Ubiquitin-mediated regulation of endocytosis by proteins of the arrestin family

This is an open access article distributed under the Creative Commons Attribution License.-- et al.In metazoans, proteins of the arrestin family are key players of G-protein-coupled receptors (GPCRS) signaling and trafficking. Following stimulation, activated receptors are phosphorylated, thus allowing the binding of arrestins and hence an >arrest> of receptor signaling. Arrestins act by uncoupling receptors from G proteins and contribute to the recruitment of endocytic proteins, such as clathrin, to direct receptor trafficking into the endocytic pathway. Arrestins also serve as adaptor proteins by promoting the recruitment of ubiquitin ligases and participate in the agonist-induced ubiquitylation of receptors, known to have impact on their subcellular localization and stability. Recently, the arrestin family has expanded following the discovery of arrestin-related proteins in other eukaryotes such as yeasts or fungi. Surprisingly, most of these proteins are also involved in the ubiquitylation and endocytosis of plasma membrane proteins, thus suggesting that the role of arrestins as ubiquitin ligase adaptors is at the core of these proteins' functions. Importantly, arrestins are themselves ubiquitylated, and this modification is crucial for their function. In this paper, we discuss recent data on the intricate connections between arrestins and the ubiquitin pathway in the control of endocytosis.This work was supported by the CNRS and by a Grant from the Fondation ARC pour la recherche sur le cancer (SFI20101201844) to S. Léon, and by a Grant from the Spanish CICYT (BFU2008-02005) to O. Vincent. M. Becuwe and A. Herrador are recipients of Ph.D. fellowships from the French Ministère de l’Enseignement Supérieur et de la Recherche and from CSIC-JAE, respectively.Peer Reviewe

Studying protein ubiquitylation in yeast

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The yeast arrestin-related protein Bul1 is a novel actor of glucose-induced endocytosis

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A metabolic switch on a yeast arrestin connects glucose signaling to transporter endocytosis

Endocytosis is a critical component of plasma membrane dynamics, by allowing the removal of proteins such as transporters or receptors in response to environmental cues. In yeast, transporter endocytosis requires their ubiquitylation at the plasma membrane by the Nedd4-like E3 ubiquitin ligase, Rsp5. Since the ubiquitylation of a given transporter occurs only in response to specific signals, this raises the question of how substrate specificity is achieved, and how it is regulated dynamically. Various "adaptor" proteins were identified, which may promote the interaction between Rsp5 and its substrates, and may provide a basis for this regulation. However, how they modulate Rsp5 function in response to extracellular stimuli is unknown. We addressed this question by studying a model transporter, Jen1, which is a lactate transporter induced in the presence of lactate and endocytosed in response to glucose (Paiva et al., JBC 2009). We identified an Rsp5 adaptor protein that belongs to the alpha-arrestin family, Art4 (also named Rod1), as essential for Jen1 ubiquitylation and endocytosis of in response to glucose. Interestingly, when cells are grown in lactate medium to induce Jen1 expression, Art4 is strongly phosphorylated by the yeast AMPK homologue, Snf1. Addition of glucose, known to trigger Jen1 endocytosis, leads to a rapid dephosphorylation of Art4, a process that requires the PP1 phosphatase regulatory subunit Reg1. This dephosphorylation allows Art4 ubiquitylation by Rsp5, and we provide details on the molecular mechanism of this regulation. We also show that Art4 ubiquitylation is required for Jen1 endocytosis. Therefore, a switch in Art4 post-translational modifications occurs in response to glucose and is required to modulate its function as an adaptor of Rsp5. This establishes yeast arrestin-like proteins as key regulators of transporter endocytosis in response to extracellular signals

Immunoselection and characterization of a human genomic PPAR binding fragment located within POTE genes

Peroxisome proliferator-activated receptors (PPARs) are ligand-inducible transcription factors and belong to the nuclear hormone receptor superfamily. They form heterodimers with retinoid X receptor (RXR) and bind to specific PPAR-response elements. To identify novel PPAR target genes, we developed an affinity method to isolate human genomic fragments containing binding sites for PPARs. For this, an antibody raised against all PPAR subtypes was used. Immunoselected fragments were amplified and sequenced. One of them, ISF1029, was mapped by BLAT and BLAST searches on different human chromosomes, downstream of several POTE genes. ISF1029 contained three hexamers strongly related to the AGGTCA motif organized according to a DR0/3 motif. The latter was found to bind to PPARΑ in gel mobility shift and supershift assays and to exhibit a downregulation potentiality in transfection experiments under clofibrate treatment. POTE genes were shown to be highly expressed in human Caco-2 colorectal adenocarcinoma cells and downregulated by fenofibrate and 9-cis-retinoic acid, as attested by RT-PCR assays. Microarray analysis confirmed and extended to the human T98-G glioblastoma cells, the downregulation of several POTE genes expression by Wy-14,643, a potent PPARΑ activator. Our data provide new insights about the pleiotropic action of PPARs

Damaged DNA Binding Protein 2 Plays a Role in Breast Cancer Cell Growth

The Damaged DNA binding protein 2 (DDB2), is involved in nucleotide excision repair as well as in other biological processes in normal cells, including transcription and cell cycle regulation. Loss of DDB2 function may be related to tumor susceptibility. However, hypothesis of this study was that DDB2 could play a role in breast cancer cell growth, resulting in its well known interaction with the proliferative marker E2F1 in breast neoplasia. DDB2 gene was overexpressed in estrogen receptor (ER)-positive (MCF-7 and T47D), but not in ER-negative breast cancer (MDA-MB231 and SKBR3) or normal mammary epithelial cell lines. In addition, DDB2 expression was significantly (3.0-fold) higher in ER-positive than in ER-negative tumor samples (P = 0.0208) from 16 patients with breast carcinoma. Knockdown of DDB2 by small interfering RNA in MCF-7 cells caused a decrease in cancer cell growth and colony formation. Inversely, introduction of the DDB2 gene into MDA-MB231 cells stimulated growth and colony formation. Cell cycle distribution and 5 Bromodeoxyuridine incorporation by flow cytometry analysis showed that the growth-inhibiting effect of DDB2 knockdown was the consequence of a delayed G1/S transition and a slowed progression through the S phase of MCF-7 cells. These results were supported by a strong decrease in the expression of S phase markers (Proliferating Cell Nuclear Antigen, cyclin E and dihydrofolate reductase). These findings demonstrate for the first time that DDB2 can play a role as oncogene and may become a promising candidate as a predictive marker in breast cancer

Seipin is required for converting nascent to mature lipid droplets

How proteins control the biogenesis of cellular lipid droplets (LDs) is poorly understood. Using Drosophila and human cells, we show here that seipin, an ER protein implicated in LD biology, mediates a discrete step in LD formation—the conversion of small, nascent LDs to larger, mature LDs. Seipin forms discrete and dynamic foci in the ER that interact with nascent LDs to enable their growth. In the absence of seipin, numerous small, nascent LDs accumulate near the ER and most often fail to grow. Those that do grow prematurely acquire lipid synthesis enzymes and undergo expansion, eventually leading to the giant LDs characteristic of seipin deficiency. Our studies identify a discrete step of LD formation, namely the conversion of nascent LDs to mature LDs, and define a molecular role for seipin in this process, most likely by acting at ER-LD contact sites to enable lipid transfer to nascent LDs. DOI: http://dx.doi.org/10.7554/eLife.16582.00

Integrated control of transporter endocytosis and recycling by the arrestin-related protein Rod1 and the ubiquitin ligase Rsp5.

International audienceAfter endocytosis, membrane proteins can recycle to the cell membrane or be degraded in lysosomes. Cargo ubiquitylation favors their lysosomal targeting and can be regulated by external signals, but the mechanism is ill-defined. Here, we studied the post-endocytic trafficking of Jen1, a yeast monocarboxylate transporter, using microfluidics-assisted live-cell imaging. We show that the ubiquitin ligase Rsp5 and the glucose-regulated arrestin-related trafficking adaptors (ART) protein Rod1, involved in the glucose-induced internalization of Jen1, are also required for the post-endocytic sorting of Jen1 to the yeast lysosome. This new step takes place at the trans-Golgi network (TGN), where Rod1 localizes dynamically upon triggering endocytosis. Indeed, transporter trafficking to the TGN after internalization is required for their degradation. Glucose removal promotes Rod1 relocalization to the cytosol and Jen1 deubiquitylation, allowing transporter recycling when the signal is only transient. Therefore, nutrient availability regulates transporter fate through the localization of the ART/Rsp5 ubiquitylation complex at the TGN

Plant tolerance to excess light energy and photooxidative damage relies on plastoquinone biosynthesis

International audiencePlastoquinone-9 is known as a photosynthetic electron carrier to which has also been attributed a role in the regulation of gene expression and enzyme activities via its redox state. Here, we show that it acts also as an antioxidant in plant leaves, playing a central photoprotective role. When Arabidopsis plants were suddenly exposed to excess light energy, a rapid consumption of plastoquinone-9 occurred, followed by a progressive increase in concentration during the acclimation phase. By overexpressing the plastoquinone-9 biosynthesis gene SPS1 (SOLANESYL DIPHOSPHATE SYNTHASE 1) in Arabidopsis, we succeeded in generating plants that specifically accumulate plastoquinone-9 and its derivative plastochromanol-8. The SPS1-overexpressing lines were much more resistant to photooxidative stress than the wild type, showing marked decreases in leaf bleaching, lipid peroxidation and PSII photoinhibition under excess light. Comparison of the SPS1 overexpressors with other prenyl quinone mutants indicated that the enhanced phototolerance of the former plants is directly related to their increased capacities for plastoquinone-9 biosynthesis

About a heat and mass transfer process reflection in phase plane

Darbā aplūkota pirmās kārtas nelineāra parasto diferenciālvienādojumu sistēma, kura radusies apskatot koksnes žāvēšanas matemātisko modeli. Šī sistēma pārnesta fāzu plaknē, noskaidrota atrisinājuma atkarība no mitras koksnes blīvuma, apkārtējā gaisa plūsmas ātrumaun koka plāksnes biezuma vērtībām. Bibliogrāfijā 8 nosaukumi Atslēgas vārdi: pirmās kārtas nelineāra parasto diferenciālvienādojumu sistēma, fāzu plaknes analīzes metode.In this work was a system of first order nonlinear ordinary differential equation examined, that came from inspecting the model of wood drying. This system was transmitted in to phase plane and established the solutions dependence of wet web density, velocity of around air and wood plate thickness values. Bibliography 8 items Keywords: first order nonlinear ordinary differential equation system, phase plane analyses method