RESUMEN
Cdc42 plays a central role in establishing polarity in yeast and animals, yet how polarization of Cdc42 is achieved in response to spatial cues is poorly understood. Using live-cell imaging, we found distinct dynamics of Cdc42 polarization in haploid budding yeast in correlation with two temporal steps of the G1 phase. The position at which the Cdc42-GTP cluster develops changes rapidly around the division site during the first step but becomes stabilized in the second step, suggesting that an axis of polarized growth is determined in mid G1. Cdc42 polarization in the first step and its proper positioning depend on Rsr1 and its GTPase-activating protein (GAP) Bud2. Interestingly, Rga1, a Cdc42 GAP, exhibits transient localization to a site near the bud neck and to the division site during cytokinesis and G1, and this temporal change of Rga1 distribution is necessary for determination of a proper growth site. Mathematical modeling suggests that a proper axis of Cdc42 polarization in haploid cells might be established through a biphasic mechanism involving sequential positive feedback and transient negative feedback.
Asunto(s)
Polaridad Celular/fisiología , Proteínas Activadoras de GTPasa/metabolismo , Saccharomycetales/metabolismo , Proteína de Unión al GTP cdc42 de Saccharomyces cerevisiae/metabolismo , Proteínas de Unión al GTP rab/metabolismo , División Celular/fisiología , Proteínas Fúngicas/metabolismo , Fase G1/fisiología , HaploidiaRESUMEN
Cells of the budding yeast Saccharomyces cerevisiae select a site for polarized growth in a specific pattern that depends on their cell type. Haploid a and α cells bud in the axial budding pattern, which requires assembly of a landmark that includes the Bud4 protein. To understand how an axial bud site is established, we performed a structure-function analysis of Bud4. Bud4 contains DUF1709 (domain of unknown function), which is similar to a part of the anillin-homology domain, and a putative Pleckstrin homology (PH) domain near to its C terminus. Although its localization depends on septins, a conserved family of GTP-binding proteins, Bud4 is necessary for the stable inheritance of septin rings during cell division. Although some anillins interact directly with septins, we find that neither DUF1709 nor the PH domain is necessary for targeting Bud4 to the mother-bud neck. Instead, this C-terminal region is crucial for association of Bud4 with Bud3 and other components of the axial landmark. Remarkably, septins colocalize with Bud4 mutant proteins that lack these C-terminal domains, forming an arc or a single ring instead of a double ring during and after cytokinesis. Interestingly, overexpression of Bud4 also induces formation of extra Bud4 rings and arcs that are associated with septins. Analyses of a series of bud4 truncation mutants suggest that at least two domains in the central region play a redundant role in targeting Bud4 to the mother-bud neck and are thus likely to interact with septins. Taken together, these results indicate that Bud4 functions as a platform that links septins to the axial landmark.
Asunto(s)
Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Septinas/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Citocinesis/genética , Citocinesis/fisiología , Proteínas de Unión al GTP/genética , Proteínas de Unión al GTP/metabolismo , Immunoblotting , Inmunoprecipitación , Microscopía Fluorescente , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Septinas/genéticaRESUMEN
ATP-binding cassette (ABC) transporters are a ubiquitous class of integral membrane proteins of immense clinical interest because of their strong association with human disease and pharmacology. To improve our understanding of these proteins, we used membrane yeast two-hybrid technology to map the protein interactome of all of the nonmitochondrial ABC transporters in the model organism Saccharomyces cerevisiae and combined this data with previously reported yeast ABC transporter interactions in the BioGRID database to generate a comprehensive, integrated 'interactome'. We show that ABC transporters physically associate with proteins involved in an unexpectedly diverse range of functions. We specifically examine the importance of the physical interactions of ABC transporters in both the regulation of one another and in the modulation of proteins involved in zinc homeostasis. The interaction network presented here will be a powerful resource for increasing our fundamental understanding of the cellular role and regulation of ABC transporters.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Mapeo de Interacción de Proteínas , Saccharomyces cerevisiae/metabolismo , Unión Proteica , Saccharomyces cerevisiae/química , Técnicas del Sistema de Dos HíbridosRESUMEN
Cell polarization occurs along a single axis that is generally determined by a spatial cue. Cells of the budding yeast Saccharomyces cerevisiae select a site for polarized growth in a specific pattern depending on cell type. Haploid a and α cells bud in the axial budding pattern, which depends on a transient marker and requires proteins Bud3, Bud4, Axl1 and Axl2. Here, we report that Bud4 functions as a platform that mediates the ordered assembly of the axial landmark at the division site during M and early G1 phase. Whereas Bud4 associates with Bud3 in all cell types and in the absence of Axl1 or Axl2, Bud4 interacts with Axl1 and Axl2 mainly in haploid cells and only in the presence of all other components of the landmark. Bud4 can bind to GTP or GDP, and a GTP-binding-defective Bud4 fails to interact with Axl1 in vitro. The same bud4 mutation leads to mis-localization of Axl1 and disrupts the axial budding pattern, indicating that GTP binding to Bud4 is important for its role in bud-site selection. We also show the cell-type-specific association of the axial landmark with Bud5, a GDP/GTP exchange factor for Rsr1. Despite their expression in all cell types, Bud4 and Axl2 associate with Bud5 specifically in haploid cells and in the presence of Axl1, whose expression is limited to a and α cells. Together, our findings suggest that Bud4 plays a critical role in the assembly of the axial landmark and its link to the Rsr1 GTPase module.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Proteínas de Unión al GTP/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Saccharomycetales/metabolismo , Secuencia de Aminoácidos , Ciclo Celular , Proteínas de Ciclo Celular/genética , División Celular/fisiología , Polaridad Celular/fisiología , Citocinesis/fisiología , Proteínas de Unión al GTP/genética , Factores de Intercambio de Guanina Nucleótido/genética , Factores de Intercambio de Guanina Nucleótido/metabolismo , Guanosina Trifosfato/metabolismo , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Metaloendopeptidasas/genética , Metaloendopeptidasas/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomycetales/genéticaRESUMEN
Cell polarization, in which intracellular substances are asymmetrically distributed, enables cells to carry out specialized functions. While cell polarity is often induced by intracellular or extracellular spatial cues, spontaneous polarization (the so-called symmetry breaking) may also occur in the absence of spatial cues. Many computational models have been used to investigate the mechanisms of symmetry breaking, and it was proved that spontaneous polarization occurs when the lateral diffusion of inactive signaling molecules is much faster than that of active signaling molecules. This conclusion leaves an important question of how, as observed in many biological systems, cell polarity emerges when active and inactive membrane-bound molecules diffuse at similar rates while cycling between cytoplasm and membrane takes place. The recent studies of Rätz and Röger showed that, when the cytosolic and membrane diffusion are very different, spontaneous polarization is possible even if the membrane-bound species diffuse at the same rate. In this paper, we formulate a two-equation non-local reaction-diffusion model with general forms of positive feedback. We apply Turing stability analysis to identify parameter conditions for achieving cell polarization. Our results show that spontaneous polarization can be achieved within some parameter ranges even when active and inactive signaling molecules diffuse at similar rates. In addition, different forms of positive feedback are explored to show that a non-local molecule-mediated feedback is important for sharping the localization as well as giving rise to fast dynamics to achieve robust polarization.
Asunto(s)
Membrana Celular/fisiología , Polaridad Celular/fisiología , Citosol/fisiología , Modelos Biológicos , Transducción de Señal/fisiología , Simulación por Computador , RetroalimentaciónRESUMEN
Cell polarization generally occurs along a single axis that is directed by a spatial cue. Cells of the budding yeast Saccharomyces cerevisiae undergo polarized growth and oriented cell division in a spatial pattern by selecting a specific bud site. Haploid a or α cells bud in the axial pattern in response to a transient landmark that includes Bud3, Bud4, Axl1, and Axl2. Septins, a family of filament-forming GTP-binding proteins, are also involved in axial budding and recruited to an incipient bud site, but the mechanism of recruitment remains unclear. Here, we show that Axl2 interacts with Bud3 and the Cdc42 GTPase in its GTP-bound state. Axl2 also interacts with Cdc10, a septin subunit, promoting efficient recruitment of septins near the cell division site. Furthermore, a cdc42 mutant defective in the axial budding pattern at a semi-permissive temperature had a reduced interaction with Axl2 and compromised septin recruitment in the G1 phase. We thus propose that active Cdc42 brings Axl2 to the Bud3-Bud4 complex and that Axl2 then interacts with Cdc10, linking septin recruitment to the axial landmark.
RESUMEN
Cdc42, a conserved Rho GTPase, plays a central role in polarity establishment in yeast and animals. Cell polarity is critical for asymmetric cell division, and asymmetric cell division underlies replicative aging of budding yeast. Yet how Cdc42 and other polarity factors impact life span is largely unknown. Here we show by live-cell imaging that the active Cdc42 level is sporadically elevated in wild type during repeated cell divisions but rarely in the long-lived bud8 deletion cells. We find a novel Bud8 localization with cytokinesis remnants, which also recruit Rga1, a Cdc42 GTPase activating protein. Genetic analyses and live-cell imaging suggest that Rga1 and Bud8 oppositely impact life span likely by modulating active Cdc42 levels. An rga1 mutant, which has a shorter life span, dies at the unbudded state with a defect in polarity establishment. Remarkably, Cdc42 accumulates in old cells, and its mild overexpression accelerates aging with frequent symmetric cell divisions, despite no harmful effects on young cells. Our findings implicate that the interplay among these positive and negative polarity factors limits the life span of budding yeast.
Asunto(s)
Saccharomycetales , Polaridad Celular/fisiología , Proteínas Activadoras de GTPasa/metabolismo , Longevidad , Saccharomyces cerevisiae/metabolismo , Saccharomycetales/metabolismo , Regulación hacia Arriba , Proteína de Unión al GTP cdc42/metabolismo , Proteína de Unión al GTP cdc42 de Saccharomyces cerevisiae/metabolismoRESUMEN
SUMMARY: The establishment of cell polarity is critical for the development of many organisms and for the function of many cell types. A large number of studies of diverse organisms from yeast to humans indicate that the conserved, small-molecular-weight GTPases function as key signaling proteins involved in cell polarization. The budding yeast Saccharomyces cerevisiae is a particularly attractive model because it displays pronounced cell polarity in response to intracellular and extracellular cues. Cells of S. cerevisiae undergo polarized growth during various phases of their life cycle, such as during vegetative growth, mating between haploid cells of opposite mating types, and filamentous growth upon deprivation of nutrition such as nitrogen. Substantial progress has been made in deciphering the molecular basis of cell polarity in budding yeast. In particular, it becomes increasingly clear how small GTPases regulate polarized cytoskeletal organization, cell wall assembly, and exocytosis at the molecular level and how these GTPases are regulated. In this review, we discuss the key signaling pathways that regulate cell polarization during the mitotic cell cycle and during mating.
Asunto(s)
Proteínas de Unión al GTP Monoméricas/metabolismo , Levaduras/fisiología , Ciclo Celular/fisiología , División Celular/fisiología , Modelos Biológicos , Proteínas de Unión al GTP Monoméricas/fisiología , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/fisiología , Levaduras/citología , Levaduras/enzimologíaRESUMEN
In both animal and yeast cells, reactive oxygen species (ROS) are produced as byproducts of metabolism and upon exposure to diverse environmental stresses. Cellular defense systems operate to avoid molecular damage caused by ROS, but the redox balance is disturbed under excessive stress. Cells of the budding yeast Saccharomyces cerevisiae undergo apoptotic-like cell death upon exposure to hydrogen peroxide (H(2)O(2)). Here, we report that the Rho5 GTPase of budding yeast is necessary for H(2)O(2)-induced cell death, which accompanies ROS accumulation and DNA fragmentation. Unlike WT, a rho5 deletion mutant (rho5Delta) exhibits little cell death, whereas the constitutively active rho5(G12V) mutant exhibits excess ROS accumulation and increased cell death upon H(2)O(2) treatment. Consistent with a role in the oxidative stress response, Rho5 interacts with the thioredoxin reductase Trr1, a key component of the cytoplasmic thioredoxin antioxidant system, in a GTP-dependent manner. This interaction occurs on the vacuolar membrane before exposure to H(2)O(2) but also in the vacuolar lumen after H(2)O(2) treatment. Trr1 levels are elevated in rho5Delta cells but are elevated only slightly in WT and not in the rho5(G12V) cells after H(2)O(2) treatment. Taken together, these data suggest that Rho5 mediates H(2)O(2)-induced cell death by regulating the level of Trr1 or by excluding Trr1 from its cytoplasmic substrate.
Asunto(s)
Apoptosis , Oxidantes/farmacología , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/efectos de los fármacos , Reductasa de Tiorredoxina-Disulfuro/metabolismo , Proteínas de Unión al GTP rho/metabolismo , Eliminación de Gen , Proteínas Fluorescentes Verdes/análisis , Proteínas Fluorescentes Verdes/genética , Guanosina Trifosfato/metabolismo , Peróxido de Hidrógeno/farmacología , Estrés Oxidativo , Especies Reactivas de Oxígeno , Saccharomyces cerevisiae/enzimología , Proteínas de Saccharomyces cerevisiae/análisis , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Unión al GTP rho/análisis , Proteínas de Unión al GTP rho/genéticaRESUMEN
The Rho GTPase Cdc42 is a central regulator of cell polarity in diverse cell types. The activity of Cdc42 is dynamically controlled in time and space to enable distinct polarization events, which generally occur along a single axis in response to spatial cues. Our understanding of the mechanisms underlying Cdc42 polarization has benefited largely from studies of the budding yeast Saccharomyces cerevisiae, a genetically tractable model organism. In budding yeast, Cdc42 activation occurs in two temporal steps in the G1 phase of the cell cycle to establish a proper growth site. Here, we review findings in budding yeast that reveal an intricate crosstalk among polarity proteins for biphasic Cdc42 regulation. The first step of Cdc42 activation may determine the axis of cell polarity, while the second step ensures robust Cdc42 polarization for growth. Biphasic Cdc42 polarization is likely to ensure the proper timing of events including the assembly and recognition of spatial landmarks and stepwise assembly of a new ring of septins, cytoskeletal GTP-binding proteins, at the incipient bud site. Biphasic activation of GTPases has also been observed in mammalian cells, suggesting that biphasic activation could be a general mechanism for signal-responsive cell polarization. Cdc42 activity is necessary for polarity establishment during normal cell division and development, but its activity has also been implicated in the promotion of aging. We also discuss negative polarity signaling and emerging concepts of Cdc42 signaling in cellular aging.
RESUMEN
The Cdc42 guanosine triphosphatase (GTPase) plays a central role in polarity development in species ranging from yeast to humans. In budding yeast, a specific growth site is selected in the G1 phase. Rsr1, a Ras GTPase, interacts with Cdc42 and its associated proteins to promote polarized growth at the proper bud site. Yet how Rsr1 regulates cell polarization is not fully understood. Here, we show that Rsr1-GDP interacts with the scaffold protein Bem1 in early G1, likely hindering the role of Bem1 in Cdc42 polarization and polarized secretion. Consistent with these in vivo observations, mathematical modeling predicts that Bem1 is unable to promote Cdc42 polarization in early G1 in the presence of Rsr1-GDP. We find that a part of the Bem1 Phox homology domain, which overlaps with a region interacting with the exocyst component Exo70, is necessary for the association of Bem1 with Rsr1-GDP. Overexpression of the GDP-locked Rsr1 interferes with Bem1-dependent Exo70 polarization. We thus propose that Rsr1 functions in spatial and temporal regulation of polarity establishment by associating with distinct polarity factors in its GTP- and GDP-bound states.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Polaridad Celular/fisiología , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Unión al GTP rab/metabolismo , División Celular , Citoplasma/metabolismo , Fase G1 , Factores de Intercambio de Guanina Nucleótido/metabolismo , Saccharomyces cerevisiae/metabolismo , Saccharomycetales/metabolismo , Transducción de Señal , Proteína de Unión al GTP cdc42/metabolismo , Proteína de Unión al GTP cdc42 de Saccharomyces cerevisiae/metabolismo , Proteínas ras/metabolismoRESUMEN
Rho GTPases play critical roles in cell proliferation and cell death in many species. As in animal cells, cells of the budding yeast Saccharomyces cerevisiae undergo regulated cell death under various physiological conditions and upon exposure to external stress. The Rho5 GTPase is necessary for oxidant-induced cell death, and cells expressing a constitutively active GTP-locked Rho5 are hypersensitive to oxidants. Yet how Rho5 regulates yeast cell death has been poorly understood. To identify genes that are involved in the Rho5-mediated cell death program, we performed two complementary genome-wide screens: one screen for oxidant-resistant deletion mutants and another screen for Rho5-associated proteins. Functional enrichment and interaction network analysis revealed enrichment for genes in pathways related to metabolism, transport, and plasma membrane organization. In particular, we find that ATG21, which is known to be involved in the CVT (Cytoplasm-to-Vacuole Targeting) pathway and mitophagy, is necessary for cell death induced by oxidants. Cells lacking Atg21 exhibit little cell death upon exposure to oxidants even when the GTP-locked Rho5 is expressed. Moreover, Atg21 interacts with Rho5 preferentially in its GTP-bound state, suggesting that Atg21 is a downstream target of Rho5 in oxidant-induced cell death. Given the high degree of conservation of Rho GTPases and autophagy from yeast to human, this study may provide insight into regulated cell death in eukaryotes in general.
Asunto(s)
Proteínas Relacionadas con la Autofagia/metabolismo , Muerte Celular , Endopeptidasas/metabolismo , Estrés Oxidativo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Transducción de Señal , Proteínas de Unión al GTP rho/metabolismo , Oxidantes/toxicidad , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/fisiologíaRESUMEN
The Cdc42 GTPase plays a central role in polarity development in many species. In budding yeast, Cdc42 is essential for polarized growth at the proper site and also for spontaneous cell polarization in the absence of spatial cues. Cdc42 polarization is critical for multiple events in the G1 phase prior to bud emergence, including bud-site assembly, polarization of the actin cytoskeleton, and septin filament assembly to form a ring at the new bud site. Yet the mechanism by which Cdc42 polarizes is not fully understood. Here we report that biphasic Cdc42 polarization in the G1 phase is coupled to stepwise assembly of the septin ring for bud emergence. We show that the Rsr1 GTPase shares a partially redundant role with Gic1 and Gic2, two related Cdc42 effectors, in the first phase of Cdc42 polarization in haploid cells. We propose that the first phase of Cdc42 polarization is mediated by positive feedback loops that function in parallel-one involving Rsr1 via local activation of Cdc42 in response to spatial cues and another involving Gic1 or Gic2 via reduction of diffusion of active Cdc42.
Asunto(s)
Polaridad Celular , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Alelos , Membrana Celular/metabolismo , Fase G1 , Proteínas Fluorescentes Verdes/metabolismo , Modelos Biológicos , Mutación/genética , Unión Proteica , Dominios Proteicos , Estabilidad Proteica , Septinas/metabolismo , Proteína de Unión al GTP cdc42 de Saccharomyces cerevisiae/metabolismo , Proteínas de Unión al GTP rab/metabolismoRESUMEN
In the budding yeast Saccharomyces cerevisiae, selection of the bud site determines the axis of polarized cell growth and eventual oriented cell division. Bud sites are selected in specific patterns depending on cell type. These patterns appear to depend on distinct types of marker proteins in the cell cortex; in particular, the bipolar budding of diploid cells depends on persistent landmarks at the birth-scar-distal and -proximal poles that involve the proteins Bud8p and Bud9p, respectively. Rax1p and Rax2p also appear to function specifically in bipolar budding, and we report here a further characterization of these proteins and of their interactions with Bud8p and Bud9p. Rax1p and Rax2p both appear to be integral membrane proteins. Although commonly used programs predict different topologies for Rax2p, glycosylation studies indicate that it has a type I orientation, with its long N-terminal domain in the extracytoplasmic space. Analysis of rax1 and rax2 mutant budding patterns indicates that both proteins are involved in selecting bud sites at both the distal and proximal poles of daughter cells as well as near previously used division sites on mother cells. Consistent with this, GFP-tagged Rax1p and Rax2p were both observed at the distal pole as well as at the division site on both mother and daughter cells; localization to the division sites was persistent through multiple cell cycles. Localization of Rax1p and Rax2p was interdependent, and biochemical studies showed that these proteins could be copurified from yeast. Bud8p and Bud9p could also be copurified with Rax1p, and localization studies provided further evidence of interactions. Localization of Rax1p and Rax2p to the bud tip and distal pole depended on Bud8p, and normal localization of Bud8p was partially dependent on Rax1p and Rax2p. Although localization of Rax1p and Rax2p to the division site did not appear to depend on Bud9p, normal localization of Bud9p appeared largely or entirely dependent on Rax1p and Rax2p. Taken together, the results indicate that Rax1p and Rax2p interact closely with each other and with Bud8p and Bud9p in the establishment and/or maintenance of the cortical landmarks for bipolar budding.
Asunto(s)
Glicoproteínas/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/fisiología , Saccharomyces cerevisiae/metabolismo , Sitios de Unión , División Celular , Membrana Celular/metabolismo , Polaridad Celular , Proliferación Celular , Clonación Molecular , Citoplasma/metabolismo , Proteínas Fúngicas/fisiología , Eliminación de Gen , Proteínas Fluorescentes Verdes/metabolismo , Proteínas de la Membrana , Mutación , Plásmidos/metabolismo , Unión Proteica , Estructura Terciaria de ProteínaRESUMEN
Polarized cell growth requires the coupling of a defined spatial site on the cell cortex to the apparatus that directs the establishment of cell polarity. In the budding yeast Saccharomyces cerevisiae, the Ras-family GTPase Rsr1p/Bud1p and its regulators select the proper site for bud emergence on the cell cortex. The Rho-family GTPase Cdc42p and its associated proteins then establish an axis of polarized growth by triggering an asymmetric organization of the actin cytoskeleton and secretory apparatus at the selected bud site. We explored whether a direct linkage exists between the Rsr1p/Bud1p and Cdc42p GTPases. Here we show specific genetic interactions between RSR1/BUD1 and particular cdc42 mutants defective in polarity establishment. We also show that Cdc42p coimmunoprecipitated with Rsr1p/Bud1p from yeast extracts. In vitro studies indicated a direct interaction between Rsr1p/Bud1p and Cdc42p, which was enhanced by Cdc24p, a guanine nucleotide exchange factor for Cdc42p. Our findings suggest that Cdc42p interacts directly with Rsr1p/Bud1p in vivo, providing a novel mechanism by which direct contact between a Ras-family GTPase and a Rho-family GTPase links the selection of a growth site to polarity establishment.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Polaridad Celular/fisiología , Factores de Intercambio de Guanina Nucleótido , Proteínas Proto-Oncogénicas/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Proteína de Unión al GTP cdc42 de Saccharomyces cerevisiae/metabolismo , Proteínas de Unión al GTP rab/metabolismo , Proteínas de Ciclo Celular/genética , Clonación Molecular , Modelos Moleculares , Unión Proteica , Estructura Terciaria de Proteína/genética , Proteínas Proto-Oncogénicas/genética , Saccharomyces cerevisiae/genética , Proteína de Unión al GTP cdc42 de Saccharomyces cerevisiae/genética , Proteínas de Unión al GTP rab/genéticaRESUMEN
Cdc42 is a small guanosine triphosphatase (GTPase) that plays a central role in polarity development in diverse cell types. Since the activity of Cdc42 is dynamically controlled in time and space, it is required to develop a biosensor to monitor its activation in vivo. In this chapter, we describe the construction and usage of a simple and robust biosensor for monitoring active Cdc42 in budding yeast. This affinity-based biosensor uses a red fluorescent protein fused to a Cdc42- and Rac-interactive binding motif from one of the Cdc42 effector proteins. Because it binds specifically to the GTP-bound Cdc42, this biosensor can be used to monitor Cdc42 activation in vivo. This or similar biosensors can be widely used for studying GTPase signaling in other cell types because of the conserved CRIB motif present among GTPase targets.
Asunto(s)
Técnicas Biosensibles/métodos , Proteínas Fúngicas/análisis , Saccharomycetales/citología , Proteína de Unión al GTP cdc42/análisis , Proteínas Fúngicas/metabolismo , Guanosina Trifosfato/metabolismo , Proteínas Luminiscentes/análisis , Proteínas Luminiscentes/metabolismo , Microscopía Fluorescente/métodos , Imagen Óptica/métodos , Proteínas Recombinantes de Fusión/análisis , Proteínas Recombinantes de Fusión/metabolismo , Saccharomycetales/química , Saccharomycetales/metabolismo , Espectrometría de Fluorescencia/métodos , Proteína de Unión al GTP cdc42/metabolismo , Proteína Fluorescente RojaRESUMEN
In yeast and animal cells, signaling pathways involving small guanosine triphosphatases (GTPases) regulate cell polarization. In budding yeast, selection of a bud site directs polarity establishment and subsequently determines the plane of cell division. Rga1, a Cdc42 GTPase-activating protein, prevents budding within the division site by inhibiting Cdc42 repolarization. A protein complex including Nba1 and Nis1 is involved in preventing rebudding at old division sites, yet how these proteins and Rga1 might function in negative polarity signaling has been elusive. Here we show that Rga1 transiently localizes to the immediately preceding and older division sites by interacting with Nba1 and Nis1. The LIM domains of Rga1 are necessary for its interaction with Nba1, and loss of this interaction results in premature delocalization of Rga1 from the immediately preceding division site and, consequently, abnormal bud-site selection in daughter cells. However, such defects are minor in mother cells of these mutants, likely because the G1 phase is shorter and a new bud site is established prior to delocalization of Rga1. Indeed, our biphasic mathematical model of Cdc42 polarization predicts that premature delocalization of Rga1 leads to more frequent Cdc42 repolarization within the division site when the first temporal step in G1 is assumed to last longer. Spatial distribution of a Cdc42 GAP in coordination with G1 progression may thus be critical for fine-tuning the orientation of the polarity axis in yeast.
Asunto(s)
Proteínas Activadoras de GTPasa/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteína de Unión al GTP cdc42 de Saccharomyces cerevisiae/metabolismo , Ciclo Celular/fisiología , División Celular/genética , División Celular/fisiología , Polaridad Celular/fisiología , Fase G1/fisiología , Proteínas Activadoras de GTPasa/genética , Orientación Espacial , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Transducción de SeñalRESUMEN
Complex protein networks are involved in nearly all cellular processes. To uncover these vast networks of protein interactions, various high-throughput screening technologies have been developed. Over the last decade, bimolecular fluorescence complementation (BiFC) assay has been widely used to detect protein-protein interactions (PPIs) in living cells. This technique is based on the reconstitution of a fluorescent protein in vivo. Easy quantification of the BiFC signals allows effective cell-based high-throughput screenings for protein binding partners and drugs that modulate PPIs. Recently, with the development of large screening libraries, BiFC has been effectively applied for genome-wide PPI studies and has uncovered novel protein interactions, providing new insight into protein functions. In this review, we describe the development of reagents and methods used for BiFC-based screens in yeast, plants, and mammalian cells. We also discuss the advantages and drawbacks of these methods and highlight the application of BiFC in large-scale studies.
Asunto(s)
Ensayos Analíticos de Alto Rendimiento/métodos , Técnicas de Sonda Molecular , Mapeo de Interacción de Proteínas/métodos , Animales , Proteínas de Arabidopsis/metabolismo , Colorantes Fluorescentes/metabolismo , Estudio de Asociación del Genoma Completo , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Mamíferos , Proteómica/métodos , Telómero/metabolismo , Levaduras/citología , Levaduras/metabolismoRESUMEN
Sho1p, an integral membrane protein, plays a vital role in the high-osmolarity glycerol (HOG) mitogen-activated protein kinase pathway in the yeast Saccharomyces cerevisiae. Activated under conditions of high osmotic stress, it interacts with other HOG pathway proteins to mediate cell signaling events, ensuring that yeast cells can adapt and remain viable. In an attempt to further understand how the function of Sho1p is regulated through its protein-protein interactions (PPIs), we identified 49 unique Sho1p PPIs through the use of membrane yeast two-hybrid (MYTH), an assay specifically suited to identify PPIs of full-length integral membrane proteins in their native membrane environment. Secondary validation by literature search, or two complementary PPI assays, confirmed 80% of these interactions, resulting in a high-quality Sho1p interactome. This set of putative PPIs included both previously characterized interactors, along with a large subset of interactors that have not been previously identified as binding to Sho1p. The SH3 domain of Sho1p was found to be important for binding to many of these interactors. One particular novel interactor of interest is the glycerol transporter Fps1p, which was shown to require the SH3 domain of Sho1p for binding via its N-terminal soluble regulatory domain. Furthermore, we found that Fps1p is involved in the positive regulation of Sho1p function and plays a role in the phosphorylation of the downstream kinase Hog1p. This study represents the largest membrane interactome analysis of Sho1p to date and complements past studies on the HOG pathway by increasing our understanding of Sho1p regulation.
Asunto(s)
Membrana Celular/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Glicerol/metabolismo , Inmunoprecipitación , Proteínas de la Membrana/genética , Proteínas Quinasas Activadas por Mitógenos/genética , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Concentración Osmolar , Fosforilación , Mapeo de Interacción de Proteínas , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Transducción de Señal , Dominios Homologos srcRESUMEN
Cell polarization occurs along a single axis that is generally determined by a spatial cue, yet the underlying mechanism is poorly understood. Using biochemical assays and live-cell imaging, we show that cell polarization to a proper growth site requires activation of Cdc42 by Bud3 in haploid budding yeast. Bud3 catalyzes the release of guanosine diphosphate (GDP) from Cdc42 and elevates intracellular Cdc42-guanosine triphosphate (GTP) levels in cells with inactive Cdc24, which has as of yet been the sole GDP-GTP exchange factor for Cdc42. Cdc42 is activated in two temporal steps in the G1 phase: the first depends on Bud3, whereas subsequent activation depends on Cdc24. Mutational analyses suggest that biphasic activation of Cdc42 in G1 is necessary for assembly of a proper bud site. Biphasic activation of Cdc42 or Rac GTPases may be a general mechanism for spatial cue-directed cell polarization in eukaryotes.