Spindle checkpoint proteins and chromosome-microtubule attachment in budding yeast

Accurate chromosome segregation depends on precise regulation of mitosis by the spindle checkpoint. This checkpoint monitors the status of kinetochore-microtubule attachment and delays the metaphase to anaphase transition until all kinetochores have formed stable bipolar connections to the mitotic spindle. Components of the spindle checkpoint include the mitotic arrest defective (MAD) genes MAD1-3, and the budding uninhibited by benzimidazole (BUB) genes BUB1 and BUB3. In animal cells, all known spindle checkpoint proteins are recruited to kinetochores during normal mitoses. In contrast, we show that whereas Saccharomyces cerevisiae Bub1p and Bub3p are bound to kinetochores early in mitosis as part of the normal cell cycle, Mad1p and Mad2p are kinetochore bound only in the presence of spindle damage or kinetochore lesions that interfere with chromosome-microtubule attachment. Moreover, although Mad1p and Mad2p perform essential mitotic functions during every division cycle in mammalian cells, they are required in budding yeast only when mitosis goes awry. We propose that differences in the behavior of spindle checkpoint proteins in animal cells and budding yeast result primarily from evolutionary divergence in spindle assembly pathways.     

Multiple pathways influence mitochondrial inheritance in budding yeast

Yeast mitochondria form a branched tubular network. Mitochondrial inheritance is tightly coupled with bud emergence, ensuring that daughter cells receive mitochondria from mother cells during division. Proteins reported to influence mitochondrial inheritance include the mitochondrial rho (Miro) GTPase Gem1p, Mmr1p, and Ypt11p. A synthetic genetic array (SGA) screen revealed interactions between gem1Delta and deletions of genes that affect mitochondrial function or inheritance, including mmr1Delta. Synthetic sickness of gem1Delta mmr1Delta double mutants correlated with defective mitochondrial inheritance by large buds. Additional studies demonstrated that GEM1, MMR1, and YPT11 each contribute to mitochondrial inheritance. Mitochondrial accumulation in buds caused by overexpression of either Mmr1p or Ypt11p did not depend on Gem1p, indicating these three proteins function independently. Physical linkage of mitochondria with the endoplasmic reticulum (ER) has led to speculation that distribution of these two organelles is coordinated. We show that yeast mitochondrial inheritance is not required for inheritance or spreading of cortical ER in the bud. Moreover, Ypt11p overexpression, but not Mmr1p overexpression, caused ER accumulation in the bud, revealing a potential role for Ypt11p in ER distribution. This study demonstrates that multiple pathways influence mitochondrial inheritance in yeast and that Miro GTPases have conserved roles in mitochondrial distribution.     

Telomere maintenance through spatial control of telomeric proteins

The six human telomeric proteins TRF1, TRF2, RAP1, TIN2, POT1, and TPP1 can form a complex called the telosome/shelterin, which is required for telomere protection and length control. TPP1 has been shown to regulate both POT1 telomere localization and telosome assembly through its binding to TIN2. It remains to be determined where such interactions take place and whether cellular compartmentalization of telomeric proteins is important for telomere maintenance. We systematically investigated here the cellular localization and interactions of human telomeric proteins. Interestingly, we found TIN2, TPP1, and POT1 to localize and interact with each other in both the cytoplasm and the nucleus. Unexpectedly, TPP1 contains a functional nuclear export signal that directly controls the amount of TPP1 and POT1 in the nucleus. Furthermore, binding of TIN2 to TPP1 promotes the nuclear localization of TPP1 and POT1. We also found that disrupting TPP1 nuclear export could result in telomeric DNA damage response and telomere length disregulation. Our findings highlight how the coordinated interactions between TIN2, TPP1, and POT1 in the cytoplasm regulate the assembly and function of the telosome in the nucleus and indicate for the first time the importance of nuclear export and spatial control of telomeric proteins in telomere maintenance.     

Replication proteins influence the maintenance of telomere length and telomerase protein stability

We investigated the effects of fission yeast replication genes on telomere length maintenance and identified 20 mutant alleles that confer lengthening or shortening of telomeres. The telomere elongation was telomerase dependent in the replication mutants analyzed. Furthermore, the telomerase catalytic subunit, Trt1, and the principal initiation and lagging-strand synthesis DNA polymerase, Polalpha, were reciprocally coimmunoprecipitated, indicating these proteins physically coexist as a complex in vivo. In a polalpha mutant that exhibited abnormal telomere lengthening and slightly reduced telomere position effect, the cellular level of the Trt1 protein was significantly lower and the coimmunoprecipitation of Trt1 and Polalpha was severely compromised compared to those in the wild-type polalpha cells. Interestingly, ectopic expression of wild-type polalpha in this polalpha mutant restored the cellular Trt1 protein to the wild-type level and shortened the telomeres to near-wild-type length. These results suggest that there is a close physical relationship between the replication and telomerase complexes. Thus, mutation of a component of the replication complex can affect the telomeric complex in maintaining both telomere length equilibrium and telomerase protein stability.     

Subtelomeric proteins negatively regulate telomere elongation in budding yeast

The Tbf1 and Reb1 proteins are present in yeast subtelomeric regions. We establish in this work that they inhibit telomerase-dependent lengthening of telomere. For example, tethering the N-terminal domain of Tbf1 and Reb1 in a subtelomeric region shortens that telomere proportionally to the number of domains bound. We further identified a 90 amino-acid long sequence within the N-terminal domain of Tbf1 that is necessary but not sufficient for its length regulation properties. The role of the subtelomeric factors in telomere length regulation is antagonized by TEL1 and does not correlate with a global telomere derepression. We show that the absence of TEL1 induces an alteration in the structure of telomeric chromatin, as defined biochemically by an increased susceptibility to nucleases and a greater heterogeneity of products. We propose that the absence of TEL1 modifies the organization of the telomeres, which allows Tbf1 and Reb1 to cis-inhibit telomerase. The involvement of subtelomeric factors in telomere length regulation provides a possible mechanism for the chromosome-specific length setting observed at yeast and human telomeres.     

Def1p is involved in telomere maintenance in budding yeast

Saccharomyces Rrm3p, a member of Pif1 5'-3' DNA helicase subfamily, helps replication forks traverse protein-DNA complexes, including the telomere. Here we have identified an Rrm3p interaction protein known to be Def1p. In def1 mutants, telomeres were approximately 200-bp shorter than that in wild-type cells. DEF1 is also required for the stable maintenance of mitochondrial DNA, and the telomere shortening phenotype seen in def1 cells is not a secondary consequence of the mitochondrion defect. A combination of DEF1 null mutation with deletion of EST2 or EST3 resulted in an accelerated senescence phenotype, suggesting that Def1p is not involved in the telomerase recruitment pathway. In the absence of telomerase, cells escape senescence by either amplifying Y' regions or TG-telomeric repeats to generate type I or type II survivors, respectively. Only type I survivors were recovered from both def1Delta est2Delta and def1Delta est3Delta double mutant cells, further suggesting that the function of Def1p in telomere maintenance is specific. Our novel findings of the functions of Def1p in telomere and mitochondria suggested that Def1p plays multiple roles in yeast.     

Structural and SAXS analysis of the budding yeast SHU-complex proteins

In Saccharomyces cerevisiae, four proteins, Shu1, Shu2, Psy3 and Csm2, form a stable SHU-complex both in vivo and in vitro. These proteins are involved in the early stages of the homologous recombination DNA damage repair process. In this paper, the crystal structure of the Psy3–Csm2 sub-complex is presented at 1.8 Å resolution and successfully fitted into our small angle X-ray scattering (SAXS) data of the SHU-complex. Taken together with our electrophoretic mobility shift assay (EMSA) results, a model is proposed for the SHU–protein complex coupled with DNA.Structured summary of protein interactions:PSY3 and CSM2 bind by X-ray crystallography (View interaction) PSY3, CSM2, Shu 1 and Shu 2 physically interact by x ray scattering (View interaction)     

The telomeric 5′ end nucleotide is regulated in the budding yeast Naumovozyma castellii

The junction between the double-stranded and single-stranded telomeric DNA (ds–ss junction) is fundamental in the maintenance of the telomeric chromatin, as it directs the assembly of the telomere binding proteins. In budding yeast, multiple Rap1 proteins bind the telomeric dsDNA, while ssDNA repeats are bound by the Cdc13 protein. Here, we aimed to determine, for the first time, the telomeric 5′ end nucleotide in a budding yeast. To this end, we developed a permutation-specific PCR-based method directed towards the regular 8-mer telomeric repeats in Naumovozyma castellii. We find that, in logarithmically growing cells, the 320 ± 30 bp long telomeres mainly terminate in either of two specific 5′ end permutations of the repeat, both corresponding to a terminal adenine nucleotide. Strikingly, two permutations are completely absent at the 5′ end, indicating that not all ds‐ss junction structures would allow the establishment of the protective telomere chromatin cap structure. Using in vitro DNA end protection assays, we determined that binding of Rap1 and Cdc13 around the most abundant ds–ss junction ensures the protection of both 5′ ends and 3′ overhangs from exonucleolytic degradation. Our results provide mechanistic insights into telomere protection, and reveal that Rap1 and Cdc13 have complementary roles.     

MRX protects telomeric DNA at uncapped telomeres of budding yeast cdc13-1 mutants

MRX, an evolutionally conserved DNA damage response complex composed of Mre11, Rad50 and Xrs2, is involved in DNA double strand break (DSB) repair, checkpoint activation and telomere maintenance. At DSBs, MRX plays a role in generating single stranded DNA (ssDNA) and signalling cell cycle arrest. Here we investigated whether MRX also contributes to generating ssDNA or signalling cell cycle arrest at uncapped telomeres. To investigate the role of MRX, we generated a conditionally degradable Rad50 protein and combined this with cdc13-1, a temperature sensitive mutation in the Cdc13 telomere capping protein. We show that Rad50 does not contribute to ssDNA generation or cell cycle arrest in response to cdcl3-1 uncapped telomeres. Instead, we find that Rad50 inhibits ssDNA accumulation and promotes cdc13-1 cell viability, consistent with a major role for MRX in telomere capping.     

Tel2p, a regulator of yeast telomeric length in vivo, binds to single-stranded telomeric DNA in vitro

The telomeres of the yeast Saccharomyces cerevisiae consist of a duplex region of TG_1–3 repeats that acquire a single-stranded 3’ extension of the TG_1–3 strand at the end of S-phase. The length of these repeats is kept within a defined range by regulators such as the TEL2-encoded protein (Tel2p). Here we show that Tel2p can specifically bind to single-stranded TG_1–3. Tel2p binding produced several shifted bands; however, only the slowest migrating band contained Tel2p. Methylation protection and interference experiments as well as gel shift experiments using inosine-containing probes indicated that the faster migrating bands resulted from Tel2p-mediated formation of DNA secondary structures held together by G-G interactions. Tel2p bound to single-stranded substrates that were at least 19 bases in length and contained 14 bases of TG_1–3, and also to double-stranded/single-stranded hybrid substrates with a 3’ TG_1–3 overhang. Tel2p binding to a hybrid substrate with a 24 base single-stranded TG_1–3 extension also produced a band characteristic of G-G-mediated secondary structures. These data suggest that Tel2p could regulate telomeric length by binding to the 3’ single-stranded TG_1–3 extension present at yeast telomeres. Received: 12 November 1998; in revised form: 6 April 1999 / Accepted: 13 April 1999     

A cytosolic NAD-dependent deacetylase, Hst2p, can modulate nucleolar and telomeric silencing in yeast

In budding yeast, the silent information regulator Sir2p is a nuclear NAD-dependent deacetylase that is essential for both telomeric and rDNA silencing. All eukaryotic species examined to date have multiple homologues of Sir two (HSTs), which share a highly conserved globular core domain. Here we report that yeast Hst2p and a mammalian Hst2p homologue, hSirT2p, are cytoplasmic in yeast and human cells, in contrast to yHst1p and ySir2p which are exclusively nuclear. Although yHst2p cannot restore silencing in a sir2 deletion, overexpression of yHst2p influences nuclear silencing events in a SIR2 strain, derepressing subtelomeric silencing while increasing repression in the rDNA. In contrast, a form of ySir2p carrying a point mutation in the conserved core domain disrupts both telomeric position effect (TPE) and rDNA repression at low expression levels. This argues that non-nuclear yHst2p can compete for a substrate or ligand specifically required for telomeric, and not rDNA repression.     

Nickel enhances telomeric silencing in Saccharomyces cerevisiae

Certain nickel compounds including crystalline nickel sulfide (NiS) and subsulfide (Ni3S2) are potent human and animal carcinogens. In Chinese hamster embryo cells, an X-linked senescence gene was inactivated following nickel-induced DNA methylation. Nickel also induced the inactivation of the gpt reporter gene by chromatin condensation and a DNA methylation process in a transgenic gpt+ Chinese hamster cell line (G12), which is located near a heterochromatic region. To determine if nickel can cause gene silencing independently of DNA methylation, based only on the induction of changes in chromatin structure, we measured its effect on gene silencing in Saccharomyces cerevisiae. Growth of yeast in the presence of nickel chloride repressed a telomeric marker gene (URA3) and resulted in a stable epigenetic switch. This phenomenon was dependent on the number of cell doubling prior to selection and also on the distance of the marker gene from the end of the chromosome. The level of TPE (telomeric position effect) increased linearly with elevations of nickel concentration. Addition of magnesium inhibited this effect, but magnesium did not silence the reporter gene by itself. The level of silencing was also assessed following treatment with other transition metals: cobalt, copper and cadmium. In the sublethal range, cobalt induced similar effects as nickel, while copper and cadmium did not change the basal level of gene expression. Silencing by copper and cadmium were evident only at concentrations of those metals where the viability was very low.     

The interaction networks of the budding yeast and human DNA replication-initiation proteins

DNA replication is a stringently regulated cellular process. In proliferating cells, DNA replication-initiation proteins (RIPs) are sequentially loaded onto replication origins during the M-to-G₁ transition to form the pre-replicative complex (pre-RC), a process known as replication licensing. Subsequently, additional RIPs are recruited to form the pre-initiation complex (pre-IC). RIPs and their regulators ensure that chromosomal DNA is replicated exactly once per cell cycle. Origin recognition complex (ORC) binds to, and marks replication origins throughout the cell cycle and recruits other RIPs including Noc3p, Ipi1-3p, Cdt1p, Cdc6p and Mcm2-7p to form the pre-RC. The detailed mechanisms and regulation of the pre-RC and its exact architecture still remain unclear. In this study, pairwise protein-protein interactions among 23 budding yeast and 16 human RIPs were systematically and comprehensively examined by yeast two-hybrid analysis. This study tested 470 pairs of yeast and 196 pairs of human RIPs, from which 113 and 96 positive interactions, respectively, were identified. While many of these interactions were previously reported, some were novel, including various ORC and MCM subunit interactions, ORC self-interactions, and the interactions of IPI3 and NOC3 with several pre-RC and pre-IC proteins. Ten of the novel interactions were further confirmed by co-immunoprecipitation assays. Furthermore, we identified the conserved interaction networks between the yeast and human RIPs. This study provides a foundation and framework for further understanding the architectures, interactions and functions of the yeast and human pre-RC and pre-IC.     

Gene activation at a distance and telomeric silencing are not affected by yeast histone H1

Until recently, it was believed that the budding yeast Saccharomyces cerevisiae has no histone H1 gene. However, a search of the yeast genome database revealed a possible H1 homologue of 258 amino acids, termed yeast histone H1 (HHO1). The protein shows 36% identity to the human H1 core domain over a stretch of 93 amino acids. Unlike other H1 proteins, Hho1p has a second possible core domain which shows 43% identity to the first core domain. Since vertebrate H1 histone had been implied in gene repression as well as gene activation at a distance, we tested the effect of deleting the yeast H1-like gene on remote activation of a modified GAL1 promoter, which contains a synthetic GAL4 binding site close to the TATA box, and the natural UAS_G, consisting of four GAL4 binding sites. Different spacing up to 1.8 kb between the proximal binding site and the distal UAS_G enhancer revealed no differences in gene activation between wild-type and knockout strains. Overexpression of a heterologous histone H1 from sea urchin showed an overall inhibition of gene activation by the GAL1 promoter, whereas overexpression of the yeast histone H1 had no effect. Also, the expression of A1, ALPHA2 or SUC2 genes, all of which are known to be responsive to an altered chromatin structure, was unchanged in HHO1 knockout or HHO1-overexpressing strains when compared to wild-type cells. We also considered the possibility that HHO1 was involved in forming the heterochromatin at telomeres. On testing for telomeric silencing of a URA reporter gene introduced 1.3 kb away from the chromosomal end, we again observed no differences between wild-type and knockout strains. Thus, the yeast histone H1-like gene appears to have no role in gene activation at a distance or in silencing under the conditions tested. It remains to be seen whether the yeast H1 histone is a gene-specific regulator rather than a general chromatin-associated protein. Received: 16 April 1997 / Accepted: 4 July 1997     

Clathrin-mediated endocytosis in budding yeast

Clathrin-mediated endocytosis in the budding yeast Saccharomyces cerevisiae involves the ordered recruitment, activity and disassembly of nearly 60 proteins at distinct sites on the plasma membrane. Two-color live-cell fluorescence microscopy has proven to be invaluable for in vivo analysis of endocytic proteins: identifying new components, determining the order of protein arrival and dissociation, and revealing even very subtle mutant phenotypes. Yeast genetics and functional genomics facilitate identification of complex interaction networks between endocytic proteins and their regulators. Quantitative datasets produced by these various analyses have made theoretical modeling possible. Here, we discuss recent findings on budding yeast endocytosis that have advanced our knowledge of how -60 endocytic proteins are recruited, perform their functions, are regulated by lipid and protein modifications, and are disassembled, all with remarkable regularity.