Maintaining protein homeostasis: early and late endosomal dual recycling for the maintenance of intracellular pools of the plasma membrane protein Chs3

[EN] The major chitin synthase activity in yeast cells, Chs3, has become a paradigm in the study of the intracellular traffic of transmembrane proteins due to its tightly regulated trafficking. This includes an efficient mechanism for the maintenance of an extensive reservoir of Chs3 at the trans-Golgi network/EE, which allows for the timely delivery of the protein to the plasma membrane. Here we show that this intracellular reservoir of Chs3 is maintained not only by its efficient AP-1-mediated recycling, but also by recycling through the retromer complex, which interacts with Chs3 at a defined region in its N-terminal cytosolic domain. Moreover, the N-terminal ubiquitination of Chs3 at the plasma membrane by Rsp5/Art4 distinctly labels the protein and regulates its retromer-mediated recycling by enabling Chs3 to be recognized by the ESCRT machinery and degraded in the vacuole. Therefore the combined action of two independent but redundant endocytic recycling mechanisms, together with distinct labels for vacuolar degradation, determines the final fate of the intracellular traffic of the Chs3 protein, allowing yeast cells to regulate morphogenesis, depending on environmental constraints

Ingression Progression Complexes Control Extracellular Matrix Remodelling during Cytokinesis in Budding Yeast

Eukaryotic cells must coordinate contraction of the actomyosin ring at the division site together with ingression of the plasma membrane and remodelling of the extracellular matrix (ECM) to support cytokinesis, but the underlying mechanisms are still poorly understood. In eukaryotes, glycosyltransferases that synthesise ECM polysaccharides are emerging as key factors during cytokinesis. The budding yeast chitin synthase Chs2 makes the primary septum, a special layer of the ECM, which is an essential process during cell division. Here we isolated a group of actomyosin ring components that form complexes together with Chs2 at the cleavage site at the end of the cell cycle, which we named ‘ingression progression complexes’ (IPCs). In addition to type II myosin, the IQGAP protein Iqg1 and Chs2, IPCs contain the F-BAR protein Hof1, and the cytokinesis regulators Inn1 and Cyk3. We describe the molecular mechanism by which chitin synthase is activated by direct association of the C2 domain of Inn1, and the transglutaminase-like domain of Cyk3, with the catalytic domain of Chs2. We used an experimental system to find a previously unanticipated role for the C-terminus of Inn1 in preventing the untimely activation of Chs2 at the cleavage site until Cyk3 releases the block on Chs2 activity during late mitosis. These findings support a model for the co-ordinated regulation of cell division in budding yeast, in which IPCs play a central role

Involvement of the exomer complex in the polarized transport of Ena1 required for Saccharomyces cerevisiae survival against toxic cations

[EN] Exomer is an adaptor complex required for the direct transport of a selected number of cargoes from the trans-Golgi network (TGN) to the plasma membrane in Saccharomyces cerevisiae However, exomer mutants are highly sensitive to increased concentrations of alkali metal cations, a situation that remains unexplained by the lack of transport of any known cargoes. Here we identify several HAL genes that act as multicopy suppressors of this sensitivity and are connected to the reduced function of the sodium ATPase Ena1. Furthermore, we find that Ena1 is dependent on exomer function. Even though Ena1 can reach the plasma membrane independently of exomer, polarized delivery of Ena1 to the bud requires functional exomer. Moreover, exomer is required for full induction of Ena1 expression after cationic stress by facilitating the plasma membrane recruitment of the molecular machinery involved in Rim101 processing and activation of the RIM101 pathway in response to stress. Both the defective localization and the reduced levels of Ena1 contribute to the sensitivity of exomer mutants to alkali metal cations. Our work thus expands the spectrum of exomer-dependent proteins and provides a link to a more general role of exomer in TGN organization.We acknowledge Emma Keck for English language revision. We also thank members of the Translucent group, J. Arino, J. Ramos, and L. Yenush, for many useful discussions throughout this work and especially L. Yenush for her generous gift of strains and reagents. The help of O. Vincent was essential for developing the work involving RIM101. We also thank R. Valle for her technical assistance at the CR Laboratory. M. Trautwein is acknowledged for data acquisition and discussions during the early stages of the project. C.A. is supported by a USAL predoctoral fellowship. Work at the Spang laboratory was supported by the University of Basel and the Swiss National Science Foundation (31003A-141207 and 310030B-163480). C.R. was supported by grant SA073U14 from the Regional Government of Castilla y Leon and by grant BFU2013-48582-C2-1-P from the CICYT/FEDER Spanish program. J.M.M. acknowledges the financial support from Universitat Politecnica de Valencia project PAID-06-10-1496.Anton, C.; Zanolari, B.; Arcones, I.; Wang, C.; Mulet, JM.; Spang, A.; Roncero, C. (2017). Involvement of the exomer complex in the polarized transport of Ena1 required for Saccharomyces cerevisiae survival against toxic cations. Molecular Biology of the Cell. 28(25):3672-3685. https://doi.org/10.1091/mbc.E17-09-0549S367236852825Ariño, J., Ramos, J., & Sychrová, H. (2010). Alkali Metal Cation Transport and Homeostasis in Yeasts. Microbiology and Molecular Biology Reviews, 74(1), 95-120. doi:10.1128/mmbr.00042-09Bard, F., & Malhotra, V. (2006). The Formation of TGN-to-Plasma-Membrane Transport Carriers. Annual Review of Cell and Developmental Biology, 22(1), 439-455. doi:10.1146/annurev.cellbio.21.012704.133126Barfield, R. 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Physiological characterization of Saccharomyces cerevisiae kha1 deletion mutants. Molecular Microbiology, 55(2), 588-600. doi:10.1111/j.1365-2958.2004.04410.xMarqués, M. C., Zamarbide-Forés, S., Pedelini, L., Llopis-Torregrosa, V., & Yenush, L. (2015). A functional Rim101 complex is required for proper accumulation of the Ena1 Na+-ATPase protein in response to salt stress in Saccharomyces cerevisiae. FEMS Yeast Research, 15(4). doi:10.1093/femsyr/fov017Mulet, J. M., Leube, M. P., Kron, S. J., Rios, G., Fink, G. R., & Serrano, R. (1999). A Novel Mechanism of Ion Homeostasis and Salt Tolerance in Yeast: the Hal4 and Hal5 Protein Kinases Modulate the Trk1-Trk2 Potassium Transporter. Molecular and Cellular Biology, 19(5), 3328-3337. doi:10.1128/mcb.19.5.3328Mulet, J. M., & Serrano, R. (2002). Simultaneous determination of potassium and rubidium content in yeast. Yeast, 19(15), 1295-1298. doi:10.1002/yea.909Murguía, J. R., Bellés, J. M., & Serrano, R. (1996). 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El reciclado endocítico en el control de la Actividad Quitin Sintasa III en Saccharomyces cerevisiae

Memoria presentada por la licenciada Irene Arcones Ríos para optar al grado de Doctor en Farmacia por la Universidad de Salamanca, que ha sido realizada en el Instituto de Biología Funcional y Genómica, centro mixto de la Universidad de Salamanca (Departamento de Microbiología y Genética) y el Consejo Superior de Investigaciones Científicas.La principal Actividad Quitín Sintasa de levaduras, Chs3, se ha convertido en un paradigma en el estudio del tráfico intracelular de proteínas transmembrana, debido a su tráfico finamente regulado. Éste incluye un mecanismo eficiente que mantiene un extenso reservorio de Chs3 en TGN / endosomas tempranos, permitiendo además el transporte controlado temporalmente de la proteína a la membrana plasmática. Aquí demostramos que este reservorio intracelular de Chs3 no se mantiene sólo gracias a un eficiente reciclado por el complejo AP-1, si no también por un reciclado mediado por el complejo retrómero, con el que Chs3 interacciona a través de una región definida de su extremo citosólico N-terminal. Además, la ubiquitinación N-terminal de Chs3 en la membrana plasmática por Rsp5 / Art4 distingue la proteína y regula su reciclado mediado por retrómero, permitiendo que Chs3 sea reconocida por el complejo ESCRT y degradada en la vacuola. Así, la acción combinada de dos mecanismos de reciclado endocítico, independientes pero redundantes, junto con el marcaje distintivo para la degradación vacuolar, determinan el destino del tráfico intracelular de la proteína Chs3, permitiendo a la levadura regular su morfogénesis dependiendo de las condiciones ambientales.Peer Reviewe

Maintaining protein homeostasis: early and late endosomal dual recycling for the maintenance of intracellular pools of the plasma membrane protein Chs3

The major chitin synthase activity in yeast cells, Chs3, has become a paradigm in the study of the intracellular traffic of transmembrane proteins due to its tightly regulated trafficking. This includes an efficient mechanism for the maintenance of an extensive reservoir of Chs3 at the trans-Golgi network/EE, which allows for the timely delivery of the protein to the plasma membrane. Here we show that this intracellular reservoir of Chs3 is maintained not only by its efficient AP-1-mediated recycling, but also by recycling through the retromer complex, which interacts with Chs3 at a defined region in its N-terminal cytosolic domain. Moreover, the N-terminal ubiquitination of Chs3 at the plasma membrane by Rsp5/Art4 distinctly labels the protein and regulates its retromer-mediated recycling by enabling Chs3 to be recognized by the ESCRT machinery and degraded in the vacuole. Therefore the combined action of two independent but redundant endocytic recycling mechanisms, together with distinct labels for vacuolar degradation, determines the final fate of the intracellular traffic of the Chs3 protein, allowing yeast cells to regulate morphogenesis, depending on environmental constraints.C.R. was supported by Grant SA073U14 from the Regional Government of Castilla y León and Grant BFU2013-48582-C2-1-P from the CICYT/FEDER program.Peer Reviewe

El reciclado endocítico en el control de la Actividad Quitín Sintasa III en Saccharomyces cerevisiae

[ES]La principal Actividad Quitín Sintasa de levaduras, Chs3, se ha convertido en un paradigma en el estudio del tráfico intracelular de proteínas transmembrana, debido a su tráfico finamente regulado. Éste incluye un mecanismo eficiente que mantiene un extenso reservorio de Chs3 en TGN / endosomas tempranos, permitiendo además el transporte controlado temporalmente de la proteína a la membrana plasmática. Aquí demostramos que este reservorio intracelular de Chs3 no se mantiene sólo gracias a un eficiente reciclado por el complejo AP-1, si no también por un reciclado mediado por el complejo retrómero, con el que Chs3 interacciona a través de una región definida de su extremo citosólico N-terminal. Además, la ubiquitinación N-terminal de Chs3 en la membrana plasmática por Rsp5 / Art4 distingue la proteína y regula su reciclado mediado por retrómero, permitiendo que Chs3 sea reconocida por el complejo ESCRT y degradada en la vacuola. Así, la acción combinada de dos mecanismos de reciclado endocítico, independientes pero redundantes, junto con el marcaje distintivo para la degradación vacuolar, determinan el destino del tráfico intracelular de la proteína Chs3, permitiendo a la levadura regular su morfogénesis dependiendo de las condiciones ambientales

Monitoring chitin deposition during septum assembly in budding yeast

The synthesis of the septum is a critical step during cytokinesis in the fungal cell. Moreover, in Saccharomyces cerevisiae septum assembly depends mostly on the proper synthesis and deposition of chitin and, accordingly, on the timely regulation of chitin synthases. In this chapter, we will see how to follow chitin synthesis by two complementary approaches: monitoring chitin deposition in vivo at the septum by calcofluor staining and fluorescence microscopy, and measuring the chitin synthase activities responsible for this synthesis.CR was supported by a grant GR31 from the Excellence Research program from the Junta de Castilla y Leon, and by grants BFU2010-18693 and BFU2013-48582- C2-1-P from the CICYT/FEDER program.Peer Reviewe

Phosphorylation of Bni4 by MAP kinases contributes to septum assembly during yeast cytokinesis

Previous work has shown that the synthetic lethality of the slt2Δrim101Δ mutant results from a combination of factors, including improper functioning of the septum assembly machinery. Here, we identify new multicopy suppressors of this lethality including Kss1, Pcl1 and Sph1, none of which seems to be linked to the upregulation of chitin synthesis. Characterization of the suppression mediated by Kss1 showed that it is independent of the transcriptional response of the CWI signaling response, but efficiently restores the Bni4 localization defects produced by the absence of Slt2. Accordingly, Bni4 interacts physically with both kinases, and its levels of phosphorylation are reduced in the slt2Δ mutant but increased after Kss1 overexpression. Using an assay based on hypersensitive cells of the cdc10-11 mutant, we have pinpointed several MAP kinase phosphorylatable residues required for Bni4 function. Our results, together with a genetic correlation analysis, strongly support a functional model linking Slt2 MAP kinase and Pcl1, a Pho85 cyclin-dependent kinase, in septum assembly through Bni4. This model, based on the coordinated phosphorylation of Bni4 by both kinases, would be able to integrate cellular signals rapidly to maintain cell integrity during cytokinesis.This research was supported by grants BFU2010-18632 and BFU2013-48582-C1 from the Spanish Ministry of Science and Innovation (Madrid, Spain). JP was partially supported by the JAEDoc program from the CSIC.Peer Reviewe

Ingression Progression Complexes Control Extracellular Matrix Remodelling during Cytokinesis in Budding Yeast

[EN]Eukaryotic cells must coordinate contraction of the actomyosin ring at the division site together with ingression of the plasma membrane and remodelling of the extracellular matrix (ECM) to support cytokinesis, but the underlying mechanisms are still poorly understood. In eukaryotes, glycosyltransferases that synthesise ECM polysaccharides are emerging as key factors during cytokinesis. The budding yeast chitin synthase Chs2 makes the primary septum, a special layer of the ECM, which is an essential process during cell division. Here we isolated a group of actomyosin ring components that form complexes together with Chs2 at the cleavage site at the end of the cell cycle, which we named ‘ingression progression complexes’ (IPCs). In addition to type II myosin, the IQGAP protein Iqg1 and Chs2, IPCs contain the F-BAR protein Hof1, and the cytokinesis regulators Inn1 and Cyk3. We describe the molecular mechanism by which chitin synthase is activated by direct association of the C2 domain of Inn1, and the transglutaminase-like domain of Cyk3, with the catalytic domain of Chs2. We used an experimental system to find a previously unanticipated role for the C-terminus of Inn1 in preventing the untimely activation of Chs2 at the cleavage site until Cyk3 releases the block on Chs2 activity during late mitosis. These findings support a model for the co-ordinated regulation of cell division in budding yeast, in which IPCs play a central rol

How do budding yeast cells coordinate late cytokinesis steps?

Resumen del trabajo presentado a la 10ª Reunión de la Red Española de Levaduras, celebrada en El Escorial (Madrid) del 16 al 18 de diciembre de 2015.During cytokinesis, cells coordinate contraction of the actomyosin ring with the ingression of the plasma membrane and remodelling of the extracellular matrix (ECM) but the underlying mechanisms are still poorly understood. In eukaryotes, glycosyltransferases that synthesise ECM polysaccharides are emerging as important players during cytokinesis. In budding yeast the chitin synthase Chs2 makes the primary septum, a special layer of ECM that is essential for cell division. To try to understand how yeast cells coordinate actomyosin ring contraction, plasma membrane ingression and remodelling of the extracellular matrix we have used budding yeast Chs2 and Inn1 proteins to isolate 'ingression progression complexes' (IPCs) that contain key actomyosin rings components. We have identified, together with Chs2 and Inn1, actomyosin ring components Cyk3, myosin type II, the IQGAP protein Iqg1 and Hof1. We propose that IPCs are central to the mechanism by which cells coordinate cytokinesis. We have found that the catalytic domain of Chs2 interacts directly with the C2 domain of Inn1 and the transglutaminase-like domain of Cyk3. We have now data indicating that Inn1, Chs2 and Cyk3 form a stable complex. We found that chitin synthase Chs2 is activated by C2 domain of Inn1, as well as the transglutaminase-like domain of Cyk3. On the other hand, it has been already described that Chs2 protein is transported in vesicles to the cleavage site at the end of mitosis, but the molecular mechanisms by which those vesicles carrying Chs2 are incorporated at the site of division are unknown. We are now characterizing some of the factors we identified interacting with IPCs during cytokinesis that could have a specific role in that process.Peer Reviewe