Two Distinct Signals Determine the Basolateral Targeting of AQP4 in the Renal Epithelial Cell Line MDCK

To fulfill their physiological role in water handling by the kidney, aquaporins are differentially targeted to the apical and the basolateral membranes of epithelial kidney cells. For example, whereas AQP1 exhibits an un-polarized distribution in the proximal tubule cells (Sabolic et al., 1992,Nielsen et al., 1993), AQP2 is specifically targeted to the apical membranes of the principal cells in the collecting duct (Fushimi et al., 1993,Nielsen et al., 1993). On the other hand, AQP3 and AQP4 are exclusively localized in the basolateral membranes of these cells (Ecelbarger et al., 1995,Frigeri et al., 1995). Although a great deal of information about the cellular distribution of aquaporins in the body has emerged from previous studies, the mechanisms underlying their targeting in polarized epithelial cells remain poorly understood.Depto. de Genética, Fisiología y MicrobiologíaFac. de Ciencias BiológicasTRUEpu

A thiol peroxidase is an H2O2 receptor and redox-transducer in gene activation.

International audienceThe Yap1 transcription factor regulates hydroperoxide homeostasis in S. cerevisiae. Yap1 is activated by oxidation when hydroperoxide levels increase. We show that Yap1 is not directly oxidized by hydroperoxide. We identified the glutathione peroxidase (GPx)-like enzyme Gpx3 as a second component of the pathway, serving the role of sensor and transducer of the hydroperoxide signal to Yap1. When oxidized by H2O2, Gpx3 Cys36 bridges Yap1 Cys598 by a disulfide bond. This intermolecular disulfide bond is then resolved into a Yap1 intramolecular disulfide bond, the activated form of the regulator. Thioredoxin turns off the pathway by reducing both sensor and regulator. These data reveal a redox-signaling function for a GPx-like enzyme and elucidate a eukaryotic hydroperoxide-sensing mechanism. Gpx3 is thus a hydroperoxide receptor and redox-transducer

Polarized trafficking and surface expression of the AQP4 water channel are coordinated by serial and regulated interactions with different clathrin–adaptor complexes

Aquaporin 4 (AQP4) is the predominant water channel in the brain. It is targeted to specific membrane domains of astrocytes and plays a crucial role in cerebral water balance in response to brain edema formation. AQP4 is also specifically expressed in the basolateral membranes of epithelial cells. However, the molecular mechanisms involved in its polarized targeting and membrane trafficking remain largely unknown. Here, we show that two independent C-terminal signals determine AQP4 basolateral membrane targeting in epithelial MDCK cells. One signal involves a tyrosine-based motif; the other is encoded by a di-leucine-like motif. We found that the tyrosine-based basolateral sorting signal also determines AQP4 clathrin-dependent endocytosis through direct interaction with the µ subunit of AP2 adaptor complex. Once endocytosed, a regulated switch in µ subunit interaction changes AP2 adaptor association to AP3. We found that the stress-induced kinase casein kinase (CK)II phosphorylates the Ser276 immediately preceding the tyrosine motif, increasing AQP4–µ3A interaction and enhancing AQP4–lysosomal targeting and degradation. AQP4 phosphorylation by CKII may thus provide a mechanism that regulates AQP4 cell surface expression

The Arabidopsis thaliana sulfiredoxin is a plastidic cysteine-sulfinic acid reductase involved in the photooxidative stress response

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Genomic analysis of Rad26 and Rad1–Rad10 reveals differences in their dependence on Mediator and RNA polymerase II

International audienceMediator is a conserved coregulator playing a key role in RNA polymerase (Pol) II transcription. Mediator also links transcription and nucleotide excision repair (NER) via a direct contact with Rad2/ERCC5(XPG) endonuclease. In this work, we analyzed the genome-wide distribution of Rad26/ERCC6(CSB) and Rad1–Rad10/ERCC4(XPF)-ERCC1, addressing the question of a potential link of these proteins with Mediator and Pol II in yeast Saccharomyces cerevisiae . Our genomic analyses reveal that Rad1–Rad10 and Rad26 are present on the yeast genome in the absence of genotoxic stress, especially at highly transcribed regions, with Rad26 binding strongly correlating with that of Pol II. Moreover, we show that Rad1–Rad10 and Rad26 colocalize with Mediator at intergenic regions and physically interact with this complex. Using kin28 TFIIH mutant, we found that Mediator stabilization on core promoters leads to an increase in Rad1–Rad10 chromatin binding, whereas Rad26 occupancy follows mainly a decrease in Pol II transcription. Combined with multivariate analyses, our results show the relationships between Rad1–Rad10, Rad26, Mediator, and Pol II, modulated by the changes in binding dynamics of Mediator and Pol II transcription. In conclusion, we extend the Mediator link to Rad1–Rad10 and Rad26 NER proteins and reveal important differences in their dependence on Mediator and Pol II. Rad2 is the most dependent on Mediator, followed by Rad1–Rad10, whereas Rad26 is the most closely related to Pol II. Our work thus contributes to new concepts of the functional interplay between transcription and DNA repair machineries, which are relevant for human diseases including cancer and XP/CS syndromes

Functional interplay between Mediator and RNA polymerase II in Rad2/XPG loading to the chromatin

International audienceTranscription and maintenance of genome integrity are fundamental cellular functions. Deregulation of transcription and defects in DNA repair lead to serious pathologies. The Mediator complex links RNA polymerase (Pol) II transcription and nucleotide excision repair via Rad2/XPG endonuclease. However, the functional interplay between Rad2/XPG, Mediator and Pol II remains to be determined. In this study, we investigated their functional dynamics using genomic and genetic approaches. In a mutant affected in Pol II phosphorylation leading to Mediator stabilization on core promoters, Rad2 genome-wide occupancy shifts towards core promoters following that of Mediator, but decreases on transcribed regions together with Pol II. Specific Mediator mutations increase UV sensitivity, reduce Rad2 recruitment to transcribed regions, lead to uncoupling of Rad2, Mediator and Pol II and to colethality with deletion of Rpb9 Pol II subunit involved in transcription-coupled repair. We provide new insights into the functional interplay between Rad2, Mediator and Pol II and propose that dynamic interactions with Mediator and Pol II are involved in Rad2 loading to the chromatin. Our work contributes to the understanding of the complex link between transcription and DNA repair machineries, dysfunction of which leads to severe diseases

Functional interplay between Mediator and TFIIB in preinitiation complex assembly in relation to promoter architecture

International audienceMediator is a large coregulator complex conserved from yeast to humans and involved in many human diseases, including cancers. Together with general transcription factors, it stimulates preinitiation complex (PIC) formation and activates RNA polymerase II (Pol II) transcription. In this study, we analyzed how Mediator acts in PIC assembly using in vivo, in vitro, and in silico approaches. We revealed an essential function of the Mediator middle module exerted through its Med10 subunit, implicating a key interaction between Mediator and TFIIB. We showed that this Mediator-TFIIB link has a global role on PIC assembly genome-wide. Moreover, the amplitude of Mediator's effect on PIC formation is gene-dependent and is related to the promoter architecture in terms of TATA elements, nucleosome occupancy, and dynamics. This study thus provides mechanistic insights into the coordinated function of Mediator and TFIIB in PIC assembly in different chromatin contexts

A Role for the Mre11-Rad50-Xrs2 Complex in Gene Expression and Chromosome Organization

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