RESUMEN
In the November 2001 issue of Developmental Cell, Vogel et al. describe that the budding yeast gamma-tubulin, Tub4p, is phosphorylated at a conserved tyrosine in G1 phase of the cell cycle. The results suggest that gamma-tubulin phosphorylation regulates the number and dynamics of microtubules.
Asunto(s)
Centro Organizador de los Microtúbulos/metabolismo , Microtúbulos/metabolismo , Proteínas de Saccharomyces cerevisiae , Tubulina (Proteína)/metabolismo , Ciclo Celular/fisiología , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Fosforilación , Tubulina (Proteína)/genéticaRESUMEN
Yeast spindle pole bodies (SPBs) duplicate once per cell cycle by a conservative mechanism resulting in a pre-existing 'old' and a newly formed SPB. The two SPBs of yeast cells are functionally distinct. It is only the SPB that migrates into the daughter cell, the bud, which carries the Bfa1p-Bub2p GTPase-activating protein (GAP) complex, a component of the spindle positioning checkpoint. We investigated whether the functional difference of the two SPBs correlates with the time of their assembly. We describe that in unperturbed cells the 'old' SPB always migrates into the bud. However, Bfa1p localization is not determined by SPB inheritance. It is the differential interaction of cytoplasmic microtubules with the mother and bud cortex that directs the Bfa1p-Bub2p GAP to the bud-ward-localized SPB. In response to defects of cytoplasmic microtubules to interact with the cell cortex, the Bfa1p-Bub2p complex binds to both SPBs. This may provide a mechanism to delay cell cycle progression when cytoplasmic microtubules fail to orient the spindle. Thus, SPBs are able to sense cytoplasmic microtubule properties and regulate the Bfa1p-Bub2p GAP accordingly.
Asunto(s)
Proteínas de Ciclo Celular , Proteínas Fúngicas/metabolismo , Proteínas de Saccharomyces cerevisiae , Antineoplásicos/farmacología , División Celular , Citoplasma/metabolismo , Proteínas del Citoesqueleto/metabolismo , Técnica del Anticuerpo Fluorescente Indirecta , Proteínas Activadoras de GTPasa/metabolismo , Genotipo , Proteínas Fluorescentes Verdes , Proteínas Luminiscentes/metabolismo , Microscopía Confocal , Microscopía Fluorescente , Microtúbulos/metabolismo , Modelos Biológicos , Nocodazol/farmacología , Fosfoproteínas/metabolismo , Plásmidos/metabolismo , Unión Proteica , Saccharomycetales/fisiología , Factores de Tiempo , Proteína Fluorescente RojaRESUMEN
Centrosomes of vertebrate cells and spindle pole bodies (SPBs) of fungi were first recognized through their ability to organize microtubules. Recent studies suggest that centrosomes and SPBs also have a function in the regulation of cell cycle progression, in particular in controlling late mitotic events. Regulators of mitotic exit and cytokinesis are associated with the SPB of budding and fission yeast. Elucidation of the molecular roles played by these regulators is helping to clarify the function of the SPB in controlling progression though mitosis.
Asunto(s)
Centrosoma/fisiología , Mitosis , Proteínas Tirosina Fosfatasas , Proteínas de Saccharomyces cerevisiae , Saccharomycetales/citología , Huso Acromático/fisiología , Animales , Proteínas de Ciclo Celular/metabolismo , División Celular , Nucléolo Celular/metabolismo , Proteínas Fúngicas/fisiología , Sustancias Macromoleculares , Proteínas de Unión al GTP Monoméricas/fisiologíaAsunto(s)
Proteínas de Saccharomyces cerevisiae , Saccharomycetales/química , Saccharomycetales/ultraestructura , Huso Acromático/ultraestructura , Técnicas del Sistema de Dos Híbridos , Anticuerpos/química , Proteínas Fúngicas/metabolismo , Glutatión Transferasa/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Modelos Biológicos , Proteínas Nucleares/metabolismo , Reacción en Cadena de la Polimerasa , Pruebas de Precipitina , Unión Proteica , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
Here, we show that the budding yeast proteins Ndc80p, Nuf2p, Spc24p and Spc25p interact at the kinetochore. Consistently, Ndc80p, Nuf2p, Spc24p and Spc25p associate with centromere DNA in chromatin immunoprecipitation experiments, and SPC24 interacts genetically with MCM21 encoding a kinetochore component. Moreover, although conditional lethal spc24-2 and spc25-7 cells form a mitotic spindle, the kinetochores remain in the mother cell body and fail to segregate the chromosomes. Despite this defect in chromosome segregation, spc24-2 and spc25-7 cells do not arrest in metaphase in response to checkpoint control. Furthermore, spc24-2 cells showed a mitotic checkpoint defect when microtubules were depolymerized with nocodazole, indicating that Spc24p has a function in checkpoint control. Since Ndc80p, Nuf2p and Spc24p are conserved proteins, it is likely that similar complexes are part of the kinetochore in other organisms.
Asunto(s)
Proteínas Fúngicas/metabolismo , Cinetocoros/metabolismo , Secuencia de Aminoácidos , Centrómero , Cromosomas Fúngicos , ADN de Hongos/metabolismo , Proteínas de Unión al ADN/aislamiento & purificación , Proteínas de Unión al ADN/metabolismo , Proteínas Fúngicas/aislamiento & purificación , Datos de Secuencia Molecular , Unión Proteica , Homología de Secuencia de AminoácidoRESUMEN
The budding yeast spindle pole body (SPB) not only organizes the astral and nuclear microtubules but is also associated with a number of cell-cycle regulators that control mitotic exit. Here, we describe that the core SPB component Nud1p is a key protein that functions in both processes. The astral microtubule organizing function of Nud1p is mediated by its interaction with the gamma-tubulin complex binding protein Spc72p. This function of Nud1p is distinct from its role in cell-cycle control: Nud1p binds the spindle checkpoint control proteins Bfa1p and Bub2p to the SPB, and is part of the mitotic exit network (MEN) in which it functions upstream of CDC15 but downstream of LTE1. In conditional lethal nud1-2 cells, the MEN component Tem1p, a GTPase, is mislocalized, whereas the kinase Cdc15p is still associated with the SPB. Thus, in nud1-2 cells the failure of Tem1p to interact with Cdc15p at the SPB probably prevents mitotic exit.
Asunto(s)
Proteínas del Citoesqueleto , Desoxirribonucleasas/fisiología , Factores de Intercambio de Guanina Nucleótido , Microtúbulos/metabolismo , Mitosis , Proteínas de Saccharomyces cerevisiae , Animales , Antineoplásicos/farmacología , Ciclo Celular , Proteínas de Ciclo Celular/metabolismo , Línea Celular , Núcleo Celular/metabolismo , Desoxirribonucleasas/genética , Citometría de Flujo , Proteínas Fúngicas/metabolismo , Proteínas de Unión al GTP/metabolismo , Proteínas Fluorescentes Verdes , Insectos , Proteínas Luminiscentes/metabolismo , Microscopía Electrónica , Microscopía Fluorescente , Proteínas de Unión al GTP Monoméricas/metabolismo , Mutación , Nocodazol/farmacología , Fenotipo , Fosforilación , Unión Proteica , Supresión Genética , Temperatura , Factores de Tiempo , Tubulina (Proteína)/metabolismo , Técnicas del Sistema de Dos Híbridos , ARNt MetiltransferasasRESUMEN
Bfa1p and Bub2p are spindle checkpoint proteins that likely have GTPase activation activity and are associated with the budding yeast spindle pole body (SPB). Here, we show that Bfa1p and Bub2p bind the Ras-like GTPase Tem1p, a component of the mitotic exit network, to the cytoplasmic face of the SPB that enters the bud, whereas the GDP/GTP exchange factor Lte1p is associated with the cortex of the bud. Migration of the SPB into the bud probably allows activation of Tem1p through Lte1p, thereby linking nuclear migration with mitotic exit. Since components of the Bub2p checkpoint are conserved in other organisms, we propose that the position of the SPB or mammalian centrosome controls the timing of mitotic exit.
Asunto(s)
Proteínas de Ciclo Celular , Proteínas del Citoesqueleto , Proteínas Fúngicas/metabolismo , Factores de Intercambio de Guanina Nucleótido , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Huso Acromático/metabolismo , Ciclo Celular , Núcleo Celular/metabolismo , Proteínas Fúngicas/genética , Microscopía Inmunoelectrónica , Microtúbulos/metabolismo , Mitosis , Proteínas de Unión al GTP Monoméricas/genética , Proteínas de Unión al GTP Monoméricas/metabolismo , Movimiento , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/genética , Intercambio de Cromátides HermanasRESUMEN
In budding yeast, microtubules are organized by the spindle pole body (SPB), which is embedded in the nuclear envelope via its central plaque structure. Here, we describe the identification of BBP1 in a suppressor screen with a conditional lethal allele of SPC29. Bbp1p was detected at the central plaque periphery of the SPB and bbp1-1 cells were found to be defective in SPB duplication. bbp1-1 cells extend their satellite into a duplication plaque like wild-type cells; however, this duplication plaque then fails to insert properly into the nuclear envelope and does not assemble a functional inner plaque. This function in SPB duplication is probably fulfilled by a stable complex of Bbp1p and Mps2p, a nuclear envelope protein that is also essential for duplication plaque insertion. In addition, we found that Bbp1p interacts with Spc29p and the half-bridge component Kar1p. These interactions are likely to play a role in connecting the SPB with the nuclear envelope and the central plaque with the half-bridge.
Asunto(s)
Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de Microtúbulos , Proteínas Nucleares/metabolismo , Proteínas de Saccharomyces cerevisiae , Huso Acromático/metabolismo , Supresión Genética , Inmunohistoquímica , Microscopía Electrónica , Mitosis/genética , Modelos Biológicos , Membrana Nuclear/metabolismo , Unión Proteica , Huso Acromático/genética , Levaduras/genética , Levaduras/metabolismoRESUMEN
Microtubule assembly is initiated in vivo by gamma-tubulin complexes. Cytoplasmic gamma-tubulin complexes are targeted to centrosomes or to other microtubule organizing centers (MTOCs) via a set of so called gamma-tubulin complex binding proteins (GTBPs) that probably interact with the conserved Spc97p/Spc98p protein family of gamma-tubulin complexes. In other cell types, gamma-tubulin complexes may initiate microtubule formation near chromosomes in a MTOC-independent manner. Recently, major advances have been achieved through the finding that gamma-tubulin, Spc97p and Spc98p form a conserved core that is probably responsible for microtubule nucleation, and by the discovery that a yeast GTBP is regulated in a cell-cycle-dependent manner and in response to an external signal. Furthermore, it was found that the small GTPase Ran in its GDP-bound state may promote spindle assembly. In addition, an essential function of gamma-tubulin in basal body duplication has been demonstrated in Paramecium.
Asunto(s)
Centrosoma/metabolismo , Microtúbulos/metabolismo , Tubulina (Proteína)/metabolismo , Animales , Centriolos/metabolismo , Humanos , Unión Proteica , Huso Acromático/metabolismo , Tubulina (Proteína)/química , Proteína de Unión al GTP ran/metabolismoAsunto(s)
Proteínas Fúngicas/aislamiento & purificación , Chaperonas Moleculares/aislamiento & purificación , Saccharomyces cerevisiae/genética , Secuencia de Bases , Cartilla de ADN , Regulación Fúngica de la Expresión Génica , Chaperonas Moleculares/genética , Datos de Secuencia Molecular , Plásmidos , Reacción en Cadena de la Polimerasa , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/genética , Saccharomyces cerevisiae/crecimiento & desarrolloRESUMEN
Group II chaperonins in the eukaryotic and archaeal cytosol assist in protein folding independently of the GroES-like cofactors of eubacterial group I chaperonins. Recently, the eukaryotic chaperonin was shown to cooperate with the hetero-oligomeric protein complex GimC (prefoldin) in folding actin and tubulins. Here we report the characterization of the first archaeal homologue of GimC, from Methanobacterium thermoautotrophicum. MtGimC is a hexamer of 87 kDa, consisting of two alpha and four beta subunits of high alpha-helical content that are predicted to contain extended coiled coils and represent two evolutionarily conserved classes of Gim subunits. Reconstitution experiments with MtGimC suggest that two subunits of the alpha class (archaeal Gimalpha and eukaryotic Gim2 and 5) form a dimer onto which four subunits of the beta class (archaeal Gimbeta and eukaryotic Gim1, 3, 4 and 6) assemble. MtGimalpha and beta can form hetero-complexes with yeast Gim subunits and MtGimbeta partially complements yeast strains lacking Gim1 and 4. MtGimC is a molecular chaperone capable of stabilizing a range of non-native proteins and releasing them for subsequent chaperonin-assisted folding. In light of the absence of Hsp70 chaperones in many archaea, GimC may fulfil an ATP-independent, Hsp70-like function in archaeal de novo protein folding.
Asunto(s)
Archaea/química , Chaperonas Moleculares/química , Chaperonas Moleculares/fisiología , Secuencia de Aminoácidos , Western Blotting , Dicroismo Circular , Proteínas HSP70 de Choque Térmico/química , Methanobacterium/química , Datos de Secuencia Molecular , Filogenia , Unión Proteica , Pliegue de Proteína , Estructura Terciaria de Proteína , Proteínas Recombinantes/química , Homología de Secuencia de Aminoácido , Factores de TiempoRESUMEN
In budding yeast microtubule organizing functions are provided by the spindle pole body (SPB), a multi-layered structure that is embedded in the nuclear envelope throughout the cell cycle. The SPB organizes the nuclear and cytoplasmic microtubules which are spatially and functionally distinct. Microtubule formation in yeast requires the Tub4p-complex, containing the gamma-tubulin Tub4p, and two additional proteins, the SPB components Spc97p and Spc98p. The Tub4p complex assembles in the cytoplasm and is then anchored to the sides of the SPB which organize microtubules. This is achieved by the binding of Spc97p and Spc98p to so-called gamma-tubulin complex binding proteins (GTBPs) at the SPB. Spc72p is the yeast GTBP at the cytoplasmic side of the SPB, while Spc110p is the nuclear GTBP. Both GTBPs control the number of Tub4p complexes associated with the SPB and thereby the number of microtubules formed. In addition, the GTBPs may regulate the activity of the Tub4p complex. Homologues of Spc97p and Spc98p have been identified from yeast to mammalian cells and these are also part of gamma-tubulin complexes, suggesting that these related proteins may also interact with GTBPs at the centrosome. Candidates for GTBPs have been identified in mammalian and insect cells.
Asunto(s)
Huso Acromático/fisiología , Levaduras/fisiología , Tubulina (Proteína)/fisiologíaRESUMEN
The spindle pole body component Kar1p has a function in nuclear fusion during conjugation, a process known as karyogamy. The molecular role of Kar1p during this process is poorly understood. Here we show that the yeast gamma-tubulin complex-binding protein Spc72p interacts directly with the N-terminal domain of Kar1p, thereby targeting the gamma-tubulin complex to the half bridge, a substructure of the spindle pole body, where it organizes microtubules. This binding of Spc72p to Kar1p has only a minor role during vegetative growth, whereas it becomes essential for karyogamy in mating cells, explaining the important role of Kar1p in this process. We also show that the localization of Spc72p within the spindle pole body changes throughout the cell cycle and even more strongly in response to mating pheromone. Taken together, these observations suggest that the relocalization of Spc72p within the spindle pole body is the 'landmark' event in the pheromone-induced reorganization of the cytoplasmic microtubules.
Asunto(s)
Proteínas Fúngicas/metabolismo , Microtúbulos/fisiología , Proteínas Nucleares/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Tubulina (Proteína)/metabolismo , Unión Proteica , Proteínas Recombinantes de Fusión/metabolismo , Saccharomyces cerevisiae/crecimiento & desarrolloRESUMEN
Epitope tagging of proteins as a strategy for the analysis of function, interactions and the subcellular distribution of proteins has become widely used. In the yeast Saccharomyces cerevisiae, molecular biological techniques have been developed that use a simple PCR-based strategy to introduce epitope tags to chromosomal loci (Wach et al., 1994). To further employ the power of this strategy, a variety of novel tags was constructed. These tags were combined with different selectable marker genes, resulting in PCR amplificable modules. Only one set of primers is required for the amplification of any module. Furthermore, convenient laboratory techniques are described that facilitate the genetic manipulations of yeast strains, as well as the analysis of the epitope-tagged proteins.
Asunto(s)
Mapeo Epitopo , Genes Fúngicos , Reacción en Cadena de la Polimerasa/métodos , Saccharomyces cerevisiae/genética , Western Blotting , Proteínas Fúngicas/genética , Proteínas Fúngicas/inmunología , Saccharomyces cerevisiae/inmunología , Huso Acromático/genética , Transformación GenéticaRESUMEN
In yeast, microtubules are organized by the spindle pole body (SPB). The SPB is a disk-like multilayered structure that is embedded in the nuclear envelope via its central plaque, whereas the outer and inner plaques are exposed to the cytoplasm and nucleoplasm, respectively. How the SPB assembles is poorly understood. We show that the inner/central plaque is composed of a stable SPB subcomplex, containing the gamma-tubulin complex-binding protein Spc110p, calmodulin, Spc42p, and Spc29p. Spc29p acts as a linker between the central plaque component Spc42p and the inner plaque protein Spc110p. Evidence is provided that the calmodulin-binding site of Spc110p influences the binding of Spc29p to Spc110p. Spc42p also was identified as a component of a cytoplasmic SPB subcomplex containing Spc94p/Nud1p, Cnm67p, and Spc42p. Spc29p and Spc42p may be part of a critical interface of nucleoplasmic and cytoplasmic assembled SPB subcomplexes that form during SPB duplication. In agreement with this, overexpressed Spc29p was found to be a nuclear protein, whereas Spc42p is cytoplasmic. In addition, an essential function of SPC29 during SPB assembly is indicated by the SPB duplication defect of conditional lethal spc29(ts) cells and by the genetic interaction of SPC29 with CDC31 and KAR1, two genes that are involved in SPB duplication.
Asunto(s)
Proteínas Fúngicas/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/fisiología , Huso Acromático/fisiología , Huso Acromático/ultraestructura , Secuencia de Aminoácidos , Sitios de Unión , Proteínas de Unión a Calmodulina , Proteínas del Citoesqueleto , Proteínas Fúngicas/aislamiento & purificación , Modelos Estructurales , Datos de Secuencia Molecular , Membrana Nuclear/fisiología , Membrana Nuclear/ultraestructura , Proteínas Nucleares/aislamiento & purificación , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/genética , Alineación de SecuenciaRESUMEN
The functional coupling of protein synthesis and chaperone-assisted folding in vivo has remained largely unexplored. Here we have analysed the chaperonin-dependent folding pathway of actin in yeast. Remarkably, overexpression of a heterologous chaperonin which traps non-native polypeptides does not interfere with protein folding in the cytosol, indicating a high-level organization of folding reactions. Newly synthesized actin avoids the chaperonin trap and is effectively channelled from the ribosome to the endogenous chaperonin TRiC. Efficient actin folding on TRiC is critically dependent on the hetero-oligomeric co-chaperone GimC. By interacting with folding intermediates and with TRiC, GimC accelerates actin folding at least 5-fold and prevents the premature release of non-native protein from TRiC. We propose that TRiC and GimC form an integrated 'folding compartment' which functions in cooperation with the translation machinery. This compartment sequesters newly synthesized actin and other aggregation-sensitive polypeptides from the crowded macromolecular environment of the cytosol, thereby allowing their efficient folding.
Asunto(s)
Actinas/química , Actinas/metabolismo , Chaperoninas/metabolismo , Péptidos y Proteínas de Señalización Intracelular , Proteínas Asociadas a Microtúbulos , Pliegue de Proteína , Saccharomyces cerevisiae/metabolismo , Actinas/genética , Animales , Bovinos , Compartimento Celular , Chaperonina 60/química , Chaperonina 60/genética , Chaperonina 60/metabolismo , Chaperoninas/química , Chaperoninas/genética , Citosol/metabolismo , Técnicas In Vitro , Sustancias Macromoleculares , Masculino , Proteínas Nucleares/química , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Ubiquitina-Proteína Ligasas , Región del Complejo T del GenomaRESUMEN
The yeast microtubule organizing centre (MTOC), known as the spindle pole body (SPB), organizes the nuclear and cytoplasmic microtubules which are functionally and spatially distinct. Microtubule organization requires the yeast gamma-tubulin complex (Tub4p complex) which binds to the nuclear side of the SPB at the N-terminal domain of Spc110p. Here, we describe the identification of the essential SPB component Spc72p whose N-terminal domain interacts with the Tub4p complex on the cytoplasmic side of the SPB. We further report that this Tub4p complex-binding domain of Spc72p is essential and that temperature-sensitive alleles of SPC72 or overexpression of a binding domain-deleted variant of SPC72 (DeltaN-SPC72) impair cytoplasmic microtubule formation. Consequently, polynucleated and anucleated cells accumulated in these cultures. In contrast, overexpression of the entire SPC72 results in more cytoplasmic microtubules compared with wild-type. Finally, exchange of the Tub4p complex-binding domains of Spc110p and Spc72p established that the Spc110p domain, when attached to DeltaN-Spc72p, was functional at the cytoplasmic site of the SPB, while the corresponding domain of Spc72p fused to DeltaN-Spc110p led to a dominant-negative effect. These results suggest that different components of MTOCs act as receptors for gamma-tubulin complexes and that they are essential for the function of MTOCs.
Asunto(s)
Proteínas Fúngicas/metabolismo , Proteínas de Microtúbulos/metabolismo , Microtúbulos/metabolismo , Proteínas de Saccharomyces cerevisiae , Tubulina (Proteína)/metabolismo , Proteínas de Unión a Calmodulina , Núcleo Celular/metabolismo , Centrosoma/metabolismo , Clonación Molecular , Citoplasma/metabolismo , Proteínas del Citoesqueleto , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas de Microtúbulos/química , Proteínas de Microtúbulos/genética , Peso Molecular , Proteínas Nucleares/metabolismo , Saccharomyces cerevisiae/citología , Eliminación de Secuencia , TemperaturaRESUMEN
In the yeast Saccharomyces cerevisiae, microtubules are organized by the spindle pole body (SPB), which is embedded in the nuclear envelope. Microtubule organization requires the gamma-tubulin complex containing the gamma-tubulin Tub4p, Spc98p, and Spc97p. The Tub4p complex is associated with cytoplasmic and nuclear substructures of the SPB, which organize the cytoplasmic and nuclear microtubules. Here we present evidence that the Tub4p complex assembles in the cytoplasm and then either binds to the cytoplasmic side of the SPB or is imported into the nucleus followed by binding to the nuclear side of the SPB. Nuclear import of the Tub4p complex is mediated by the essential nuclear localization sequence of Spc98p. Our studies also indicate that Spc98p in the Tub4p complex is phosphorylated at the nuclear, but not at the cytoplasmic, side of the SPB. This phosphorylation is cell cycle dependent and occurs after SPB duplication and nucleation of microtubules by the new SPB and therefore may have a role in mitotic spindle function. In addition, activation of the mitotic checkpoint stimulates Spc98p phosphorylation. The kinase Mps1p, which functions in SPB duplication and mitotic checkpoint control, seems to be involved in Spc98p phosphorylation. Our results also suggest that the nuclear and cytoplasmic Tub4p complexes are regulated differently.
Asunto(s)
Núcleo Celular/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Huso Acromático/fisiología , Tubulina (Proteína)/metabolismo , Secuencia de Aminoácidos , Transporte Biológico , Ciclo Celular/genética , Citoplasma , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Microtúbulos , Datos de Secuencia Molecular , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Fosforilación , Saccharomyces cerevisiae/metabolismo , Tubulina (Proteína)/genéticaRESUMEN
We describe the identification of GIM1/YKE2, GIM2/PAC10, GIM3, GIM4 and GIM5 in a screen for mutants that are synthetically lethal with tub4-1, encoding a mutated yeast gamma-tubulin. The cytoplasmic Gim proteins encoded by these GIM genes are present in common complexes as judged by co-immunoprecipitation and gel filtration experiments. The disruption of any of these genes results in similar phenotypes: the gim null mutants are synthetically lethal with tub4-1 and super-sensitive towards the microtubule-depolymerizing drug benomyl. All except Deltagim4 are cold-sensitive and their microtubules disassemble at 14 degrees C. The Gim proteins have one function related to alpha-tubulin and another to Tub4p, supported by the finding that the benomyl super-sensitivity is caused by a reduced level of alpha-tubulin while the synthetic lethality with tub4-1 is not. In addition, GIM1/YKE2 genetically interacts with two distinct classes of genes, one of which is involved in tubulin folding and the other in microtubule nucleation. We show that the Gim proteins are important for Tub4p function and bind to overproduced Tub4p. The mammalian homologues of GIM1/YKE2 and GIM2/PAC10 rescue the synthetically lethal phenotype with tub4-1 as well as the cold-sensitivity and benomyl super-sensitivity of the yeast deletion mutants. We suggest that the Gim proteins form a protein complex that promotes formation of functional alpha- and gamma-tubulin.