RESUMEN
Mutants of the yeast Kar3 protein are defective in nuclear fusion, or karyogamy, during mating and show slow mitotic growth, indicating a requirement for the protein both during mating and in mitosis. DNA sequence analysis predicts that Kar3 is a microtubule motor protein related to kinesin, but with the motor domain at the C-terminus of the protein rather than the N-terminus as in kinesin heavy chain. We have expressed Kar3 as a fusion protein with glutathione S-transferase (GST) and determined the in vitro motility properties of the bacterially expressed protein. The GST-Kar3 fusion protein bound to a coverslip translocates microtubules in gliding assays with a velocity of 1-2 microns/min and moves towards microtubule minus ends, unlike kinesin but like kinesin-related Drosophila ncd. Taxol-stabilized microtubules bound to GST-Kar3 on a coverslip shorten as they glide, resulting in faster lagging end, than leading end, velocities. Comparison of lagging and leading end velocities with velocities of asymmetrical axoneme-microtubule complexes indicates that microtubules shorten preferentially from the lagging or minus ends. The minus end-directed translocation and microtubule bundling of GST-Kar3 is consistent with models in which the Kar3 protein crosslinks internuclear microtubules and mediates nuclear fusion by moving towards microtubule minus ends, pulling the two nuclei together. In mitotic cells, the minus end motility of Kar3 could move chromosomes polewards, either by attaching to kinetochores and moving them polewards along microtubules, or by attaching to kinetochore microtubules and pulling them polewards along other polar microtubules.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Proteínas Fúngicas/metabolismo , Proteínas de Microtúbulos/metabolismo , Proteínas Asociadas a Microtúbulos , Microtúbulos/fisiología , Proteínas de Saccharomyces cerevisiae , Adenosina Trifosfato/farmacología , Proteínas Fúngicas/genética , Guanosina Trifosfato/farmacología , Microscopía de Interferencia/métodos , Proteínas de Microtúbulos/genética , Microtúbulos/efectos de los fármacos , Modelos Biológicos , Paclitaxel , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
The Kar3 protein (Kar3p), a protein related to kinesin heavy chain, and the Cik1 protein (Cik1p) appear to participate in the same cellular processes in S. cerevisiae. Phenotypic analysis of mutants indicates that both CIK1 and KAR3 participate in spindle formation and karyogamy. In addition, the expression of both genes is induced by pheromone treatment. In vegetatively growing cells, both Cik1::beta-gal and Kar3::beta-gal fusions localize to the spindle pole body (SPB), and after pheromone treatment both fusion proteins localize to the spindle pole body and cytoplasmic microtubules. The dependence of Cik1p and Kar3p localization upon one another was investigated by indirect immunofluorescence of fusion proteins in pheromone-treated cells. The Cik1p::beta-gal fusion does not localize to the SPB or microtubules in a kar3 delta strain, and the Kar3p::beta-gal fusion protein does not localize to microtubule-associated structures in a cik1 delta strain. Thus, these proteins appear to be interdependent for localization to the SPB and microtubules. Analysis by both the two-hybrid system and co-immunoprecipitation experiments indicates that Cik1p and kar3p interact, suggesting that they are part of the same protein complex. These data indicate that interaction between a putative kinesin heavy chain-related protein and another protein can determine the localization of motor activity and thereby affect the functional specificity of the motor complex.
Asunto(s)
Proteínas Fúngicas/metabolismo , Proteínas de Microtúbulos , Proteínas Asociadas a Microtúbulos , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Citoplasma/metabolismo , Técnica del Anticuerpo Fluorescente , Proteínas Fúngicas/genética , Inmunohistoquímica , Cariotipificación , Microtúbulos/metabolismo , Fenotipo , Feromonas , Pruebas de PrecipitinaRESUMEN
The last year has seen dramatic progress in the use of genetic and biochemical approaches to identify microtubule-organizing center components. The use of vertebrate and invertebrate egg extracts has allowed the development of novel assays for centrosome duplication and activation. A variety of mutations in fungi are being used to sort out the pathway of spindle pole body duplication.
Asunto(s)
Microtúbulos/ultraestructura , Huso Acromático/ultraestructura , Animales , División Celular , Ciclinas/metabolismo , Células Eucariotas/ultraestructura , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Hongos/genética , Hongos/ultraestructura , Invertebrados/genética , Invertebrados/metabolismo , Proteínas de Microtúbulos/genética , Proteínas de Microtúbulos/metabolismo , Mitosis , Fosforilación , Procesamiento Proteico-PostraduccionalRESUMEN
To understand how cytokinesis is regulated during mitosis, we tested cyclin-p34cdc2 for myosin-II kinase activity, and investigated the mitotic-specific phosphorylation of myosin-II in lysates of Xenopus eggs. Purified cyclin-p34cdc2 phosphorylated the regulatory light chain of cytoplasmic and smooth muscle myosin-II in vitro on serine-1 or serine-2 and threonine-9, sites known to inhibit the actin-activated myosin ATPase activity of smooth muscle and nonmuscle myosin (Nishikawa, M., J. R. Sellers, R. S. Adelstein, and H. Hidaka. 1984. J. Biol. Chem. 259:8808-8814; Bengur, A. R., A. E. Robinson, E. Appella, and J. R. Sellers. 1987. J. Biol. Chem. 262:7613-7617; Ikebe, M., and S. Reardon. 1990. Biochemistry. 29:2713-2720). Serine-1 or -2 of the regulatory light chain of Xenopus cytoplasmic myosin-II was also phosphorylated in Xenopus egg lysates stabilized in metaphase, but not in interphase. Inhibition of myosin-II by cyclin-p34cdc2 during prophase and metaphase could delay cytokinesis until chromosome segregation is initiated and thus determine the timing of cytokinesis relative to earlier events in mitosis.
Asunto(s)
Proteína Quinasa CDC2/metabolismo , Ciclinas/metabolismo , Miosinas/metabolismo , Secuencia de Aminoácidos , Animales , Ciclo Celular , División Celular , Electroforesis en Gel de Poliacrilamida , Factor Promotor de Maduración/metabolismo , Datos de Secuencia Molecular , Miosinas/antagonistas & inhibidores , Fosforilación , Especificidad por Sustrato , Factores de Tiempo , XenopusRESUMEN
The actomyosin contractile-ring mechanism remains the paradigm for cytokinesis after 20 years of experimental testing. Recent evidence suggests that Ca2+ triggers the contraction and that cell-cycle kinases regulate the timing of cytokinesis. New work is required to identify the signals from the mitotic spindle that specify the position of the furrow.