RESUMO
We identified two new Saccharomyces cerevisiae kinesin-related genes, KIP1 and KIP2, using polymerase chain reaction primers corresponding to highly conserved regions of the kinesin motor domain. Both KIP proteins are expressed in vivo, but deletion mutations conferred no phenotype. Moreover, kip1 kip2 double mutants and a triple mutant with kinesin-related kar3 had no synthetic phenotype. Using a genetic screen for mutations that make KIP1 essential, we identified another gene, KSL2, which proved to be another kinesin-related gene, CIN8. KIP1 and CIN8 are functionally redundant: double mutants arrested in mitosis whereas the single mutants did not. The microtubule organizing centers of arrested cells were duplicated but unseparated, indicating that KIP1 or CIN8 is required for mitotic spindle assembly. Consistent with this role, KIP1 protein was found to colocalize with the mitotic spindle.
Assuntos
Cinesinas/metabolismo , Família Multigênica/genética , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Fuso Acromático/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Imuno-Histoquímica , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Proteínas Motores Moleculares , Dados de Sequência Molecular , Morfogênese , Mutagênese , Mutação , Fenótipo , Saccharomyces cerevisiae/genética , Homologia de Sequência do Ácido Nucleico , Relação Estrutura-AtividadeRESUMO
To investigate the relationship between structure and function of kinesin-like proteins, we have identified by polymerase chain reaction (PCR) a new kinesin-like protein in the filamentous fungus Aspergillus nidulans, which we have designated KLPA. DNA sequence analysis showed that the predicted KLPA protein contains a COOH terminal kinesin-like motor domain. Despite the structural similarity of KLPA to the KAR3 and NCD kinesin-like proteins of Saccharomyces cerevisiae and Drosophila melanogaster, which also posses COOH-terminal kinesin-like motor domains, there are no significant sequence similarities between the nonmotor or tail portions of these proteins. Nevertheless, expression studies in S. cerevisiae showed that klpA can complement a null mutation in KAR3, indicating that primary amino acid sequence conservation between the tail domains of kinesin-like proteins is not necessarily required for conserved function. Chromosomal deletion of the klpA gene exerted no observable mutant phenotype, suggesting that in A. nidulans there are likely to be other proteins functionally redundant with KLPA. Interestingly, the temperature sensitive phenotype of a mutation in another gene, bimC, which encodes a kinesin-like protein involved in mitotic spindle function in A. nidulans, was suppressed by deletion of klpA. We hypothesize that the loss of KLPA function redresses unbalanced forces within the spindle caused by mutation in bimC, and that the KLPA and BIMC kinesin-like proteins may play opposing roles in spindle function.
Assuntos
Aspergillus nidulans/genética , Cinesinas/genética , Mitose , Sequência de Aminoácidos , Aspergillus nidulans/ultraestrutura , Sequência de Bases , Clonagem Molecular , Deleção de Genes , Expressão Gênica , Genes Fúngicos , Teste de Complementação Genética , Cinesinas/fisiologia , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos/química , Reação em Cadeia da Polimerase , RNA Fúngico/genética , RNA Mensageiro/genética , Mapeamento por Restrição , Fuso Acromático/ultraestruturaRESUMO
The MIF2 gene of Saccharomyces cerevisiae has been implicated in mitosis. Here we provide genetic evidence that MIF2 encodes a centromere protein. Specifically, we found that mutations in MIF2 stabilize dicentric minichromosomes and confer high instability (i.e., a synthetic acentric phenotype) to chromosomes that bear a cis-acting mutation in element I of the yeast centromeric DNA (CDEI). Similarly, we observed synthetic phenotypes between mutations in MIF2 and trans-acting mutations in three known yeast centromere protein genes-CEP1/CBF1/CPF1, NDC10/CBF2, and CEP3/CBF3B. In addition, the mif2 temperature-sensitive phenotype can be partially rescued by increased dosage of CEP1. Synthetic lethal interactions between a cep1 null mutation and mutations in either NDC10 or CEP3 were also detected. Taken together, these data suggest that the Mif2 protein interacts with Cep1p at the centromere and that the yeast centromere indeed exists as a higher order protein-DNA complex. The Mif2 and Cep1 proteins contain motifs of known transcription factors, suggesting that assembly of the yeast centromere is analogous to that of eukaryotic enhancers and origins of replication. We also show that the predicted Mif2 protein shares two short regions of homology with the mammalian centromere Ag CENP-C and that two temperature-sensitive mutations in MIF2 lie within these regions. These results provide evidence for structural conservation between yeast and mammalian centromeres.
Assuntos
Centrômero/química , Proteínas Cromossômicas não Histona/genética , Proteínas Fúngicas/genética , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Sequência de Aminoácidos , Animais , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos , Proteínas Cromossômicas não Histona/química , Cromossomos Fúngicos , DNA Fúngico/genética , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Genes Fúngicos/genética , Genes Supressores/genética , Humanos , Cinetocoros , Camundongos , Dados de Sequência Molecular , Mutação , Proteínas Nucleares/genética , Saccharomyces cerevisiae/química , Homologia de Sequência de Aminoácidos , TemperaturaRESUMO
We showed recently that a complex between Duo1p and Dam1p is required for both spindle integrity and kinetochore function in the budding yeast Saccharomyces cerevisiae. To extend our understanding of the functions and interactions of the Duo1p/Dam1p complex, we analyzed the novel gene product Dad1p (for Duo1 and Dam1 interacting). Dad1p physically associates with Duo1p by two-hybrid analysis, coimmunoprecipitates with Duo1p and Dam1p out of yeast protein extracts, and shows interdependent localization with Duo1p and Dam1p to the mitotic spindle. These results indicate that Dad1p functions as a component of the Duo1p/Dam1p complex. Like Duo1p and Dam1p, Dad1p also localizes to kinetochore regions in chromosomes spreads. Here, we also demonstrate by chromatin immunoprecipitation that Duo1p, Dam1p, and Dad1p associate specifically with centromeric DNA in a manner that is dependent upon Ndc10 and partially dependent upon the presence of microtubules. To explore the functions of Dad1p in vivo, we generated a temperature-sensitive allele, dad1-1. This allele shows spindle defects and a mitotic arrest phenotype that is dependent upon the spindle assembly checkpoint. In addition, dad1-1 mutants undergo chromosome mis-segregation at the restrictive temperature, resulting in a dramatic decrease in viability.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Cinetocoros/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/metabolismo , Fuso Acromático/metabolismo , Alelos , Sequência de Aminoácidos , Proteínas de Ciclo Celular/genética , Cromatina/metabolismo , Segregação de Cromossomos/efeitos dos fármacos , Proteínas do Citoesqueleto , Epistasia Genética , Regulação Fúngica da Expressão Gênica , Cinetocoros/efeitos dos fármacos , Microscopia de Fluorescência , Proteínas Associadas aos Microtúbulos/genética , Dados de Sequência Molecular , Mutação/genética , Nocodazol/farmacologia , Testes de Precipitina , Ligação Proteica , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/genética , Homologia de Sequência de Aminoácidos , Fuso Acromático/efeitos dos fármacos , Temperatura , Técnicas do Sistema de Duplo-HíbridoAssuntos
Cromatina/ultraestrutura , DNA Fúngico/isolamento & purificação , Saccharomyces cerevisiae/ultraestrutura , Fracionamento Celular/métodos , Centrômero/química , Centrômero/ultraestrutura , Cromatina/química , Cromatina/genética , DNA Fúngico/química , Genoma Fúngico , Indicadores e Reagentes , Testes de Precipitina/métodos , Saccharomyces cerevisiae/genética , Esferoplastos/ultraestruturaRESUMO
The KAR3 gene is essential for yeast nuclear fusion during mating, and its expression is strongly induced by alpha factor. The predicted KAR3 protein sequence contains two globular domains separated by an alpha-helical coiled coil. The carboxy-terminal domain is homologous to the amino-terminal mechanochemical domain of Drosophila kinesin heavy chain. Mutation of the putative ATP binding site produces a dominant "poison" of nuclear fusion. The mutant protein shows enhanced microtubule association in vivo, as predicted for a kinesin-like protein in a state of rigor binding. Localization of hybrid proteins to cytoplasmic microtubules in shmoos indicates that the amino-terminal domain also contains determinants for microtubule association. Thus, KAR3 is a member of a larger family of kinesin-like proteins characterized by the presence of the mechanochemical domain tethered to different protein binding domains. The phenotypes of kar3 mutants suggest that the protein mediates microtubule sliding during nuclear fusion and possibly mitosis.
Assuntos
Adenosina Trifosfatases/genética , Núcleo Celular/ultraestrutura , Proteínas Fúngicas/genética , Genes Fúngicos , Proteínas Associadas aos Microtúbulos , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Sítios de Ligação , Proteínas Fúngicas/metabolismo , Teste de Complementação Genética , Genótipo , Cinesinas , Dados de Sequência Molecular , Mutação , Fenótipo , Plasmídeos , Conformação Proteica , RNA Mensageiro/genética , Mapeamento por Restrição , Saccharomyces cerevisiae/citologia , Homologia de Sequência do Ácido NucleicoRESUMO
The centromere-kinetochore complex is a specialized chromatin structure that mediates bipolar attachment of replicated chromosomes to the mitotic spindle, thereby ensuring proper sister chromatid separation during anaphase. The manner in which this important multimeric structure is specified and assembled within chromatin is unknown. Using in vivo cross-linking followed by immunoprecipitation, we show that the Mif2 protein of the budding yeast Saccharomyces cerevisiae, previously implicated in centromere function by genetic criteria, resides specifically at centromeric loci in vivo. This provides definitive evidence for structural conservation between yeast and mammalian centromeres, as Mif2p shares homology with CENP-C, a mammalian centromere protein. Ndc10p and Cbf1p, previously implicated in centromere function by genetic and in vitro biochemical assays, were also found to interact with centromeric DNA in vivo. By examining Mif2p, Ndc10p, and Cbf1p association with centromeric DNA derivatives, we demonstrate the existence of centromeric subcomplexes that may correspond to assembly intermediates. Based on these observations, we provide a simple model for centromere assembly. Finally, given the sensitivity of this technique, its application to other sequence-specific protein-DNA complexes within the cell, such as origins of replication and enhancer-promoter regions, could be of significant value.
Assuntos
Centrômero/metabolismo , Proteínas Fúngicas/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos , Reagentes de Ligações Cruzadas , DNA Fúngico/metabolismo , Proteínas de Ligação a DNA/metabolismo , Técnica Indireta de Fluorescência para Anticorpo , Cinetocoros , Proteínas Nucleares/metabolismo , Testes de PrecipitinaRESUMO
Histones are fundamental structural components of chromatin and are expected to play important roles in chromosome dynamics. Here, we present direct evidence that Cse4p, a histone H3 variant, is a structural component of the core centromere of S. cerevisiae. In histone H4 and Cse4p mutants, the core centromere chromatin structure is disrupted at restrictive temperature. Overexpression of Cse4p suppresses this defect in the H4 mutant, implying that the two proteins act together in centromere structure. We show by chromatin immunoprecipitation experiments that Cse4p is specifically cross-linked to centromeric DNA. Furthermore, by immunofluorescence microscopy, Cse4p is found in discrete foci consistent with that expected for centromeres. These results suggest the kinetochore is assembled on a specialized centromeric nucleosome containing Cse4p.