The Mps1 Kinase Modulates the Recruitment and Activity of Cnn1CENP-T at Saccharomyces cerevisiae Kinetochores

Kinetochores are conserved protein complexes that bind the replicated chromosomes to the mitotic spindle and then direct their segregation. To better comprehend Saccharomyces cerevisiae kinetochore function, we dissected the phospho-regulated dynamic interaction between conserved kinetochore protein Cnn1^CENP-T, the centromere region, and the Ndc80 complex through the cell cycle. Cnn1 localizes to kinetochores at basal levels from G1 through metaphase but accumulates abruptly at anaphase onset. How Cnn1 is recruited and which activities regulate its dynamic localization are unclear. We show that Cnn1 harbors two kinetochore-localization activities: a C-terminal histone-fold domain (HFD) that associates with the centromere region and a N-terminal Spc24/Spc25 interaction sequence that mediates linkage to the microtubule-binding Ndc80 complex. We demonstrate that the established Ndc80 binding site in the N terminus of Cnn1, Cnn1^60–84, should be extended with flanking residues, Cnn1^25–91, to allow near maximal binding affinity to Ndc80. Cnn1 localization was proposed to depend on Mps1 kinase activity at Cnn1–S74, based on in vitro experiments demonstrating the Cnn1–Ndc80 complex interaction. We demonstrate that from G1 through metaphase, Cnn1 localizes via both its HFD and N-terminal Spc24/Spc25 interaction sequence, and deletion or mutation of either region results in anomalous Cnn1 kinetochore levels. At anaphase onset (when Mps1 activity decreases) Cnn1 becomes enriched mainly via the N-terminal Spc24/Spc25 interaction sequence. In sum, we provide the first in vivo evidence of Cnn1 preanaphase linkages with the kinetochore and enrichment of the linkages during anaphase.     

Redundant Regulation of Cdk1 Tyrosine Dephosphorylation in Saccharomyces cerevisiae

Cdk1 activity drives both mitotic entry and the metaphase-to-anaphase transition in all eukaryotes. The kinase Wee1 and the phosphatase Cdc25 regulate the mitotic activity of Cdk1 by the reversible phosphorylation of a conserved tyrosine residue. Mutation of cdc25 in Schizosaccharomyces pombe blocks Cdk1 dephosphorylation and causes cell cycle arrest. In contrast, deletion of MIH1, the cdc25 homolog in Saccharomyces cerevisiae, is viable. Although Cdk1-Y19 phosphorylation is elevated during mitosis in mih1∆ cells, Cdk1 is dephosphorylated as cells progress into G₁, suggesting that additional phosphatases regulate Cdk1 dephosphorylation. Here we show that the phosphatase Ptp1 also regulates Cdk1 dephosphorylation in vivo and can directly dephosphorylate Cdk1 in vitro. Using a novel in vivo phosphatase assay, we also show that PP2A bound to Rts1, the budding yeast B56-regulatory subunit, regulates dephosphorylation of Cdk1 independently of a function regulating Swe1, Mih1, or Ptp1, suggesting that PP2A^Rts1 either directly dephosphorylates Cdk1-Y19 or regulates an unidentified phosphatase.     

New Regulators of Clathrin-Mediated Endocytosis Identified in Saccharomyces cerevisiae by Systematic Quantitative Fluorescence Microscopy

Despite the importance of clathrin-mediated endocytosis (CME) for cell biology, it is unclear if all components of the machinery have been discovered and many regulatory aspects remain poorly understood. Here, using Saccharomyces cerevisiae and a fluorescence microscopy screening approach we identify previously unknown regulatory factors of the endocytic machinery. We further studied the top scoring protein identified in the screen, Ubx3, a member of the conserved ubiquitin regulatory X (UBX) protein family. In vivo and in vitro approaches demonstrate that Ubx3 is a new coat component. Ubx3-GFP has typical endocytic coat protein dynamics with a patch lifetime of 45 ± 3 sec. Ubx3 contains a W-box that mediates physical interaction with clathrin and Ubx3-GFP patch lifetime depends on clathrin. Deletion of the UBX3 gene caused defects in the uptake of Lucifer Yellow and the methionine transporter Mup1 demonstrating that Ubx3 is needed for efficient endocytosis. Further, the UBX domain is required both for localization and function of Ubx3 at endocytic sites. Mechanistically, Ubx3 regulates dynamics and patch lifetime of the early arriving protein Ede1 but not later arriving coat proteins or actin assembly. Conversely, Ede1 regulates the patch lifetime of Ubx3. Ubx3 likely regulates CME via the AAA-ATPase Cdc48, a ubiquitin-editing complex. Our results uncovered new components of the CME machinery that regulate this fundamental process.     

The Anaphase Promoting Complex Regulates Yeast Lifespan and rDNA Stability by Targeting Fob1 for Degradation

Genomic stability, stress response, and nutrient signaling all play critical, evolutionarily conserved roles in lifespan determination. However, the molecular mechanisms coordinating these processes with longevity remain unresolved. Here we investigate the involvement of the yeast anaphase promoting complex (APC) in longevity. The APC governs passage through M and G1 via ubiquitin-dependent targeting of substrate proteins and is associated with cancer and premature aging when defective. Our two-hybrid screen utilizing Apc5 as bait recovered the lifespan determinant Fob1 as prey. Fob1 is unstable specifically in G1, cycles throughout the cell cycle in a manner similar to Clb2 (an APC target), and is stabilized in APC (apc5^CA) and proteasome (rpn10∆) mutants. Deletion of FOB1 increased replicative lifespan (RLS) in wild type (WT), apc5^CA, and apc10∆ cells, and suppressed apc5^CA cell cycle progression and rDNA recombination defects. Alternatively, increased FOB1 expression decreased RLS in WT cells, but did not reduce the already short apc5^CA RLS, suggesting an epistatic interaction between apc5^CA and fob1∆. Mutation to a putative L-Box (Fob1^E420V), a Destruction Box-like motif, abolished Fob1 modifications, stabilized the protein, and increased rDNA recombination. Our work provides a mechanistic role played by the APC to promote replicative longevity and genomic stability in yeast.     

Remodeling of the Rad51 DNA Strand-Exchange Protein by the Srs2 Helicase

Homologous recombination is associated with the dynamic assembly and disassembly of DNA–protein complexes. Assembly of a nucleoprotein filament comprising ssDNA and the RecA homolog, Rad51, is a key step required for homology search during recombination. The budding yeast Srs2 DNA translocase is known to dismantle Rad51 filament in vitro. However, there is limited evidence to support the dismantling activity of Srs2 in vivo. Here, we show that Srs2 indeed disrupts Rad51-containing complexes from chromosomes during meiosis. Overexpression of Srs2 during the meiotic prophase impairs meiotic recombination and removes Rad51 from meiotic chromosomes. This dismantling activity is specific for Rad51, as Srs2 Overexpression does not remove Dmc1 (a meiosis-specific Rad51 homolog), Rad52 (a Rad51 mediator), or replication protein A (RPA; a single-stranded DNA-binding protein). Rather, RPA replaces Rad51 under these conditions. A mutant Srs2 lacking helicase activity cannot remove Rad51 from meiotic chromosomes. Interestingly, the Rad51-binding domain of Srs2, which is critical for Rad51-dismantling activity in vitro, is not essential for this activity in vivo. Our results suggest that a precise level of Srs2, in the form of the Srs2 translocase, is required to appropriately regulate the Rad51 nucleoprotein filament dynamics during meiosis.     

Integrity and Function of the Saccharomyces cerevisiae Spindle Pole Body Depends on Connections Between the Membrane Proteins Ndc1, Rtn1, and Yop1

The nuclear envelope in Saccharomyces cerevisiae harbors two essential macromolecular protein assemblies: the nuclear pore complexes (NPCs) that enable nucleocytoplasmic transport, and the spindle pole bodies (SPBs) that mediate chromosome segregation. Previously, based on metazoan and budding yeast studies, we reported that reticulons and Yop1/DP1 play a role in the early steps of de novo NPC assembly. Here, we examined if Rtn1 and Yop1 are required for SPB function in S. cerevisiae. Electron microscopy of rtn1Δ yop1Δ cells revealed lobular abnormalities in SPB structure. Using an assay that monitors lateral expansion of the SPB central layer, we found that rtn1Δ yop1Δ SPBs had decreased connections to the NE compared to wild type, suggesting that SPBs are less stable in the NE. Furthermore, large budded rtn1Δ yop1Δ cells exhibited a high incidence of short mitotic spindles, which were frequently misoriented with respect to the mother–daughter axis. This correlated with cytoplasmic microtubule defects. We found that overexpression of the SPB insertion factors NDC1, MPS2, or BBP1 rescued the SPB defects observed in rtn1Δ yop1Δ cells. However, only overexpression of NDC1, which is also required for NPC biogenesis, rescued both the SPB and NPC associated defects. Rtn1 and Yop1 also physically interacted with Ndc1 and other NPC membrane proteins. We propose that NPC and SPB biogenesis are altered in cells lacking Rtn1 and Yop1 due to competition between these complexes for Ndc1, an essential common component of both NPCs and SPBs.     

DNA Replication Checkpoint Signaling Depends on a Rad53–Dbf4 N-Terminal Interaction in Saccharomyces cerevisiae

Dbf4-dependent kinase (DDK) and cyclin-dependent kinase (CDK) are essential to initiate DNA replication at individual origins. During replication stress, the S-phase checkpoint inhibits the DDK- and CDK-dependent activation of late replication origins. Rad53 kinase is a central effector of the replication checkpoint and both binds to and phosphorylates Dbf4 to prevent late-origin firing. The molecular basis for the Rad53–Dbf4 physical interaction is not clear but occurs through the Dbf4 N terminus. Here we found that both Rad53 FHA1 and FHA2 domains, which specifically recognize phospho-threonine (pT), interacted with Dbf4 through an N-terminal sequence and an adjacent BRCT domain. Purified Rad53 FHA1 domain (but not FHA2) bound to a pT Dbf4 peptide in vitro, suggesting a possible phospho-threonine-dependent interaction between FHA1 and Dbf4. The Dbf4–Rad53 interaction is governed by multiple contacts that are separable from the Cdc5- and Msa1-binding sites in the Dbf4 N terminus. Importantly, abrogation of the Rad53–Dbf4 physical interaction blocked Dbf4 phosphorylation and allowed late-origin firing during replication checkpoint activation. This indicated that Rad53 must stably bind to Dbf4 to regulate its activity.     

Novel Connections Between DNA Replication,Telomere Homeostasis,and the DNA Damage Response Revealed by a Genome-Wide Screen for TEL1/ATM Interactions in Saccharomyces cerevisiae

Tel1 is the budding yeast ortholog of the mammalian tumor suppressor and DNA damage response (DDR) kinase ATM. However, tel1-Δ cells, unlike ATM-deficient cells, do not exhibit sensitivity to DNA-damaging agents, but do display shortened (but stably maintained) telomere lengths. Neither the extent to which Tel1p functions in the DDR nor the mechanism by which Tel1 contributes to telomere metabolism is well understood. To address the first question, we present the results from a comprehensive genome-wide screen for genetic interactions with tel1-Δ that cause sensitivity to methyl methanesulfonate (MMS) and/or ionizing radiation, along with follow-up characterizations of the 13 interactions yielded by this screen. Surprisingly, many of the tel1-Δ interactions that confer DNA damage sensitivity also exacerbate the short telomere phenotype, suggesting a connection between these two phenomena. Restoration of normal telomere length in the tel1-Δ xxx-Δ mutants results in only minor suppression of the DNA damage sensitivity, demonstrating that the sensitivity of these mutants must also involve mechanisms independent of telomere length. In support of a model for increased replication stress in the tel1-Δ xxx-Δ mutants, we show that depletion of dNTP pools through pretreatment with hydroxyurea renders tel1-Δ cells (but not wild type) MMS-sensitive, demonstrating that, under certain conditions, Tel1p does indeed play a critical role in the DDR.     

Coordination of Recombination with Meiotic Progression in the Caenorhabditis elegans Germline by KIN-18, a TAO Kinase That Regulates the Timing of MPK-1 Signaling

Meiosis is a tightly regulated process requiring coordination of diverse events. A conserved ERK/MAPK-signaling cascade plays an essential role in the regulation of meiotic progression. The Thousand And One kinase (TAO) kinase is a MAPK kinase kinase, the meiotic role of which is unknown. We have analyzed the meiotic functions of KIN-18, the homolog of mammalian TAO kinases, in Caenorhabditis elegans. We found that KIN-18 is essential for normal meiotic progression; mutants exhibit accelerated meiotic recombination as detected both by analysis of recombination intermediates and by crossover outcome. In addition, ectopic germ-cell differentiation and enhanced levels of apoptosis were observed in kin-18 mutants. These defects correlate with ectopic activation of MPK-1 that includes premature, missing, and reoccurring MPK-1 activation. Late progression defects in kin-18 mutants are suppressed by inhibiting an upstream activator of MPK-1 signaling, KSR-2. However, the acceleration of recombination events observed in kin-18 mutants is largely MPK-1-independent. Our data suggest that KIN-18 coordinates meiotic progression by modulating the timing of MPK-1 activation and the progression of recombination events. The regulation of the timing of MPK-1 activation ensures the proper timing of apoptosis and is required for the formation of functional oocytes. Meiosis is a conserved process; thus, revealing that KIN-18 is a novel regulator of meiotic progression in C. elegans would help to elucidate TAO kinase’s role in germline development in higher eukaryotes.     

Too Much of a Good Thing: The Unique and Repeated Paths Toward Copper Adaptation

Copper is a micronutrient essential for growth due to its role as a cofactor in enzymes involved in respiration, defense against oxidative damage, and iron uptake. Yet too much of a good thing can be lethal, and yeast cells typically do not have tolerance to copper levels much beyond the concentration in their ancestral environment. Here, we report a short-term evolutionary study of Saccharomyces cerevisiae exposed to levels of copper sulfate that are inhibitory to the initial strain. We isolated and identified adaptive mutations soon after they arose, reducing the number of neutral mutations, to determine the first genetic steps that yeast take when adapting to copper. We analyzed 34 such strains through whole-genome sequencing and by assaying fitness within different environments; we also isolated a subset of mutations through tetrad analysis of four lines. We identified a multilayered evolutionary response. In total, 57 single base-pair mutations were identified across the 34 lines. In addition, gene amplification of the copper metallothionein protein, CUP1-1, was rampant, as was chromosomal aneuploidy. Four other genes received multiple, independent mutations in different lines (the vacuolar transporter genes VTC1 and VTC4; the plasma membrane H+-ATPase PMA1; and MAM3, a protein required for normal mitochondrial morphology). Analyses indicated that mutations in all four genes, as well as CUP1-1 copy number, contributed significantly to explaining variation in copper tolerance. Our study thus finds that evolution takes both common and less trodden pathways toward evolving tolerance to an essential, but highly toxic, micronutrient.     

The Principal Role of Ku in Telomere Length Maintenance Is Promotion of Est1 Association with Telomeres

Telomere length is tightly regulated in cells that express telomerase. The Saccharomyces cerevisiae Ku heterodimer, a DNA end-binding complex, positively regulates telomere length in a telomerase-dependent manner. Ku associates with the telomerase RNA subunit TLC1, and this association is required for TLC1 nuclear retention. Ku–TLC1 interaction also impacts the cell-cycle-regulated association of the telomerase catalytic subunit Est2 to telomeres. The promotion of TLC1 nuclear localization and Est2 recruitment have been proposed to be the principal role of Ku in telomere length maintenance, but neither model has been directly tested. Here we study the impact of forced recruitment of Est2 to telomeres on telomere length in the absence of Ku’s ability to bind TLC1 or DNA ends. We show that tethering Est2 to telomeres does not promote efficient telomere elongation in the absence of Ku–TLC1 interaction or DNA end binding. Moreover, restoration of TLC1 nuclear localization, even when combined with Est2 recruitment, does not bypass the role of Ku. In contrast, forced recruitment of Est1, which has roles in telomerase recruitment and activation, to telomeres promotes efficient and progressive telomere elongation in the absence of Ku–TLC1 interaction, Ku DNA end binding, or Ku altogether. Ku associates with Est1 and Est2 in a TLC1-dependent manner and enhances Est1 recruitment to telomeres independently of Est2. Together, our results unexpectedly demonstrate that the principal role of Ku in telomere length maintenance is to promote the association of Est1 with telomeres, which may in turn allow for efficient recruitment and activation of the telomerase holoenzyme.     

Genetic Analysis of the Ribosome Biogenesis Factor Ltv1 of Saccharomyces cerevisiae

Ribosome biogenesis has been studied extensively in the yeast Saccharomyces cerevisiae. Yeast Ltv1 is a conserved 40S-associated biogenesis factor that has been proposed to function in small subunit nuclear export. Here we show that Ltv1 has a canonical leucine-rich nuclear export signal (NES) at its extreme C terminus that is both necessary for Crm1 interaction and Ltv1 export. The C terminus of Ltv1 can substitute for the NES in the 60S-export adapter Nmd3, demonstrating that it is a functional NES. Overexpression of an Ltv1 lacking its NES (Ltv1∆C13) was strongly dominant negative and resulted in the nuclear accumulation of RpS3-GFP; however, export of the pre-40S was not affected. In addition, expression of endogenous levels of Ltv1∆C protein complemented both the slow-growth phenotype and the 40S biogenesis defect of an ltv1 deletion mutant. Thus, if Ltv1 is a nuclear export adapter for the pre-40S subunit, its function must be fully redundant with additional export factors. The dominant negative phenotype of Ltv1∆NES overexpression was suppressed by co-overexpressing RpS3 and its chaperone, Yar1, or by deletion of the RpS3-binding site in Ltv1∆NES, suggesting that titration of RpS3 by Ltv1∆NES is deleterious in yeast. The dominant-negative phenotype did not correlate with a decrease in 40S levels but rather with a reduction in the polysome-to-monosome ratio, indicating reduced rates of translation. We suggest that titration of RpS3 by excess nuclear Ltv1 interferes with 40S function or with a nonribosomal function of RpS3.     

Fertility and Polarized Cell Growth Depends on eIF5A for Translation of Polyproline-Rich Formins in Saccharomyces cerevisiae

eIF5A is an essential and evolutionary conserved translation elongation factor, which has recently been proposed to be required for the translation of proteins with consecutive prolines. The binding of eIF5A to ribosomes occurs upon its activation by hypusination, a modification that requires spermidine, an essential factor for mammalian fertility that also promotes yeast mating. We show that in response to pheromone, hypusinated eIF5A is required for shmoo formation, localization of polarisome components, induction of cell fusion proteins, and actin assembly in yeast. We also show that eIF5A is required for the translation of Bni1, a proline-rich formin involved in polarized growth during shmoo formation. Our data indicate that translation of the polyproline motifs in Bni1 is eIF5A dependent and this translation dependency is lost upon deletion of the polyprolines. Moreover, an exogenous increase in Bni1 protein levels partially restores the defect in shmoo formation seen in eIF5A mutants. Overall, our results identify eIF5A as a novel and essential regulator of yeast mating through formin translation. Since eIF5A and polyproline formins are conserved across species, our results also suggest that eIF5A-dependent translation of formins could regulate polarized growth in such processes as fertility and cancer in higher eukaryotes.