RESUMEN

The 26S proteasome is the multiprotein complex that degrades proteins that have been marked for destruction by the ubiquitin pathway. It is made up of two multisubunit complexes, the 20S catalytic core and the 19S regulatory complex. We describe the isolation and characterization of conditional mutants in the regulatory complex and their use to investigate interactions between different subunits. In addition we have investigated the localization of the 26S proteasome in fission yeast, by immunofluorescence in fixed cells and live cells with the use of a GFP-tagged subunit. Surprisingly, we find that in mitotic cells the 26S proteasome occupies a discrete intracellular compartment, the nuclear periphery. Electron microscopic analysis demonstrates that the complex resides inside the nuclear envelope. During meiosis the localization showed a more dynamic distribution. In meiosis I the proteasome remained around the nuclear periphery. However, during meiosis II there was a dramatic relocalization: initially, the signal occupied the area between the dividing nuclei, but at the end of mitosis the signal dispersed, returning to the nuclear periphery on ascospore formation. This observation implies that the nuclear periphery is a major site of proteolysis in yeast during mitotic growth and raises important questions about the function of the 26S proteasome in protein degradation.

Asunto(s)

Péptido Hidrolasas/metabolismo , Complejo de la Endopetidasa Proteasomal , Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces/enzimología , Animales , Núcleo Celular/metabolismo , Proteínas Fúngicas/metabolismo , Proteínas Fluorescentes Verdes , Proteínas Luminiscentes/genética , Meiosis , Mitosis , Mutación , Schizosaccharomyces/genética , Especificidad por Sustrato , Temperatura , Transactivadores/metabolismo

RESUMEN

Fission yeast centromeres are transcriptionally silent and form a heterochromatin-like structure essential for normal centromere function; this appears analogous to heterochromatin and position effect variegation in other eukaryotes. Conditional mutations in three genes designated cep (centromere enhancer of position effect) were found to enhance transcriptional silencing within centromeres. Cloning of the cep1(+) and cep2(+) genes by functional complementation revealed that they are identical to the previously described genes pad1(+) and mts2(+), respectively, which both encode subunits of the proteasome 19S cap. Like Mts2 and Mts4, epitope-tagged Cep1/Pad1 localizes to or near the nuclear envelope throughout the cell cycle. The cep mutants display a range of phenotypes depending on the temperature. Silencing within the central domain of centromeres is increased at 36 degrees C. This suggests that the proteasome is involved in regulating silencing and thus centromeric chromatin architecture, possibly by lowering the level of some chromatin-associated protein by ubiquitin-dependent degradation. This is the first report of defective proteasome function affecting heterochromatin-mediated transcriptional silencing. At 36 and 32 degrees C, the cep mutants lose chromosomes at an elevated rate, and at 18 degrees C, the mutants are cryosensitive for growth. Cytological analysis at 18 degrees C revealed a defect in sister chromatid separation while other mitotic events occurred normally, indicating that cep mutations might interfere specifically with the degradation of inhibitor(s) of sister chromatid separation. These observations suggest that 19S subunits confer a level of substrate specificity on the proteasome and raise the possibility of a link between components involved in centromere architecture and sister chromatid cohesion.

Asunto(s)

Centrómero , Segregación Cromosómica , Cisteína Endopeptidasas/metabolismo , Proteínas de Unión al ADN/genética , Proteínas Fúngicas/genética , Regulación Fúngica de la Expresión Génica , Complejos Multienzimáticos/metabolismo , Proteínas Nucleares/genética , Proteínas de Saccharomyces cerevisiae , Schizosaccharomyces/genética , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice , Proteínas de Unión al ADN/metabolismo , Elementos de Facilitación Genéticos , Proteínas Fúngicas/metabolismo , Genes Fúngicos , Cinetocoros , Mitosis , Mutagénesis , Complejo de la Endopetidasa Proteasomal , Temperatura , Transcripción Genética

RESUMEN

We have isolated a conditional lethal mutant mts3 in the fission yeast Schizosaccharomyces pombe which at the permissive temperature is resistant to the mitotic poison MBC and at the restrictive temperature is defective in metaphase to anaphase transition. The predicted amino acid sequence of mts3+ is 36% identical with the budding yeast gene NIN1. NIN1 cloned into a fission yeast expression vector can rescue both mts3 temperature-sensitive and null alleles demonstrating that NIN1 is the budding yeast homologue of the fission yeast mts3+ gene. The phenotype of the mts3 null is identical with the mts3 ts mutant demonstrating that the phenotype of the mts3 ts mutant is due to loss of mts3+ function. The deduced amino acid sequences of both mts3+ and NIN1 show homology to peptide sequences obtained from subunit 14 of the 26 S protease purified from bovine or human cells.

Asunto(s)

Ciclo Celular/genética , Proteínas Fúngicas/genética , Genes Fúngicos , Mutación , Péptido Hidrolasas/genética , Complejo de la Endopetidasa Proteasomal , Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces/genética , Secuencia de Aminoácidos , Anafase , Secuencia de Bases , Clonación Molecular , Cartilla de ADN , ADN Complementario , Citometría de Flujo , Biblioteca de Genes , Genes Letales , Cinética , Metafase , Datos de Secuencia Molecular , Péptido Hidrolasas/metabolismo , Fenotipo , Reacción en Cadena de la Polimerasa , Protamina Quinasa/metabolismo , Schizosaccharomyces/citología , Schizosaccharomyces/enzimología , Homología de Secuencia de Aminoácido , Temperatura

Asunto(s)

Proteínas Bacterianas/genética , Endorribonucleasas , Escherichia coli/genética , Genes Bacterianos/genética , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Secuencia de Bases , Datos de Secuencia Molecular

RESUMEN

We have isolated a mutant, mts2, in the fission yeast Schizosaccharomyces pombe which is defective in chromosome segregation. The predicted amino-acid sequence of the cloned mts2+ gene product is 75% identical to the S4 subunit of the human 26S ATP/ubiquitin-dependent protease. The human S4 subunit complementary DNA expressed from an S. pombe expression plasmid can rescue an S. pombe mts2 gene disruption. Both observations demonstrate that the mts2+ gene is the S. pombe homologue of the human S4 subunit. In addition, we provide genetic evidence for a physical interaction between the S4 and the related S7 subunit in the 26S multiprotein protease. We show that polyubiquitin-conjugated proteins accumulate in the mts2 mutant at the restrictive temperature, demonstrating that the mutant has an in vivo defect in the ubiquitin-dependent proteolysis pathway. Finally, the phenotype for the mts2 mutant indicates that protein degradation by the 26S protease is essential not for entry into but for the completion of mitosis.

Asunto(s)

Endopeptidasas/genética , Genes Fúngicos/fisiología , Mitosis/genética , Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces/genética , Secuencia de Aminoácidos , Endopeptidasas/fisiología , Prueba de Complementación Genética , Células HeLa , Humanos , Mitosis/efectos de los fármacos , Datos de Secuencia Molecular , Mutación , Schizosaccharomyces/citología , Schizosaccharomyces/enzimología , Homología de Secuencia de Aminoácido , Temperatura

RESUMEN

We have used an antibody to a previously identified 180 kDa (Hmp1) protein in Escherichia coli to clone the corresponding gene, which encodes a polypeptide of 114 kDa that has a mobility equivalent to 180 kDa in SDS/PAGE. We have demonstrated that the 180 kDa polypeptide is the primary gene product and not due to aggregation with other molecules. Moreover, our data indicate that the highly charged C-terminal region of the protein is responsible for its anomalous behaviour when analysed by SDS/PAGE. The hmp1 gene is in fact identical to ams (abnormal mRNA stability), also designated rne (RnaseE), and reported to have an ORF of 91 kDa. This discrepancy with the data in this paper can be ascribed to the omission of two bases in the previously reported sequence, generating an apparent stop codon. We previously demonstrated that the 180 kDa Hmp1/Ams protein cross reacted with both a polyclonal antibody and a monoclonal antibody raised against a yeast heavy chain myosin. However, we could detect no homology with myosin genes in the ams/hmp1 sequence. From the DNA sequence data, we identified a putative nucleotide binding site and a transmembrane domain in the N-terminal half of the molecule. In the C-terminal half, which appears to constitute a separate domain dominated by proline and charged amino acids, we also identified a region homologous to the highly conserved 70 kDa snRNP protein, involved in RNA splicing in eukaryotes. This feature would be consistent with reports that ams encodes RNaseE, an enzyme required for the processing of several stable RNAs in E. coli.

Asunto(s)

Proteínas Bacterianas/genética , Proteínas Cromosómicas no Histona , Endorribonucleasas , Proteínas de Escherichia coli , Escherichia coli/genética , Genes Bacterianos , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , ADN Bacteriano , Datos de Secuencia Molecular , Mapeo Restrictivo

RESUMEN

Calcium and calcium-binding proteins including those resembling calmodulin are implicated in numerous diverse processes in bacteria. These processes include chemotaxis, sporulation, virulence, the transport of sugars and proteins, phosphorylation, heat shock, the initiation of DNA replication, septation, nucleoid structure, nuclease activity and recombination, the stability of the envelope, and phospholipid synthesis and configuration. That such varied processes should have a common factor, calcium, suggests major underlying principles of calcium metabolism which have yet to be discovered.

Asunto(s)

Bacterias/metabolismo , Calcio/metabolismo , Proteínas de Unión al Calcio/metabolismo , Ciclo Celular , Pared Celular/metabolismo , Mutación

RESUMEN

This report is a final presentation of data collected in a drought monitoring partnership which was set up by UNICEF with the Centre for Social Research after recognizing that information on a regular basis was needed in order for the vulnerable sectors of society to be targeted effectively. The project operated primarily in the southern region and collected data on household incomes; food consumption and sources; water supplies as well as anthropometric measurements of children

Asunto(s)

Desnutrición , Ciencias de la Nutrición