Search

[ES] El potasio es un catión monovalente clave en múltiples aspectos del crecimiento y de la supervivencia celular, como la compensación de cargas negativas de las macromoléculas, el mantenimiento del volumen celular y la turgencia, la actividad enzimática, la síntesis de proteínas, el mantenimiento del potencial de membrana y el control de pH intracelular. En las plantas, además de las funciones fisiológicas básicas a nivel celular mencionadas, este catión está implicado en un importante proceso fisiológico, el control de la apertura estomática. A través del estoma la planta controla la transpiración y el intercambio de gases, además de ser una vía de entrada para patógenos. Los canales de entrada de potasio (Kin) regulan la entrada de este ion en las células oclusivas y participan en el control de la apertura estomática. KAT1 y su ortólogo KAT2 son los principales canales de entrada de potasio localizados en la membrana plasmática de las células oclusivas, las cuales forman el poro estomático. La identificación y caracterización funcional de reguladores de canales de potasio es una aproximación prometedora de cara a mejorar la tolerancia a sequía, así como la susceptibilidad a patógenos. En el presente proyecto caracterizamos la función de MEE31 y la interacción entre MEE31 y KAT1. MEE31 es una proteína que surgió como posible interactor de KAT1 en un rastreo Split-Ubiquitin realizado en nuestro laboratorio. Para ello se realizaron estudios de complementación funcional en distintos fondos mutantes de S. cerevisiae. Adicionalmente, se realizaron ensayos de interacción proteína-proteína y estudios de localización celular en plantas modelo. Además se presenta un análisis de los estudios previos de la función de MEE31 con el objetivo de desarrollar hipótesis contrastables sobre los posibles roles de MEE31 como regulador de KAT1., [EN] Potassium is a key monovalent cation in multiple aspects of cell growth and survival, such as the compensation of negatives charges of macromolecules, maintenance of cell volume and turgor, enzymatic activity, protein synthesis, maintenance of membrane potential and intracellular pH control. In plants, in addition to the basic physiological functions mentioned at the cellular level, this cation is involved in an important physiological process, the control of stomatal opening. Through the stomata the plant controls transpiration and gas exchange, in addition to being a path of entry for pathogens. The inward rectifying potassium channels (Kin) regulate the entry of this ion into the guard cells thus participate in the control of stomatal opening. KAT1, and its orthologue KAT2, are the main inward rectifying channels located in the plasma membrane of the guard cells that form the stomatal pore, where they form homo- or heterotetrameric type channels. The identification and functional characterization of potassium channel regulators is a promising approach to improve drought tolerance, as well as susceptibility to pathogens. In the present project, we functionally characterized the function and interaction between MEE31 and KAT1. MEE31 is a protein that emerged as a possible KAT1 interactor in a Split-Ubiquitin screen carried out in our laboratory. For this, functional complementation studies were carried out in different S. cerevisiae mutant backgrounds. In addition, we performed protein-protein interaction assays and cell localization studies in a plant model. We also present an analysis of the prior studies of MEE31 function in order to develop testable hypotheses about the possible roles of MEE31 as a KAT1 regulator.

[ES] El correcto funcionamiento de cualquier organismo requiere el mantenimiento de la homeostasis iónica, un proceso dinámico esencial que debe garantizar la presencia de iones a niveles adecuados, evitando su acumulación. Para ello, las células han desarrollado sistemas altamente eficientes que se encargan de captar y almacenar estos elementos, así como de permitir respuestas rápidas frente a posibles cambios ambientales. El potasio es el catión más abundante en las plantas, siendo también un nutriente esencial para estas. Tal es su importancia que juega un papel clave en funciones como la síntesis proteica, la activación enzimática o la turgencia celular. En relación con esta última, el flujo de potasio a través de las membranas de las células oclusivas regula su turgencia y, con ello, también la apertura o cierre estomáticos. Es a través de estas estructuras celulares, los estomas, que la célula es capaz de realizar tanto el intercambio gaseoso como la transpiración. La entrada de potasio en las células oclusivas viene regulada por distintos canales de entrada de potasio, entre estos se encuentra la familia Shaker de canales dependientes de voltaje. Pertenecientes a esta familia, en la membrana de las células oclusivas, se encuentran los principales responsables de dicha entrada de potasio: KAT1 y su homólogo KAT2. Dada su implicación directa en la apertura estomática, estos canales ganan importancia frente a un posible estrés abiótico como la sequía. En estudios previos, nuestro grupo identificó diferentes proteínas de Arabidopsis thaliana capaces de interaccionar con KAT1 mediante un rastreo basado en el sistema Split-Ubiquitina. Una de las proteínas identificadas fue BAG4, miembro de la familia BAG, que se caracteriza por un dominio BAG conservado cerca de la región C-terminal de sus proteínas. Este dominio interacciona directamente con una clase de las proteínas de choque térmico Hsp70, regulando así su actividad. En base a la función de sus homólogos en mamíferos y a distintas pruebas experimentales, se cree que esta familia está implicada en la regulación de procesos como la resistencia a patógenos o a estrés abiótico. También se demostró que BAG4 regula KAT1, de forma que su co-expresión mejora la actividad de KAT1, pero la falta de expresión de BAG4 lleva a un retraso en la apertura estomática. Por tanto, la caracterización detallada de la interacción entre KAT1 y BAG4, así como el estudio de los efectos que tiene en la capacidad reguladora de BAG4, pueden tener gran repercusión en los estudios de las respuestas de las plantas frente a la sequía. Mediante el sistema de clonación MoClo se diseñaron las construcciones necesarias para llevar a cabo esta caracterización. Las construcciones obtenidas se probaron, y los resultados preliminares muestran que las construcciones MoClo presentan una actividad reducida en el ensayo de interacción proteína-proteína Split-Ubiquitina, en comparación con los plásmidos control en base a los que se diseñaron. Sin embargo, observamos interacción entre KAT1 y BAG4, demostrando así la capacidad de estos plásmidos de ser usados para un análisis detallado de esta interacción., [EN] The correct functioning of any type of organism requires the maintenance of ionic homeostasis. This is an essential, dynamic process that must guarantee the presence of ions at The correct functioning of any organism requires the maintenance of ionic homeostasis, an essential, dynamic process that must guarantee the presence of ions at adequate levels, preventing them from accumulating. To this end, cells have developed highly efficient systems that capture and store these elements, as well as allowing rapid responses to possible environmental changes. Potassium is the most abundant cation in plants, and is also an essential nutrient for them. This cation plays a key role in functions such as protein synthesis, enzyme activation or cell turgor. In relation to the latter, the flow of potassium through the membranes of the guard cells regulates their turgidity and, therefore, the opening or closing of the stomata. It is through these pore structures, the stomata, that the cell is able to perform both gas exchange and transpiration. The entry of potassium into the guard cells is regulated by different potassium entry channels, including the Shaker family of voltage-dependent channels. KAT1 and its close homologue KAT2 are Shaker family channels highly expressed in the guard cell membrane, that play a major role in potassium uptake in this cell type. Given their direct involvement in the stomatal opening, the regulation of the activity of these channels is crucial for the response to abiotic stresses such as drought. In previous studies, our group identified different proteins capable of interacting with KAT1 using a Split-ubiquitin screening in yeast. One of the identified proteins was BAG4, a member of the BAG family, which is characterized by a common BAG domain near the C-terminal region of these proteins. This characteristic domain interacts directly with the Heat Shock 70 (Hsp70) proteins, thus regulating their activity. Based on the role of their mammalian counterparts and various experimental tests, this family is believed to be involved in the regulation of processes such as resistance to pathogens or abiotic stress. In the same study, our group also proved that BAG4 regulates the KAT1 channel, so that its co-expression improves the KAT1 channel activity, whereas the lack of BAG4 expression caused altered stomatal movement. Thus, the characterization of the interaction between KAT1 and BAG4, as well as the study of its effects on the regulatory capacity of BAG4, could have great repercussions in the study of drought responses. Using the modular cloning system MoClo, the constructs needed to perform this characterization were designed. The completed plasmids were tested. Our preliminary results show that the MoClo constructs present a reduced activity in the Split-Ubiquitin protein-protein interaction assay, as compared to the control plasmids upon which our design was based. However, we could observe the protein-protein interaction between KAT1 and BAG4, demonstrating the utility os these constructs for the detailed analysis of this interaction.

Cdc42 organizes cellular polarity and directs the formation of cellular structures in many organisms. By locating Cdc24, the source of active Cdc42, to the growing front of the yeast cell, the scaffold protein Bem1, is instrumental in shaping the cellular gradient of Cdc42. This gradient instructs bud formation, bud growth, or cytokinesis through the actions of a diverse set of effector proteins. To address how Bem1 participates in these transformations, we systematically tracked its protein interactions during one cell cycle to define the ensemble of Bem1 interaction states for each cell cycle stage. Mutants of Bem1 that interact with only a discrete subset of the interaction partners allowed to assign specific functions to different interaction states and identified the determinants for their cellular distributions. The analysis characterizes Bem1 as a cell cycle–specific shuttle that distributes active Cdc42 from its source to its effectors. It further suggests that Bem1 might convert the PAKs Cla4 and Ste20 into their active conformations., publishedVersion

The polarisome comprises a network of proteins that organizes polar growth in yeast and filamentous fungi. The yeast formin Bni1 and the actin nucleation-promoting factor Bud6 are subunits of the polarisome that together catalyze the formation of actin cables below the tip of yeast cells. We identified YFR016c (Aip5) as an interaction partner of Bud6 and the polarisome scaffold Spa2. Yeast cells lacking Aip5 display a reduced number of actin cables. Aip5 binds with its N-terminal region to Spa2 and with its C-terminal region to Bud6. Both interactions collaborate to localize Aip5 at bud tip and neck, and are required to stimulate the formation of actin cables. Our experiments characterize Aip5 as a novel subunit of a complex that regulates the number of actin filaments at sites of polar growth., Summary: YFR016c/Aip5 binds to the polarisome components Bud6 and Spa2 and supports the polarisome in the formation of actin cables in yeast cells.

Yeast two-hybrid (Y2H) is a well-established genetics-based system that uses yeast to selectively display binary protein-protein interactions (PPIs). To meet the current need to unravel complex PPI networks, several adaptations have been made to establish medium- to high-throughput Y2H screening platforms, with several having successfully incorporated the use of the next-generation sequencing (NGS) technology to increase the scale and sensitivity of the method. However, these have been to date mainly restricted to the use of fully annotated custom-made open reading frame (ORF) libraries and subject to complex downstream data processing. Here, a streamlined Y2H library screening strategy, based on integration of Y2H with NGS, called Y2H-seq, was developed, which allows efficient and reliable screening of Y2H cDNA libraries. To generate proof of concept, the method was applied to screen for interaction partners of two key components of the jasmonate signaling machinery in the model plant Arabidopsis thaliana, resulting in the identification of several previously reported as well as hitherto unknown interactors. Our Y2H-seq method offers a user-friendly, specific and sensitive screening method that allows identification of PPIs without prior knowledge of the organism’s ORFs, thereby extending the method to organisms of which the genome has not entirely been annotated yet. The quantitative NGS readout allows to increase genome coverage, thereby overcoming some of the bottlenecks of current Y2H technologies, which will further strengthen the value of the Y2H technology as a discovery platform.

[ES] La homeostasis del ion potasio es clave para la supervivencia y el crecimiento de los seres vivos. El flujo de entrada de este ion en la célula está regulado por unos canales localizados en su membrana plasmática. Dada la gran importancia de su correcto funcionamiento estos transportadores se encuentran altamente regulados. En este proyecto nos hemos centrado en dos canales de potasio prototipo: el canal rectificador de entrada KAT1 de Arabidopsis thaliana y el trasportador de alta afinidad de potasio de Saccharomyces cerevisiae Trk1. El canal rectificador de entrada KAT1 pertenece a la familia Shaker y participa en la apertura y el cierre de estomas. Por esta razón su regulación es fundamental para la resistencia de la planta al estrés hídrico. En estudios anteriores utilizando el ensayo de interacción proteína-proteína Split-Ubiquitin nuestro grupo identificó 18 genes de A. thaliana cuyas proteínas interaccionan físicamente con KAT1. Sin embargo, para confirmar la especificidad de estas interacciones son necesarios más análisis. Con este objetivo, el fin de este trabajo consistió en detectar entre estos 18 interactores posibles falsos positivos. Los falsos positivos pueden aparecer cuando las proteínas bait y las prey se acumulan en compartimentos intracelulares dando lugar a la activación inespecífica del sistema. Por ello, los análisis realizados para su detección consistieron en repetir el análisis de interacción proteína-proteína Split-Ubiquitin con las 18 proteínas candidatas utilizando la subunidad gamma del complejo oligosacariltransferasa de levadura, Ost3, como proteína bait. Con este ensayo se identificaron como falsos positivos tres de las proteínas candidatas debido a su interacción con la proteína Ost3, la cual no está relacionada con ellas. Éstas fueron descartadas para futuros análisis. Pero, lo que es más importante, se confirmó la especificidad con KAT1 de 13 de ellas. Con éstas se llevarán a cabo futuros ensayos en planta para determinar si son verdaderas reguladoras de KAT1. El otro objetivo fue generar herramientas para poder aplicar el análisis de interacción proteínaproteína Split-Ubiquitin al transportador de potasio de levadura Trk1 y al homólogo de KAT1, KAT2. En este trabajo se describe la construcción de los plásmidos bait para estos dos transportadores mediante recombinación en levadura. Con los plásmidos generados se realizó un análisis funcional inicial con el fin de comprobar que eran adecuados para próximos estudios. Entre estos estudios se incluyen rastreos propios para la identificación de posibles interactores. A pesar de que en KAT2 no observamos actividad como transportador ni capacidad de dimerizar en este sistema, sí que observamos que Trk1 era funcional e interaccionaba consigo mismo. Este resultado, aunque preliminar, es la primera confirmación experimental de la tetramerizacón de Trk1 propuesta por Guy y Durell en 1999., [EN] Potassium homeostasis is crucial for survival and development of essentially all living organisms. Inward fluxes of this ion are mediated by channels located in the plasma membrane. Due to the importance of the correct operation of these transporters, they are highly regulated. In this project, we have focused on two prototypic potassium channels: the rectifying inward cannel KAT1 from Arabidopsis thaliana and the Trk1 high affinity potassium transporter from Saccharomyces cerevisiae. The inward rectifying channel, KAT1 belongs to the Shaker family and its activity participates in stomatal opening and closing. For this reason its regulation is proposed to be fundamental for plant drought resistance. In previous studies using the Split-Ubiquitin protein-protein interaction assay, our group has identified 18 A. thaliana genes whose proteins interact physically with KAT1. However, in order to confirm the specificity of these interactions it is necessary to carry out further analyses. To this end, in this project our aim was to identify possible false positives among the 18 clones we identified. False positives may arise in cases where the prey and bait proteins artifactually accumulate in intracellular compartments leading to non-specific activation of the system. The analysis presented here consisted of repeating Split-Ubiquitin protein-protein interaction analysis with the 18 candidate clones using the gamma subunit of the yeast oligosaccharyltransferase complex, Ost3 as the bait protein. Three candidate proteins that interact with the unrelated Ost3 bait protein were identified as false positives and will be discarded from future analyses. But, more importantly, the specificity of 13 was confirmed. These proteins will be the subject of further studies in plants to determine if they are bona fide KAT1 regulators. In addition, we aimed to generate tools to apply the Split-Ubiquitin protein-protein interaction assay to the Trk1 yeast potassium transporter and the KAT1 homologue, KAT2. Here, we describe the construction of the bait vectors for both transporters using in vivo recombination in yeast. We performed an initial analysis of the function of these newly generated plasmids to determine whether they are appropriate for further studies including library screenings to identify novel interactors. Although we failed to observe KAT2 transport activity or dimerization in this system, we did observe that Trk1 is functional and that is interacts with itself. This observation, although preliminary, is the first experimental confirmation of the tetramerization of Trk1 proposed by Durell and Guy in 1999.

International audience; Scaffold proteins are known to regulate important cellular processes by interacting with multiple proteins to modulate molecular responses. RACK1 (Receptor for Activated C Kinase 1) is a WD-40 type scaffold protein, conserved in eukaryotes, from Chlamydymonas to plants and humans, expresses ubiquitously and plays regulatory roles in diverse signal transduction and stress response pathways. Here we present the use of Arabidopsis RACK1A, the predominant isoform of a 3-member family, as a bait to screen a split-ubiquitin based cDNA library. In total 97 proteins from dehydration, salt stress, ribosomal and photosynthesis pathways are found to potentially interact with RACK1A. False positive interactions were eliminated following extensive selection based growth potentials. Confirmation of a sub-set of selected interactions is demonstrated through the co-transformation with individual plasmid containing cDNA and the respective bait. Interaction of diverse proteins points to a regulatory role of RACK1A in the cross-talk between signaling pathways. Promoter analysis of the stress and photosynthetic pathway genes revealed conserved transcription factor binding sites. RACK1A is known to be a multifunctional protein and the current identification of potential interacting proteins and future in vivo elucidations of the physiological basis of such interactions will shed light on the possible molecular mechanisms that RACK1A uses to regulate diverse signaling pathways.

"Las dehidrinas (DHNs) o el grupo 2 de las proteínas LEA (Late Embryogenesis Abundant), son proteínas intrínsecamente desordenadas y altamente hidrofílicas, que típicamente se acumulan durante los estadios tardíos del desarrollo de la semilla o se inducen en tejidos vegetativos durante condiciones de déficit hídrico. Se ha reportado que las DHNs se distribuyen en diversos compartimentos celulares como: el citosol, núcleo, cloroplastos, mitocondria, retículo endoplásmico y membrana plasmática. Diversos estudios apuntan a que sus funciones comunes son: la unión a fosfolípidos ácidos, iones, crioprotección y la estabilización de membranas o proteínas durante condiciones de estrés abiótico Sin embargo, su mecanismo de acción aún no está completamente entendido. Recientemente, nuestro grupo reportó que plantas transgénicas de Arabidopsis que sobreexpresan una dehidrina de Opuntia streptacantha (OpsDHN1) muestran un incremento en la tolerancia a estrés por congelamiento. Para identificar las proteínas que interactúan con OpsDHN1 a través de las cuales ejerce dicho efecto, llevamos a cabo un ensayo de doble híbrido basado en la técnica splitubiquitin, a través del cual identificamos posibles proteínas interactoras de la dehidrina de nopal (OpsDHN1). Dentro de estos interactores encontramos proteínas de membrana como: RPT2B, PIP2;2 y ARA-5 y proteínas de cloroplasto como: NPQ4. Además, identificamos mayoritariamente una proteína de función desconocida (At1g44920) que se localiza en el cloroplasto. Por otro lado, demostramos que la OpsDHN1 puede interactuar consigo misma, lo que sugiere la formación de dímeros de ésta. La identificación de estos posibles interactores, incluyendo a la propia OpsDHN1, nos proporciona una valiosa información respecto a la identidad de las proteínas con las cuales puede participar esta dehidrina; lo que nos podría llevar a entender mejor los mecanismos moleculares mediante los cuales llevan a cabo su función las dehidrinas." "Dehydrins (DHNs) or group 2 LEA (Late Embryogenesis Abundant) proteins are intrinsically unstructured and highly hydrophilic proteins, they typically accumulate during the last stages of seed development or are induced in vegetative tissues under water stress conditions. It has been reported that DHNs are distributed in diverse cell compartments such as cytosol, nucleus, chloroplasts, mitochondria, endoplasmic reticulum and plasma membrane. Based on diverse genetic and transgenic studies it has been suggested that the common roles for dehydrins are binding to acidic phospholipids, to ions, cryoprotection and to stabilize membrane structures or proteins during abiotic stress conditions. Previously, our research group reported that transgenic Arabidopsis plants overexpressing a dehydrin from Opuntia streptacantha (OpsDHN1) show enhanced tolerance to freezing stress. In order to identify the OpsDHN1 interacting proteins through which it exerts this effect, we performed a yeast two-hybrid assay based on the split-ubiquitin technique, by which we identified potential interacting proteins of the dehydrin from cactus (OpsDHN1). Within these interactors are membrane proteins such as: RPT2B, PIP2;2 and ARA-5, and the chloroplast protein NPQ4. In addition, we identified an abundantly protein of unknown function (At1g44920) located in the chloroplast. Furthermore, we demonstrated that OpsDHN1 can interact with itself, suggesting the formation of homo-dimers. The identification of these potential interactors, including OpsDHN1 provides us with valuable information regarding the identity of the proteins with which dehydrin can participate and this could lead us to a better understanding of the molecular mechanisms by which dehydrins could perform their function."

In higher plants, cellulose is synthesized by so-called rosette protein complexes with cellulose synthases (CESAs) as catalytic subunits of the complex. The CESAs are divided into two distinct families, three of which are thought to be specialized for the primary cell wall and three for the secondary cell wall. In this article, the potential of primary and secondary CESAs forming a functional rosette complex has been investigated. The membrane-based yeast two-hybrid and biomolecular fluorescence systems were used to assess the interactions between three primary (CESA1, CESA3, CESA6), and three secondary (CESA4, CESA7, CESA8) Arabidopsis (Arabidopsis thaliana) CESAs. The results showed that all primary CESAs can physically interact both in vitro and in planta with all secondary CESAs. Although CESAs are broadly capable of interacting in pairwise combinations, they are not all able to form functional complexes in planta. Analysis of transgenic lines showed that CESA7 can partially rescue defects in the primary cell wall biosynthesis in a weak cesa3 mutant. Green fluorescent protein-CESA protein fusions revealed that when CESA3 was replaced by CESA7 in the primary rosette, the velocity of the mixed complexes was slightly faster than the native primary complexes. CESA1 in turn can partly rescue defects in secondary cell wall biosynthesis in a cesa8ko mutant, resulting in an increase of cellulose content relative to cesa8ko. These results demonstrate that sufficient parallels exist between the primary and secondary complexes for cross-functionality and open the possibility that mixed complexes of primary and secondary CESAs may occur at particular times.

Journal of Experimental Botany, 60 (1), ISSN:1460-2431, ISSN:0022-0957

The Shaker potassium channels are tetrameric proteins formed by the assembly of four alpha-subunits. The oligomerization can occur among both homo- and hetero-alpha-subunits. KDC1 is a carrot Shaker-like potassium channel expressed in the epidermis of plantlet roots and the protoderm of somatic embryos. KDC1 was previously characterised electrophysiologically in CHO and Xenopus oocytes cells, but the experiments performed in these systems did not provide conclusive evidence that KDC1 forms a functional homomeric channel in plant cells. In this report, we show that KDC1 localizes to the plasma membrane of root cells in transgenic tobacco plants transformed with a KDC1::GFP fusion construct. In tobacco mesophyll protoplasts, transiently transformed with KDC1::GFP, KDC1 was present on the endomembrane and the protoplasts did not show any inward potassium current, as demonstrated by patch-clamp experiments. The co-expression of KDC1::GFP with the Arabidopsis thaliana potassium channel AKT1 in tobacco mesophyll protoplasts has the effect of shifting KDC1 localization from endomembranes to the plasma membrane. Patch-clamp experiments performed on tobacco mesophyll protoplasts expressing AKT1 alone or in combination with KDC1::GFP showed voltage-activated inward potassium currents with different properties. In particular, the addition of Zn2+ to the bath solution induced a clear decrease of the potassium currents in protoplasts transformed with AKT1 alone, whereas a current potentiation (indicative of KDC1 presence) was observed in protoplasts co-transformed with AKT1 + KDC1::GFP. Split-Ubiquitin assay experiments performed in yeast cells confirmed the interaction between AKT1 and KDC1.

Upon uracil depletion, the transcriptional activator Ppr1p stimulates expression of the Saccharomyces cerevisiae URA3 gene only four-fold. We performed a split-ubiquitin screen with Tup1p as bait, and we found that the global repressor Tup1p interacts with the transcriptional activator Ppr1p both in vivo and in vitro. The interaction is biologically significant, since the deletion of the TUP1 gene as well as the removal of the Tup1p-binding domain from Ppr1p results in an increased expression of the URA3 gene. Our results suggest that Tup1p blocks Ppr1p directly, and that Ppr1p is a weak activator of transcription because of its interaction with Tup1p. Thus we were able to demonstrate that the global repressor Tup1p can modulate transcription by interacting with an activator.

A method based on a combination of the Split-Ubiquitin system with two spectrally different fluorescent proteins (SPLIFF) is shown to enable measurement of protein interactions in vivo with high spatial and temporal resolution in yeast., SPLIFF visualizes protein interactions with high spatial and temporal resolution. Spc72p and Kar9p interact with the MAP Stu2p at opposite poles of microtubules. Histone chaperone Nap1p and Kcc4 kinase interact preferentially at the bud site. F-BAR protein Hof1p associates with the polarisome during cell fusion and cytokinesis., We introduce a fluorescent reporter for monitoring protein–protein interactions in living cells. The method is based on the Split-Ubiquitin method and uses the ratio of two auto-fluorescent reporter proteins as signal for interaction (SPLIFF). The mating of two haploid yeast cells initiates the analysis and the interactions are followed online by two-channel time-lapse microscopy of the diploid cells during their first cell cycle. Using this approach we could with high spatio-temporal resolution visualize the differences between the interactions of the microtubule binding protein Stu2p with two of its binding partners, monitor the transient association of a Ran-GTPase with its receptors at the nuclear pore, and distinguish between protein interactions at the polar cortical domain at different phases of polar growth. These examples further demonstrate that protein–protein interactions identified from large-scale screens can be effectively followed up by high-resolution single-cell analysis.

Interaction induced changes in the conformation of proteins are frequently the molecular basis for the modulation of their activities. Although proteins perform their functions in cells, surrounded by many potential interaction partners, the studies of their conformational changes have been mainly restricted to in vitro studies. Ste4p (G beta) and Ste18p (G gamma) are the subunits of a heterotrimeric G-protein in the yeast Saccharomyces cerevisiae. A split-ubiquitin based conformational sensor was used to detect a major structural rearrangement in Ste18p upon binding to Ste4p. Based on these in vivo results and the solved structure of the mammalian G beta gamma, we propose that G gamma of yeast adopts an equally extended structure, which is only induced upon association with G beta.