repress in a promoter-specific manner and does not spread. Our results imply that after the duplication, SIR2 and HST1 subfunctionalized. The single SIR2/HST1 gene from Kluyveromyces lactis, a closely related species that diverged prior to the duplication, can suppress an hst1Δ mutation in S. cerevisiae as well as interact with Sir4p in S. cerevisiae. In addition, the existence of two distinct protein interaction domains for the Sir and Sum1 complexes was revealed through the analysis of a chimeric Sir2–Hst1 molecule. Therefore, the ability of Sir2p to substitute for Hst1p probably results from a retained but reduced affinity for the Sum1 complex that is a consequence of subfunctionalization via the duplication, degeneration, and complementation mechanism. These results suggest that the evolutionary path of duplicate gene preservation may be an important indicator for the ability of duplicated genes to contribute to genetic robustness.     

Bax Forms an Oligomer via Separate,Yet Interdependent,Surfaces

Interactions of Bcl-2 family proteins regulate permeability of the mitochondrial outer membrane and apoptosis. In particular, Bax forms an oligomer that permeabilizes the membrane. To map the interface of the Bax oligomer we used Triton X-100 as a membrane surrogate and performed site-specific photocross-linking. Bax-specific adducts were formed through photo-reactive probes at multiple sites that can be grouped into two surfaces. The first surface overlaps with the BH1–3 groove formed by Bcl-2 Homology motif 1, 2, and 3; the second surface is a rear pocket located on the opposite side of the protein from the BH1–3 groove. Further cross-linking experiments using Bax BH3 peptides and mutants demonstrated that the two surfaces interact with their counterparts in neighboring proteins to form two separated interfaces and that interaction at the BH1–3 groove primes the rear pocket for further interaction. Therefore, Bax oligomerization proceeds through a series of interactions that occur at separate, yet allosterically, coupled interfaces.     

SOCS-6 Negatively Regulates T Cell Activation through Targeting p56lck to Proteasomal Degradation

The T cell-specific tyrosine kinase, p56^lck, plays crucial roles in T cell receptor (TCR)-mediated T cell activation. Here, we report that SOCS-6 (suppressor of cytokine signaling-6) is a negative regulator of p56^lck. SOCS-6 was identified as a protein binding to the kinase domain of p56^lck through yeast two-hybrid screening. SOCS-6 bound specifically to p56^lck (F505), which mimics the active form of p56^lck, but not to wild type p56^lck. In Jurkat T cells, SOCS-6 binding to p56^lck was detected 1–2 h after TCR stimulation. Confocal microscopy showed that upon APC-T cell conjugation, SOCS-6 was recruited to the immunological synapse and colocalized with the active form of p56^lck. SOCS-6 promoted p56^lck ubiquitination and its subsequent targeting to the proteasome. Moreover, SOCS-6 overexpression led to repression of TCR-dependent interleukin-2 promoter activity. These results establish that SOCS-6 acts as a negative regulator of T cell activation by promoting ubiquitin-dependent proteolysis.     

DNA polymerization-independent functions of DNA polymerase epsilon in assembly and progression of the replisome in fission yeast

DNA polymerase epsilon (Pol ε) synthesizes the leading strands, following the CMG (Cdc45, Mcm2-7, and GINS [Go-Ichi-Nii-San]) helicase that translocates on the leading-strand template at eukaryotic replication forks. Although Pol ε is essential for the viability of fission and budding yeasts, the N-terminal polymerase domain of the catalytic subunit, Cdc20/Pol2, is dispensable for viability, leaving the following question: what is the essential role(s) of Pol ε? In this study, we investigated the essential roles of Pol ε using a temperature-sensitive mutant and a recently developed protein-depletion (off-aid) system in fission yeast. In cdc20-ct1 cells carrying mutations in the C-terminal domain of Cdc20, the CMG components, RPA, Pol α, and Pol δ were loaded onto replication origins, but Cdc45 did not translocate from the origins, suggesting that Pol ε is required for CMG helicase progression. In contrast, depletion of Cdc20 abolished the loading of GINS and Cdc45 onto origins, indicating that Pol ε is essential for assembly of the CMG complex. These results demonstrate that Pol ε plays essential roles in both the assembly and progression of CMG helicase.     

DNA replication is completed in Saccharomyces cerevisiae cells that lack functional Cdc14, a dual-specificity protein phosphatase

The Cdc14 protein encodes a dual-specificity protein phosphatase which functions in late mitosis, and considerable genetic evidence suggests a role in DNA replication. We find that cdc14 mutants arrested in late mitosis maintain persistent levels of mitotic kinase activity, suggesting that Cdc14 controls inactivation of this kinase. Overexpression of Sic1, a cyclin-dependent protein kinase inhibitor, is able to suppress telophase mutants such as dbf2, cdc5 and cdc15, but not cdc14. It does, however, force cdc14-arrested cells into the next cell cycle, in which an apparently normal S phase occurs as judged by FACS and pulsed-field gel electrophoretic analysis. Furthermore, in a promoter shut-off experiment, cells lacking Cdc14 appear to carry out a normal S phase. Thus Cdc14 functions mainly in late mitosis and it has no essential role in S phase.