RESUMO
We have identified a novel centromere-associated gene product from Saccharomyces cerevisiae that plays a role in spindle assembly and stability. Strains with a deletion of SLK19 (synthetic lethal Kar3p gene) exhibit abnormally short mitotic spindles, increased numbers of astral microtubules, and require the presence of the kinesin motor Kar3p for viability. When cells are deprived of both Slk19p and Kar3p, rapid spindle breakdown and mitotic arrest is observed. A functional fusion of Slk19p to green fluorescent protein (GFP) localizes to kinetochores and, during anaphase, to the spindle midzone, whereas Kar3p-GFP was found at the nuclear side of the spindle pole body. Thus, these proteins seem to play overlapping roles in stabilizing spindle structure while acting from opposite ends of the microtubules.
Assuntos
Centrômero/metabolismo , Proteínas Fúngicas/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/citologia , Fuso Acromático/metabolismo , Anáfase , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Proteínas Fúngicas/genética , Genes Letais , Cinesinas , Cinetocoros/metabolismo , Microscopia de Fluorescência , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Mitose , Mutação , Ácido Orótico/análogos & derivados , Fenótipo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMO
Three unlinked genes, TDH1, TDH2 and TDH3, encode the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (triose-phosphate dehydrogenase; TDH) in the yeast Saccharomyces cerevisiae. We demonstrate that the synthesis of the three encoded TDH polypeptides (TDHa, TDHb and TDHc, respectively) is not co-ordinately regulated and that TDHa is only synthesised as cells enter stationary phase, due to glucose starvation, or in heat-shocked cells. Furthermore, the synthesis of TDHb, but not TDHc, is strongly repressed by a heat shock. Hence, the TDHa enzyme may play a cellular role, distinct from glycolysis, that is required by stressed cells.
Assuntos
Genes Fúngicos , Gliceraldeído-3-Fosfato Desidrogenases/biossíntese , Saccharomyces cerevisiae/enzimologia , Autorradiografia , Eletroforese em Gel Bidimensional , Eletroforese em Gel de Poliacrilamida , Regulação Enzimológica da Expressão Gênica , Regulação Fúngica da Expressão Gênica , Gliceraldeído-3-Fosfato Desidrogenases/genética , Gliceraldeído-3-Fosfato Desidrogenases/isolamento & purificação , Metionina/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Radioisótopos de EnxofreRESUMO
Hsp26 is one of the major small heat-shock proteins (Hsp) of the yeast Saccharomyces cerevisiae, yet its cellular role remains to be discovered. To examine the cellular consequences of overexpression of Hsp26, the gene encoding this protein (HSP26) was overexpressed from a multicopy plasmid using either its own promoter or by coupling it to the efficient constitutive PGK promoter. The PGK promoter provided the opportunity to overexpress Hsp26 under non-stress conditions and such high level synthesis, prior to a lethal heat shock (50 degrees C), gave a small but reproducible elevation in thermotolerance. In transformed strains overexpressing Hsp26 under either stressed or non-stress conditions, the Hsp26 polypeptide was recovered almost exclusively as a high molecular weight aggregate. This high molecular weight aggregate (or heat-shock granule; HSG) was purified by differential centrifugation and sucrose gradient density centrifugation and shown, by electron microscopic analysis, to be of a uniform size (15-25 nm diameter). Analysis of the purified HSG demonstrated that it had a molecular weight of 550 kDa, yet contained no other integral polypeptides or other macromolecules.
Assuntos
Proteínas Fúngicas/metabolismo , Proteínas de Choque Térmico/metabolismo , Saccharomyces cerevisiae/fisiologia , Proteínas Fúngicas/biossíntese , Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica , Proteínas de Choque Térmico/biossíntese , Proteínas de Choque Térmico/genética , Temperatura Alta , Peso Molecular , Plasmídeos , Regiões Promotoras Genéticas , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoAssuntos
Proteínas de Choque Térmico , Saccharomyces cerevisiae/análise , Sequência de Aminoácidos , Animais , Endopeptidases/metabolismo , Proteínas de Choque Térmico/biossíntese , Proteínas de Choque Térmico/química , Proteínas de Choque Térmico/fisiologia , Temperatura Alta , Dados de Sequência Molecular , Peso Molecular , Saccharomyces cerevisiae/fisiologiaRESUMO
The nucleotide sequence of the Saccharomyces cerevisiae gene encoding a small heat-shock protein (Hsp26) has been determined. It reveals a 213-amino acid protein (27 kDa) that contains no methionine (Met) residues. Radiolabelling studies demonstrate the N-terminal Met residue is cleaved post-translationally. The Hsp26 amino acid sequence shows significant homology with both a range of eukaryotic small Hsps and with vertebrate alpha-crystallins. Particularly highly conserved among these proteins is a hydrophobic tetrapeptide sequence Gly-Val-Leu-Thr. These findings are discussed in relation to the structure and function of small Hsps.