RESUMO
Concentration gradients regulate many cell biological and developmental processes. In rod-shaped fission yeast cells, polar cortical gradients of the DYRK family kinase Pom1 couple cell length with mitotic commitment by inhibiting a mitotic inducer positioned at midcell. However, how Pom1 gradients are established is unknown. Here, we show that Tea4, which is normally deposited at cell tips by microtubules, is both necessary and, upon ectopic cortical localization, sufficient to recruit Pom1 to the cell cortex. Pom1 then moves laterally at the plasma membrane, which it binds through a basic region exhibiting direct lipid interaction. Pom1 autophosphorylates in this region to lower lipid affinity and promote membrane release. Tea4 triggers Pom1 plasma membrane association by promoting its dephosphorylation through the protein phosphatase 1 Dis2. We propose that local dephosphorylation induces Pom1 membrane association and nucleates a gradient shaped by the opposing actions of lateral diffusion and autophosphorylation-dependent membrane detachment.
Assuntos
Membrana Celular/metabolismo , Proteínas Quinases/metabolismo , Schizosaccharomyces/metabolismo , Sequência de Aminoácidos , Ciclo Celular , Proteínas Associadas aos Microtúbulos/metabolismo , Dados de Sequência Molecular , Fosforilação , Proteínas Quinases/química , Schizosaccharomyces/citologia , Proteínas de Schizosaccharomyces pombe/metabolismo , Alinhamento de SequênciaRESUMO
Transcription enables the production of RNA from a DNA template. Due to the highly dynamic nature of transcription, live-cell imaging methods play a crucial role in measuring the kinetics of this process. For instance, transcriptional bursts have been visualized using fluorescent phage-coat proteins that associate tightly with messenger RNA (mRNA) stem loops formed on nascent transcripts. To convert the signal emanating from a transcription site into meaningful estimates of transcription dynamics, the influence of various parameters on the measured signal must be evaluated. Here, the effect of gene length on the intensity of the transcription site focus was analyzed. Intuitively, a longer gene can support a larger number of transcribing polymerases, thus leading to an increase in the measured signal. However, measurements of transcription induced by hyper-osmotic stress responsive promoters display independence from gene length. A mathematical model of the stress-induced transcription process suggests that the formation of gene loops that favor the recycling of polymerase from the terminator to the promoter can explain the observed behavior. One experimentally validated prediction from this model is that the amount of mRNA produced from a short gene should be higher than for a long one as the density of active polymerase on the short gene will be increased by polymerase recycling. Our data suggest that this recycling contributes significantly to the expression output from a gene and that polymerase recycling is modulated by the promoter identity and the cellular state.
Assuntos
Transcrição Gênica , Regiões Promotoras Genéticas , RNA Mensageiro/genéticaRESUMO
Local activity of the small GTPase Cdc42 is critical for cell polarization. Whereas scaffold-mediated positive feedback was proposed to break symmetry of budding yeast cells and produce a single zone of Cdc42 activity, the existence of similar regulation has not been probed in other organisms. Here, we address this problem using rod-shaped cells of fission yeast Schizosaccharomyces pombe, which exhibit zones of active Cdc42-GTP at both cell poles. We implemented the CRY2-CIB1 optogenetic system for acute light-dependent protein recruitment to the plasma membrane, which allowed to directly demonstrate positive feedback. Indeed, optogenetic recruitment of constitutively active Cdc42 leads to co-recruitment of the guanine nucleotide exchange factor (GEF) Scd1 and endogenous Cdc42, in a manner dependent on the scaffold protein Scd2. We show that Scd2 function is dispensable when the positive feedback operates through an engineered interaction between the GEF and a Cdc42 effector, the p21-activated kinase 1 (Pak1). Remarkably, this rewired positive feedback confers viability and allows cells to form 2 zones of active Cdc42 even when otherwise essential Cdc42 activators are lacking. These cells further revealed that the small GTPase Ras1 plays a role in both localizing the GEF Scd1 and promoting its activity, which potentiates the positive feedback. We conclude that scaffold-mediated positive feedback, gated by Ras activity, confers robust polarization for rod-shape formation.
Assuntos
Matriz Nuclear/fisiologia , Schizosaccharomyces , Proteína cdc42 de Ligação ao GTP/metabolismo , Proteínas ras/fisiologia , Polaridade Celular/genética , Retroalimentação Fisiológica/fisiologia , Optogenética , Organismos Geneticamente Modificados , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Proteína cdc42 de Ligação ao GTP/genéticaRESUMO
In non-motile fungi, sexual reproduction relies on strong morphogenetic changes in response to pheromone signaling. We report here on a systematic screen for morphological abnormalities of the mating process in fission yeast Schizosaccharomyces pombe. We derived a homothallic (self-fertile) collection of viable deletions, which, upon visual screening, revealed a plethora of phenotypes affecting all stages of the mating process, including cell polarization, cell fusion and sporulation. Cell fusion relies on the formation of the fusion focus, an aster-like F-actin structure that is marked by strong local accumulation of the myosin V Myo52, which concentrates secretion at the fusion site. A secondary screen for fusion-defective mutants identified the myosin V Myo51-associated coiled-coil proteins Rng8 and Rng9 as critical for the coalescence of the fusion focus. Indeed, rng8Δ and rng9Δ mutant cells exhibit multiple stable dots at the cell-cell contact site, instead of the single focus observed in wildtype. Rng8 and Rng9 accumulate on the fusion focus, dependent on Myo51 and tropomyosin Cdc8. A tropomyosin mutant allele, which compromises Rng8/9 localization but not actin binding, similarly leads to multiple stable dots instead of a single focus. By contrast, myo51 deletion does not strongly affect fusion focus coalescence. We propose that focusing of the actin filaments in the fusion aster primarily relies on Rng8/9-dependent cross-linking of tropomyosin-actin filaments.
Assuntos
Proteínas de Ciclo Celular/genética , Miosina Tipo V/genética , Miosinas/genética , Proteínas de Schizosaccharomyces pombe/genética , Citoesqueleto de Actina/genética , Citoesqueleto de Actina/metabolismo , Actinas/genética , Sequência de Aminoácidos/genética , Citoesqueleto/genética , Citoesqueleto/metabolismo , Miosina Tipo V/metabolismo , Fenótipo , Ligação Proteica , Reprodução/genética , Schizosaccharomyces/genética , Deleção de SequênciaRESUMO
In mating fission yeast cells, sensing and response to extracellular pheromone concentrations occurs through an exploratory Cdc42 patch that stochastically samples the cell cortex before stabilizing towards a mating partner. Active Ras1 (Ras1-GTP), an upstream regulator of Cdc42, and Gap1, the GTPase-activating protein for Ras1, localize at the patch. We developed a reaction-diffusion model of Ras1 patch appearance and disappearance with a positive feedback by a Guanine nucleotide Exchange Factor (GEF) and Gap1 inhibition. The model is based on new estimates of Ras1-GDP, Ras1-GTP and Gap1 diffusion coefficients and rates of cytoplasmic exchange studied by FRAP. The model reproduces exploratory patch behavior and lack of Ras1 patch in cells lacking Gap1. Transition to a stable patch can occur by change of Gap1 rates constants or local increase of the positive feedback rate constants. The model predicts that the patch size and number of patches depend on the strength of positive and negative feedbacks. Measurements of Ras1 patch size and number in cells overexpressing the Ras1 GEF or Gap1 are consistent with the model.
Assuntos
Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/fisiologia , Proteínas ras/metabolismo , Actinas/metabolismo , Proteínas Ativadoras de GTPase/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Modelos Biológicos , Feromônios/metabolismo , Ligação Proteica , Reprodução , Schizosaccharomyces/enzimologia , Schizosaccharomyces/metabolismo , Transdução de Sinais , Processos Estocásticos , Proteína cdc42 de Ligação ao GTP/metabolismoRESUMO
The small Rho-family GTPase Cdc42 is critical for cell polarization and polarizes spontaneously in absence of upstream spatial cues. Spontaneous polarization is thought to require dynamic Cdc42 recycling through Guanine nucleotide Dissociation Inhibitor (GDI)-mediated membrane extraction and vesicle trafficking. Here, we describe a functional fluorescent Cdc42 allele in fission yeast, which demonstrates Cdc42 dynamics and polarization independent of these pathways. Furthermore, an engineered Cdc42 allele targeted to the membrane independently of these recycling pathways by an amphipathic helix is viable and polarizes spontaneously to multiple sites in fission and budding yeasts. We show that Cdc42 is highly mobile at the membrane and accumulates at sites of activity, where it displays slower mobility. By contrast, a near-immobile transmembrane domain-containing Cdc42 allele supports viability and polarized activity, but does not accumulate at sites of activity. We propose that Cdc42 activation, enhanced by positive feedback, leads to its local accumulation by capture of fast-diffusing inactive molecules.
Assuntos
Actinas/metabolismo , Polaridade Celular , Inibidores de Dissociação do Nucleotídeo Guanina/metabolismo , Schizosaccharomyces/metabolismo , Proteína cdc42 de Ligação ao GTP/metabolismo , Alelos , Corantes Fluorescentes , Transporte Proteico , Schizosaccharomyces/citologia , Proteína cdc42 de Ligação ao GTP/genéticaRESUMO
Plants strictly regulate the uptake and distribution of Zn, which is essential for plant growth and development. Here, we show that Arabidopsis thaliana PCR2 is essential for Zn redistribution and Zn detoxification. The pcr2 loss-of-function mutant was compromised in growth, both in Zn-excessive and -deficient conditions. The roots of pcr2 accumulated more Zn than did control plants, whereas the roots of plants overexpressing PCR2 contained less Zn, indicating that PCR2 removes Zn from the roots. Consistent with a role for PCR2 as a Zn-efflux transporter, PCR2 reduced the intracellular concentration of Zn when expressed in yeast cells. PCR2 is located mainly in epidermal cells and in the xylem of young roots, while it is expressed in epidermal cells in fully developed roots. Zn accumulated in the epidermis of the roots of pcr2 grown under Zn-limiting conditions, whereas it was found in the stele of wild-type roots. The transport pathway mediated by PCR2 does not seem to overlap with that mediated by the described Zn translocators (HMA2 and HMA4) since the growth of pcr2 hma4 double and pcr2 hma2 hma4 triple loss-of-function mutants was more severely inhibited than the individual single knockout mutants, both under conditions of excess or deficient Zn. We propose that PCR2 functions as a Zn transporter essential for maintaining an optimal Zn level in Arabidopsis.
Assuntos
Proteínas de Arabidopsis/fisiologia , Arabidopsis/metabolismo , Zinco/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Transporte Biológico , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Local Cdc42 GTPase activation promotes polarized exocytosis, resulting in membrane flows that deplete low-mobility membrane-associated proteins from the growth region. To investigate the self-organizing properties of the Cdc42 secretion-polarization system under membrane flow, we developed a reaction-diffusion particle model. The model includes positive feedback activation of Cdc42, hydrolysis by GTPase-activating proteins (GAPs), and flow-induced displacement by exo/endocytosis. Simulations show how polarization relies on flow-induced depletion of low mobility GAPs. To probe the role of Cdc42 mobility in the fission yeast Schizosaccharomyces pombe, we changed its membrane binding properties by replacing its prenylation site with 1, 2 or 3 repeats of the Rit1 C terminal membrane binding domain (ritC), yielding alleles with progressively lower unbinding and diffusion rates. Concordant modelling predictions and experimental observations show that lower Cdc42 mobility results in lower Cdc42 activation level and wider patches. Indeed, while Cdc42-1ritC cells are viable and polarized, Cdc42-2ritC polarize poorly and Cdc42-3ritC is inviable. The model further predicts that GAP depletion increases Cdc42 activity at the expense of loss of polarization. Experiments confirm this prediction, as deletion of Cdc42 GAPs restores viability to Cdc42-3ritC cells. Our combined experimental and modelling studies demonstrate how membrane flows are an integral part of Cdc42-driven pattern formation.
RESUMO
Plant development and physiology are widely determined by the polar transport of the signaling molecule auxin. This process is controlled on the cellular efflux level catalyzed by members of the PIN (pin-formed) and ABCB (ATP-binding cassette protein subfamily B)/P-glycoprotein family that can function independently and coordinately. In this study, we have identified by means of chemical genomics a novel auxin transport inhibitor (ATI), BUM (2-[4-(diethylamino)-2-hydroxybenzoyl]benzoic acid), that efficiently blocks auxin-regulated plant physiology and development. In many respects, BUM resembles the functionality of the diagnostic ATI, 1-N-naphtylphtalamic acid (NPA), but it has an IC(50) value that is roughly a factor 30 lower. Physiological analysis and binding assays identified ABCBs, primarily ABCB1, as key targets of BUM and NPA, whereas PIN proteins are apparently not directly affected. BUM is complementary to NPA by having distinct ABCB target spectra and impacts on basipetal polar auxin transport in the shoot and root. In comparison with the recently identified ATI, gravacin, it lacks interference with ABCB membrane trafficking. Individual modes or targets of action compared with NPA are reflected by apically shifted root influx maxima that might be the result of altered BUM binding preferences or affinities to the ABCB nucleotide binding folds. This qualifies BUM as a valuable tool for auxin research, allowing differentiation between ABCB- and PIN-mediated efflux systems. Besides its obvious application as a powerful weed herbicide, BUM is a bona fide human ABCB inhibitor with the potential to restrict multidrug resistance during chemotherapy.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Genômica , Ácidos Indolacéticos/metabolismo , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/química , Arabidopsis/efeitos dos fármacos , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/metabolismo , Ácido Benzoico/química , Ácido Benzoico/metabolismo , Ácido Benzoico/farmacologia , Transporte Biológico/efeitos dos fármacos , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Modelos Moleculares , Conformação Proteica , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Especificidade por SubstratoRESUMO
The fission yeast cells Schizosaccharomyces pombe divide at constant cell size regulated by environmental stimuli. An important pathway of cell size control involves the membrane-associated DYRK-family kinase Pom1, which forms decreasing concentration gradients from cell poles and inhibits mitotic inducers at midcell. Here, we identify the phosphatase 2C Ptc1 as negative regulator of Pom1. Ptc1 localizes to cell poles in a manner dependent on polarity and cell-wall integrity factors. We show that Ptc1 directly binds Pom1 and can dephosphorylate it in vitro but modulates Pom1 localization indirectly upon growth in low-glucose conditions by influencing microtubule stability. Thus, Ptc1 phosphatase plays both direct and indirect roles in the Pom1 cell size control pathway.
Assuntos
Proteínas Quinases/metabolismo , Proteína Fosfatase 2C/metabolismo , Proteína Fosfatase 2/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Proteínas de Ciclo Celular/metabolismo , Membrana Celular/metabolismo , Polaridade Celular , Tamanho Celular , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Mitose , Fosforilação , Proteínas Quinases/genética , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/genéticaRESUMO
Cells self-organize using reaction-diffusion and fluid-flow principles. Whether bulk membrane flows contribute to cell patterning has not been established. Here, using mathematical modeling, optogenetics, and synthetic probes, we show that polarized exocytosis causes lateral membrane flows away from regions of membrane insertion. Plasma membraneassociated proteins with sufficiently low diffusion and/or detachment rates couple to the flows and deplete from areas of exocytosis. In rod-shaped fission yeast cells, zones of Cdc42 GTPase activity driving polarized exocytosis are limited by GTPase activating proteins (GAPs). We show that membrane flows pattern the GAP Rga4 distribution and that coupling of a synthetic GAP to membrane flows is sufficient to establish the rod shape. Thus, membrane flows induced by Cdc42-dependent exocytosis form a negative feedback restricting the zone of Cdc42 activity.
RESUMO
Sterols are crucial components of biological membranes, which are synthetized in the ER and accumulate in the plasma membrane (PM). Here, by applying a genetically encoded sterol biosensor (D4H), we visualize a sterol flow between PM and endosomes in the fission yeast Schizosaccharomyces pombe. Using time-lapse and correlative light-electron microscopy, we found that inhibition of Arp2/3-dependent F-actin assembly promotes the reversible relocalization of D4H from the PM to internal sterol-rich compartments (STRIC) labeled by synaptobrevin Syb1. Retrograde sterol internalization to STRIC is independent of endocytosis or an intact Golgi, but depends on Ltc1, a LAM/StARkin-family protein localized to ER-PM contact sites. The PM in ltc1Δ cells over-accumulates sterols and upon Arp2/3 inhibition forms extended ER-interacting invaginations, indicating that sterol transfer contributes to PM size homeostasis. Anterograde sterol movement from STRIC is independent of canonical vesicular trafficking but requires Arp2/3, suggesting a novel role for this complex. Thus, transfer routes orthogonal to vesicular trafficking govern the flow of sterols in the cell.
Assuntos
Complexo 2-3 de Proteínas Relacionadas à Actina/antagonistas & inibidores , Antiporters/metabolismo , Membrana Celular/metabolismo , Endossomos/metabolismo , Schizosaccharomyces/metabolismo , Esteróis/metabolismo , Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Actinas/metabolismo , Antiporters/genética , Técnicas Biossensoriais , Membrana Celular/efeitos dos fármacos , Membrana Celular/genética , Endocitose/efeitos dos fármacos , Endocitose/genética , Retículo Endoplasmático/genética , Retículo Endoplasmático/metabolismo , Endossomos/efeitos dos fármacos , Endossomos/ultraestrutura , Genes Reporter , Complexo de Golgi/metabolismo , Microscopia Eletrônica de Transmissão , Oxigenases de Função Mista/genética , Oxigenases de Função Mista/metabolismo , Mutação , Proteínas R-SNARE/metabolismoRESUMO
In the fission yeast Schizosaccharomyces pombe, pheromone signaling engages a signaling pathway composed of a G protein-coupled receptor, Ras, and a mitogen-activated protein kinase (MAPK) cascade that triggers sexual differentiation and gamete fusion. Cell-cell fusion requires local cell wall digestion, which relies on an initially dynamic actin fusion focus that becomes stabilized upon local enrichment of the signaling cascade on the structure. We constructed a live-reporter of active Ras1 (Ras1-guanosine triphosphate [GTP]) that shows Ras activity at polarity sites peaking on the fusion structure before fusion. Remarkably, constitutive Ras1 activation promoted fusion focus stabilization and fusion attempts irrespective of cell pairing, leading to cell lysis. Ras1 activity was restricted by the guanosine triphosphatase-activating protein Gap1, which was itself recruited to sites of Ras1-GTP and was essential to block untimely fusion attempts. We propose that negative feedback control of Ras activity restrains the MAPK signal and couples fusion with cell-cell engagement.
Assuntos
Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/enzimologia , Proteínas ras/metabolismo , Polaridade Celular , Ativação Enzimática , Retroalimentação Fisiológica , Proteínas Ativadoras de GTPase/genética , Proteínas Ativadoras de GTPase/metabolismo , Regulação Enzimológica da Expressão Gênica , Regulação Fúngica da Expressão Gênica , Cinética , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/genética , Schizosaccharomyces/genética , Schizosaccharomyces/crescimento & desenvolvimento , Proteínas de Schizosaccharomyces pombe/genética , Transdução de Sinais , Proteínas ras/genéticaRESUMO
Previously, the immunophilin-like protein TWD1 from Arabidopsis has been demonstrated to interact with the ABC transporters AtPGP1 and its closest homologue, AtPGP19. Physiological and biochemical investigation of pgp1/pgp19 and of twd1 plants suggested a regulatory role of TWD1 on AtPGP1/AtPGP19 transport activities. To further understand the dramatic pleiotropic phenotype that is caused by loss-of-function mutation of the TWD1 gene, we were interested in other TWD1 interacting proteins. AtMRP1, a multidrug resistance-associated (MRP/ABCC)-like ABC transporter, has been isolated in a yeast two-hybrid screen. We demonstrate molecular interaction between TWD1 and ABC transporters AtMRP1 and its closest homologue, AtMRP2. Unlike AtPGP1, AtMRP1 binds to the C-terminal tetratricopeptide repeat domain of TWD1, which is well known to mediate protein-protein interactions. Domain mapping proved that TWD1 binds to a motif of AtMRP1 that resembles calmodulin-binding motifs; and calmodulin binding to the C-terminus of MRP1 was verified. By membrane fractionation and GFP-tagging, we localized AtMRP1 to the central vacuolar membrane and the TWD1-AtMRP1 complex was verified in vivo by coimmunoprecipitation. We were able to demonstrate that TWD1 binds to isolated vacuoles and has a significant impact on the uptake of metolachlor-GS and estradiol-beta-glucuronide, well-known substrates of vacuolar transporters AtMRP1 and AtMRP2.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Proteínas de Ligação a Tacrolimo/metabolismo , Vacúolos/metabolismo , Transportadores de Cassetes de Ligação de ATP/análise , Transportadores de Cassetes de Ligação de ATP/genética , Acetamidas/análise , Acetamidas/metabolismo , Sequência de Aminoácidos , Proteínas de Arabidopsis/análise , Proteínas de Arabidopsis/genética , Proteínas de Ligação a Calmodulina/genética , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Alinhamento de Sequência , Proteínas de Ligação a Tacrolimo/genética , Técnicas do Sistema de Duplo-Híbrido , Vacúolos/químicaRESUMO
Cell pairing is central for many processes, including immune defense, neuronal connection, hyphal fusion, and sexual reproduction. How does a cell orient toward a partner, especially when faced with multiple choices? Fission yeast Schizosaccharomyces pombe P and M cells, which respectively express P and M factor pheromones [1, 2], pair during the mating process induced by nitrogen starvation. Engagement of pheromone receptors Map3 and Mam2 [3, 4] with their cognate pheromone ligands leads to activation of the Gα protein Gpa1 to signal sexual differentiation [3, 5, 6]. Prior to cell pairing, the Cdc42 GTPase, a central regulator of cell polarization, forms dynamic zones of activity at the cell periphery at distinct locations over time [7]. Here we show that Cdc42-GTP polarization sites contain the M factor transporter Mam1, the general secretion machinery, which underlies P factor secretion, and Gpa1, suggesting that these are sub-cellular zones of pheromone secretion and signaling. Zone lifetimes scale with pheromone concentration. Computational simulations of pair formation through a fluctuating zone show that the combination of local pheromone release and sensing, short pheromone decay length, and pheromone-dependent zone stabilization leads to efficient pair formation. Consistently, pairing efficiency is reduced in the absence of the P factor protease. Similarly, zone stabilization at reduced pheromone levels, which occurs in the absence of the predicted GTPase-activating protein for Ras, leads to reduction in pairing efficiency. We propose that efficient cell pairing relies on fluctuating local signal emission and perception, which become locked into place through stimulation.
Assuntos
Feromônios/metabolismo , Proteínas de Schizosaccharomyces pombe/química , Schizosaccharomyces/citologia , Transportadores de Cassetes de Ligação de ATP/metabolismo , Fusão Celular , Polaridade Celular , Schizosaccharomyces/fisiologia , Proteínas de Schizosaccharomyces pombe/metabolismo , Transdução de Sinais , Proteína cdc42 de Ligação ao GTP/metabolismoRESUMO
Previous data have suggested an involvement of MDR/PGP-like ABC transporters in transport of the plant hormone auxin and, recently, AtPGP1 has been demonstrated to catalyze the primary active export of auxin. Here we show that related isoform AtPGP4 is expressed predominantly during early root development. AtPGP4 loss-of-function plants reveal enhanced lateral root initiation and root hair lengths both known to be under the control of auxin. Further, atpgp4 plants show altered sensitivities toward auxin and the auxin transport inhibitor, NPA. Finally, mutant roots reveal elevated free auxin levels and reduced auxin transport capacities. These results together with yeast growth assays suggest a direct involvement of AtPGP4 in auxin transport processes controlling lateral root and root hair development.
Assuntos
Transportadores de Cassetes de Ligação de ATP/fisiologia , Proteínas de Arabidopsis/fisiologia , Arabidopsis/crescimento & desenvolvimento , Ácidos Indolacéticos/fisiologia , Raízes de Plantas/fisiologia , Sequência de Bases , Primers do DNARESUMO
The exocyst complex is essential for many exocytic events, by tethering vesicles at the plasma membrane for fusion. In fission yeast, polarized exocytosis for growth relies on the combined action of the exocyst at cell poles and myosin-driven transport along actin cables. We report here the identification of fission yeast Schizosaccharomyces pombe Sec3 protein, which we identified through sequence homology of its PH-like domain. Like other exocyst subunits, sec3 is required for secretion and cell division. Cells deleted for sec3 are only conditionally lethal and can proliferate when osmotically stabilized. Sec3 is redundant with Exo70 for viability and for the localization of other exocyst subunits, suggesting these components act as exocyst tethers at the plasma membrane. Consistently, Sec3 localizes to zones of growth independently of other exocyst subunits but depends on PIP(2) and functional Cdc42. FRAP analysis shows that Sec3, like all other exocyst subunits, localizes to cell poles largely independently of the actin cytoskeleton. However, we show that Sec3, Exo70 and Sec5 are transported by the myosin V Myo52 along actin cables. These data suggest that the exocyst holocomplex, including Sec3 and Exo70, is present on exocytic vesicles, which can reach cell poles by either myosin-driven transport or random walk.
Assuntos
Actinas/metabolismo , Polaridade Celular , Complexos Multiproteicos/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/citologia , Proteínas de Transporte Vesicular/metabolismo , Sequência de Aminoácidos , Modelos Biológicos , Dados de Sequência Molecular , Miosinas/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Transporte Proteico , Proteínas de Schizosaccharomyces pombe/química , Proteínas de Transporte Vesicular/química , Proteína cdc42 de Ligação ao GTP/metabolismoRESUMO
The immunophilin-like FKBP42 TWISTED DWARF1 (TWD1) has been shown to control plant development via the positive modulation of ABCB/P-glycoprotein (PGP)-mediated transport of the plant hormone auxin. TWD1 functionally interacts with two closely related proteins, ABCB1/PGP1 and ABCB19/PGP19/MDR1, both of which exhibit the ability to bind to and be inhibited by the synthetic auxin transport inhibitor N-1-naphylphtalamic acid (NPA). They are also inhibited by flavonoid compounds, which are suspected modulators of auxin transport. The mechanisms by which flavonoids and NPA interfere with auxin efflux components are unclear. We report here the specific disruption of PGP1-TWD1 interaction by NPA and flavonoids using bioluminescence resonance energy transfer with flavonoids functioning as a classical established inhibitor of mammalian and plant PGPs. Accordingly, TWD1 was shown to mediate modulation of PGP1 efflux activity by these auxin transport inhibitors. NPA bound to both PGP1 and TWD1 but was excluded from the PGP1-TWD1 complex expressed in yeast, suggesting a transient mode of action in planta. As a consequence, auxin fluxes and gravitropism in twd1 roots are less affected by NPA treatment, whereas TWD1 gain-of-function promotes root bending. Our data support a novel model for the mode of drug-mediated P-glycoprotein regulation mediated via protein-protein interaction with immunophilin-like TWD1.
Assuntos
Subfamília B de Transportador de Cassetes de Ligação de ATP/química , Arabidopsis/metabolismo , Imunofilinas/química , Ácidos Indolacéticos/química , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/química , Transportadores de Cassetes de Ligação de ATP/química , Proteínas de Arabidopsis/química , Transporte Biológico , Membrana Celular/metabolismo , Flavonoides/química , Homozigoto , Luminescência , Modelos Biológicos , Raízes de Plantas/metabolismo , Ligação Proteica , Proteínas de Ligação a Tacrolimo/químicaRESUMO
The rate, polarity, and symmetry of the flow of the plant hormone auxin are determined by the polar cellular localization of PIN-FORMED (PIN) auxin efflux carriers. Flavonoids, a class of secondary plant metabolites, have been suspected to modulate auxin transport and tropic responses. Nevertheless, the identity of specific flavonoid compounds involved and their molecular function and targets in vivo are essentially unknown. Here we show that the root elongation zone of agravitropic pin2/eir1/wav6/agr1 has an altered pattern and amount of flavonol glycosides. Application of nanomolar concentrations of flavonols to pin2 roots is sufficient to partially restore root gravitropism. By employing a quantitative cell biological approach, we demonstrate that flavonoids partially restore the formation of lateral auxin gradients in the absence of PIN2. Chemical complementation by flavonoids correlates with an asymmetric distribution of the PIN1 protein. pin2 complementation probably does not result from inhibition of auxin efflux, as supply of the auxin transport inhibitor N-1-naphthylphthalamic acid failed to restore pin2 gravitropism. We propose that flavonoids promote asymmetric PIN shifts during gravity stimulation, thus redirecting basipetal auxin streams necessary for root bending.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/crescimento & desenvolvimento , Flavonoides/metabolismo , Gravitropismo/fisiologia , Ácidos Indolacéticos/metabolismo , Raízes de Plantas/crescimento & desenvolvimento , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Flavonoides/genética , Gravitropismo/efeitos dos fármacos , Ácidos Indolacéticos/farmacologia , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Ftalimidas/farmacologia , Reguladores de Crescimento de Plantas/genética , Reguladores de Crescimento de Plantas/metabolismo , Reguladores de Crescimento de Plantas/farmacologia , Raízes de Plantas/genética , Raízes de Plantas/metabolismoRESUMO
The immunophilin-like protein TWISTED DWARF1 (TWD1/FKBP42) has been shown to physically interact with the multidrug resistance/P-glycoprotein (PGP) ATP-binding cassette transporters PGP1 and PGP19 (MDR1). Overlapping phenotypes of pgp1/pgp19 and twd1 mutant plants suggested a positive regulatory role of TWD1 in PGP-mediated export of the plant hormone auxin, which controls plant development. Here, we provide evidence at the cellular and plant levels that TWD1 controls PGP-mediated auxin transport. twd1 and pgp1/pgp19 cells showed greatly reduced export of the native auxin indole-3-acetic acid (IAA). Constitutive overexpression of PGP1 and PGP19, but not TWD1, enhanced auxin export. Coexpression of TWD1 and PGP1 in yeast and mammalian cells verified the specificity of the regulatory effect. Employing an IAA-specific microelectrode demonstrated that IAA influx in the root elongation zone was perturbed and apically shifted in pgp1/pgp19 and twd1 roots. Mature roots of pgp1/pgp19 and twd1 plants revealed elevated levels of free IAA, which seemed to account for agravitropic root behavior. Our data suggest a novel mode of PGP regulation via FK506-binding protein-like immunophilins, implicating possible alternative strategies to overcome multidrug resistance.