Arrestin-like proteins mediate ubiquitination and endocytosis of the yeast metal transporter Smf1

Many plasma membrane proteins in yeast are ubiquitinated and endocytosed, but how they are recognized for modification has remained unknown. Here, we show that the manganese transporter Smf1 is endocytosed when cells are exposed to cadmium ions, that this endocytosis depends on Rsp5-dependent ubiquitination of specific lysines and that it also requires phosphorylation at nearby sites. This phosphorylation is, however, constitutive rather than stress-induced. Efficient ubiquitination requires Ecm21 or Csr2, two members of a family of arrestin-like yeast proteins that contain several PY motifs and bind to Rsp5. Ecm21 also binds to phosphorylated Smf1, providing a link between Rsp5 and its substrate. PY motif-containing arrestin-like proteins are found in many species, including humans, and might have a general role as ubiquitin ligase adaptors

Yeast Npi3/Bro1 is involved in ubiquitin-dependent control of permease trafficking

AbstractThe membrane traffic and stability of the general amino acid permease Gap1 of Saccharomyces cerevisiae are under nitrogen control. Addition of a preferential nitrogen source such as ammonium to cells growing on a poor nitrogen source induces internalization of the permease and its subsequent degradation in the vacuole. This down-regulation requires ubiquitination of Gap1 through a process involving ubiquitin ligase Npi1/Rsp5, ubiquitin hydrolase Npi2/Doa4, and Bul1/2, two Npi1/Rsp5 interacting proteins. Here we report that yet another protein, Npi3, is involved in the regulation of Gap1 trafficking. We show that Npi3 is required for NH4+-induced down-regulation of Gap1, and particularly for efficient ubiquitination of the permease. Npi3 plays a pleiotropic role in permease down-regulation, since it is also involved in ubiquitination and stress-induced down-regulation of the uracil permease Fur4 and in glucose-induced degradation of hexose transporters Hxt6/7. We further provide evidence that Npi3 is required for direct vacuolar sorting of neosynthesized Gap1 permease as it occurs in npr1 mutant cells. NPI3 is identical to BRO1, a gene encoding a protein of unknown biochemical function and recently proposed to be involved in protein turnover. Npi3/Bro1 homologues include fungal proteins required for proteolytic cleavage of zinc finger proteins and the mouse Aip1 protein involved in apoptosis. We propose that proteins of the Npi3/Bro1 family, including homologues from higher species, may play a conserved role in ubiquitin-dependent control of membrane protein trafficking

Role of Bro1, the yeast homologue of Mammalian Alix, in ubiquitin-dependent protein sorting into the multivesicular body (MVB) pathway

Degradation of membrane proteins in the vacuole/lysosome is dependent on their prior sorting into the multivesicular body (MVB) pathway. This sorting process involves incorporation of proteins into vesicles that are formed by budding of the limiting membrane of the endosome into the lumen of the organelle. The MVB sorting process on the whole is highly conserved from yeast to human, and depends on the Vps27/Hrs, ESCRT-I, -II, and -III protein complexes functioning sequentially on the endosomal membrane, as well as on additional factors, such as the ubiquitinating enzyme Rsp5/Nedd4. It has now been established that ubiquitin serves as a sorting signal for many cargoes into the MVB pathway. In this thesis work, we provide evidence that Bro1 is not required for protein ubiquitination or early steps of endocytosis, but functions at the late endosome level as an integral component of the MVB pathway. Similarly to its human homologue Alix, Bro1 interacts with components of the ESCRT-I and ESCRT-III complexes. The putative role of Bro1/Alix in bridging an interaction between ESCRT-I and –III might be important to strengthen an association of these protein complexes to allow efficient sorting of cargo proteins. Deficiency in Bro1 results in recycling of the endocytosed Gap1 permease back to the plasma membrane, a process coupled to deubiquitination of the permease. This recycling is a non-classical phenotype for cells impaired in MVB pathway thus suggesting Bro1 to have a particular role in this sorting process. Furthermore, the conserved C-terminal proline-rich domain (PRD) of Bro1 is specifically important for MVB sorting of cargo proteins that are subject to ubiquitination. We show Bro1 (via its PRD) to play a highly important role in recruitment of the deubiquitinating enzyme Doa4 to the endosome. Consistent with this, Bro1 is required for deubiquitination of cargo proteins, a step occurring just before cargo incorporation into the endosomal vesicles, and similarly to Doa4, for ubiquitin recycling. In contrast to previous interpretations, we show that Doa4 has a direct role in sorting of ubiquitinated cargo proteins into the MVB pathway. We propose that Doa4 – via its association to Bro1 - achieves this role by catalyzing deubiquitination of cargo proteins and/or some components of the MVB sorting machinery. We further show Bro1 to interact with the ubiquitin ligase Rsp5, which, in addition to being required for cargo protein ubiquitination at the plasma membrane, apparently contributes to multiple steps of endocytosis and MVB sorting. Also the Bro1-Rsp5 interaction is dependent on the C-terminal PRD region of Bro1. We propose that this interaction is conserved. A role for ubiquitin in regulation of the MVB sorting machinery is emerging: the function of factors recognizing and sorting ubiquitinated cargo proteins in the MVB pathway is suggested to be coupled to their cycling between ubiquitinated and deubiquitinated stages. A growing body of evidence indicates that ubiquitin ligases of the Rsp5/Nedd4 family play a central role in this regulation. We speculate the Bro1/Alix protein, through its ability to simultaneously interact with factors of the MVB sorting machinery and with ubiquitinating and deubiquitinating enzymes to play a central role in the successive rounds of ubiquitination and deubiquitination of specific factors along the MVB pathway. Doctorat en sciences, Spécialisation biologie moléculaireinfo:eu-repo/semantics/nonPublishe

Hepatic maturation of human pluripotent stem cell-derived hepatoblasts in three-dimensional in vitro cultures

There is a great demand of cultured human hepatocytes for hepatotoxicity studies, drug testing, disease modelling and liver transplantation purposes. The current gold standard, primary human hepatocytes (PHHs), suffer from poor availability and high variability. Furthermore, PHHs are short-lived in in vitro cultures. Pluripotent stem cell (PSC)-derived hepatocyte-like cells (HLCs) have emerged as potential substitutes for PHHs in in vitro studies. PSCs are widely available, and additionally allow studies of hepatogenesis and open possibilities for personalized medicine. However, obtaining HLCs with mature hepatocyte functions in vitro has turned out to be challenging, and the differentiated cells have remained immature compared to PHHs. In vivo, hepatic differentiation and maturation of PSCs is guided by cues from the environment. Mimicking the 3D cellular environment in vitro has already shown encouraging results, but today, HLCs are still awaiting to fulfil their promise as a new gold standard. The aim of this study was first to select a new working human PSC line for in vitro hepatic differentiation and maturation. Hepatic differentiation and maturation of the selected cell line, embryonic stem cell line ESI-017, was next studied in five different 3D culture conditions (spheroids in suspension culture, four different hydrogels: Matrigel, collagen type I, mixture of Matrigel and collagen type I and alginate) with the aim to find the most favourable culture condition for later studies. For this, the PSCs were first differentiated to definitive endoderm cells and then to hepatoblasts in 2D cultures, on Matrigel- and laminin-521-coated plates, respectively. The PSC-derived hepatoblasts were then transferred for 16 days to the different 3D culture conditions for hepatic maturation. Of the conditions, suspension culture and mixture of Matrigel and collagen type I -hydrogel were estimated most promising and were selected for further studies. Hepatic maturation of the PSC-derived HLCs was estimated by analysing protein and mRNA expression levels of key marker genes, such as CYP3A4, AAT, MRP2, HNF4A, ALB, AFP, CK-8/18 and CK-19 by immunofluorescence staining and qPCR, respectively, and by cell morphology. Based on cell morphology and noticeable level of CYP3A4 expression, suspension culture shows most potential of the studied conditions in hepatic maturation of PSC-derived hepatoblasts. However, given that expression level of many other hepatocyte marker genes in these HLCs remained low compared to PHHs or human fetal liver samples, it is evident that adjustments to protocol and culturing conditions are still needed.Laboratorio-olosuhteissa eli in vitro viljeltyjä hepatosyyttejä tarvitaan enenevissä määrin uusien lääkeaineiden testaamisessa, maksatoksisuustutkimuksissa, maksasairauksien mallinnukseen sekä maksansiirtoihin. Maksasta eristetyt hepatosyytit, eli primaarisolut ovat pitkään olleet paras ihmisen maksan toimintaa jäljittelevistä in vitro viljellyistä solumalleista, nk. "kultainen standardi". Niiden saatavuus on kuitenkin heikko ja solujen välillä esiintyy suurta vaihtelua. Laboratorio-olosuhteissa ne ovat lisäksi melko lyhytikäisiä. Primaarisolujen korvaajiksi on ehdotettu pluripotenteista eli monikykyisistä kantasoluista erilaistettuja hepatosyyttien kaltaisia soluja. Pluripotenttien solujen etuna verrattuna maksan primaarisoluihin on niiden hyvä saatavuus. Lisäksi ne mahdollistavat maksan kehityksen tutkimisen sekä avaavat uusia mahdollisuuksia yksilölliselle lääkinnälle. Pluripotenttien kantasolujen erilaistaminen toiminnallisiksi maksasoluiksi on kuitenkin osoittautunut haasteelliseksi; erilaistetut solut ovat epäkypsiä eivätkä toiminnaltaan näin vastaa primaarisoluja. Kudoksessa kantasolujen erilaistumista ja kypsymistä ohjaavat ympäristöstä tulevat erilaiset viestit, ja matkimalla erilaistuvien solujen luonnollista, maksakudoksen kolmiulotteista eli 3D kasvuympäristöä onkin jo lupaavasti edistytty pluripotenttien kantasolujen erilaistamisessa hepatosyyteiksi. Työ on kuitenkin vielä kesken, eivätkä pluripotenteista kantasoluista erilaistetut hepatosyyttien kaltaiset solut ole vielä korvanneet primaarisoluja "kultaisena standardina". Työn tarkoituksena oli ensin valita uusi pluripotentti kantasolulinja, jota voidaan käyttää hepatosyyteiksi erilaistamiseen. Valittua alkion kantasolulinjaa (ESI-017) käytettiin työn seuraavassa vaiheessa arvioimaan viiden eri 3D kasvualustan (suspensioviljelmä ja neljä eri hydrogeeliä: Matrigel, tyypin I kollageeni, Matrigelin ja tyyppi I kollageenin sekoitus ja alginaatti) vaikutusta hepatosyytiksi erilaistamisessa. Alkion kantasolut erilaistettiin ensin definitiivisen endodermin (DE) soluiksi ja tuotetut DE solut sitten hepatoblasteiksi eli hepatosyyttien progenitorisoluiksi kaksiulotteisilla kasvualustoilla. DE erilaistamisen aikana soluja viljeltiin Matrigel- ja hepatoblasteiksi erilaistamisen aikana laminiini-521 -päällystetyillä alustoilla. Hepatoblastit siirrettiin 16 päivän ajaksi 3D kasvualustalle hepatosyytiksi erilaistamiseksi. 3D kasvuolosuhteista kaksi, suspensioviljelmä ja Matrigelin ja tyypin I kollageenin yhdistelmä, valittiin työn viimeiseen vaiheeseen, jossa hepatoblastien kypsymistä hepatosyyttien kaltaisiksi soluiksi seurattiin tarkastelemalla usean merkkigeenin (CYP3A4, AAT, MRP2, HNF4A, ALB, AFP, CK-8/18 and CK-19) mRNAn ja proteiininen ilmentymistä qPCR:n ja immunofluoresenssivärjäysten avulla sekä tarkastelemalla solujen morfologiaa. CYP3A4 entsyymin ilmentymisen sekä solumorfologian perusteella, hepatoblastien suspensioviljelmä vaikuttaa tukevan parhaiten hepatoblastien kypsymistä hepatosyyteiksi. Tuotetut hepatosyyttien kaltaiset solut eivät kuitenkaan olleet täysin kypsiä ja muiden hepatosyyttien merkkigeenien ilmentyminen jäi alhaisemmaksi kuin maksan primaarisoluissa tai sikiön maksasoluissa. On ilmeistä, että käytettyjä menetelmiä ja olosuhteita on kehitettävä ja tutkittava edelleen

Role of Bro1, the yeast homologue of Mammalian Alix, in ubiquitin-dependent protein sorting into the multivesicular body (MVB) pathway

Degradation of membrane proteins in the vacuole/lysosome is dependent on their prior sorting into the multivesicular body (MVB) pathway. This sorting process involves incorporation of proteins into vesicles that are formed by budding of the limiting membrane of the endosome into the lumen of the organelle. The MVB sorting process on the whole is highly conserved from yeast to human, and depends on the Vps27/Hrs, ESCRT-I, -II, and -III protein complexes functioning sequentially on the endosomal membrane, as well as on additional factors, such as the ubiquitinating enzyme Rsp5/Nedd4. It has now been established that ubiquitin serves as a sorting signal for many cargoes into the MVB pathway. In this thesis work, we provide evidence that Bro1 is not required for protein ubiquitination or early steps of endocytosis, but functions at the late endosome level as an integral component of the MVB pathway. Similarly to its human homologue Alix, Bro1 interacts with components of the ESCRT-I and ESCRT-III complexes. The putative role of Bro1/Alix in bridging an interaction between ESCRT-I and –III might be important to strengthen an association of these protein complexes to allow efficient sorting of cargo proteins. Deficiency in Bro1 results in recycling of the endocytosed Gap1 permease back to the plasma membrane, a process coupled to deubiquitination of the permease. This recycling is a non-classical phenotype for cells impaired in MVB pathway thus suggesting Bro1 to have a particular role in this sorting process. Furthermore, the conserved C-terminal proline-rich domain (PRD) of Bro1 is specifically important for MVB sorting of cargo proteins that are subject to ubiquitination. We show Bro1 (via its PRD) to play a highly important role in recruitment of the deubiquitinating enzyme Doa4 to the endosome. Consistent with this, Bro1 is required for deubiquitination of cargo proteins, a step occurring just before cargo incorporation into the endosomal vesicles, and similarly to Doa4, for ubiquitin recycling. In contrast to previous interpretations, we show that Doa4 has a direct role in sorting of ubiquitinated cargo proteins into the MVB pathway. We propose that Doa4 – via its association to Bro1 - achieves this role by catalyzing deubiquitination of cargo proteins and/or some components of the MVB sorting machinery. We further show Bro1 to interact with the ubiquitin ligase Rsp5, which, in addition to being required for cargo protein ubiquitination at the plasma membrane, apparently contributes to multiple steps of endocytosis and MVB sorting. Also the Bro1-Rsp5 interaction is dependent on the C-terminal PRD region of Bro1. We propose that this interaction is conserved. A role for ubiquitin in regulation of the MVB sorting machinery is emerging: the function of factors recognizing and sorting ubiquitinated cargo proteins in the MVB pathway is suggested to be coupled to their cycling between ubiquitinated and deubiquitinated stages. A growing body of evidence indicates that ubiquitin ligases of the Rsp5/Nedd4 family play a central role in this regulation. We speculate the Bro1/Alix protein, through its ability to simultaneously interact with factors of the MVB sorting machinery and with ubiquitinating and deubiquitinating enzymes to play a central role in the successive rounds of ubiquitination and deubiquitination of specific factors along the MVB pathway. Doctorat en sciences, Spécialisation biologie moléculaireinfo:eu-repo/semantics/nonPublishe

Split-Ubiquitin Two-Hybrid Assay To Analyze Protein-Protein Interactions at the Endosome: Application to Saccharomyces cerevisiae Bro1 Interacting with ESCRT Complexes, the Doa4 Ubiquitin Hydrolase, and the Rsp5 Ubiquitin Ligase▿ †

Targeting of membrane proteins into the lysosomal/vacuolar lumen for degradation requires their prior sorting into multivesicular bodies (MVB). The MVB sorting pathway depends on ESCRT-0, -I, -II, and -III protein complexes functioning on the endosomal membrane and on additional factors, such as Bro1/Alix and the ubiquitin ligase Rsp5/Nedd4. We used the split-ubiquitin two-hybrid assay to analyze the interaction partners of yeast Bro1 at its natural cellular location. We show that Bro1 interacts with ESCRT-I and -III components, including Vps23, the Saccharomyces cerevisiae homologue of human Tsg101. These interactions do not require the C-terminal proline-rich domain (PRD) of Bro1. Rather, this PRD interacts with the Doa4 deubiquitinating enzyme to recruit it to the endosome. This interaction is disrupted by a single amino acid substitution in the conserved ELC box motif in Doa4. The PRD of Bro1 also mediates an association with Rsp5, and this interaction appears to be conserved, as Alix, the human homologue of Bro1, coimmunoprecipitates with Nedd4 in yeast lysates. We further show that the Bro1 PRD domain is essential to MVB sorting of only cargo proteins whose sorting to the vacuolar lumen is dependent on their own ubiquitination and Doa4. The Bro1 region preceding the PRD, however, is required for MVB sorting of proteins irrespective of whether their targeting to the vacuole is dependent on their ubiquitination and Doa4. Our data indicate that Bro1 interacts with several ESCRT components and contributes via its PRD to associating ubiquitinating and deubiquitinating enzymes with the MVB sorting machinery

Retinoic acid down-regulates the expression of EmH-3 homeobox-containing gene in the freshwater sponge Ephydatia muelleri

The effects of retinoic acid (RA), a common morphogen and gene expression regulator in vertebrates, were studied in the freshwater sponge Ephydatia muelleri, both on morphogenesis and on the expression of EmH-3 homeobox-containing gene. At 0.3 μM, RA had no noticeable influence on sponge development, slightly up-regulating EmH-3 expression. In contrast, in sponges reared in 10, 8 μM and to a lesser extent 2 μM RA, there was a strong down-regulation of EmH-3 expression after hatching. This induced modifications in cell composition and morphology, greatly disturbing normal development. Archaeocytes kept the features found in newly hatched sponges while choanocytes and a functional aquiferous system were completely absent. The inhibition of morphogenesis and down-regulation of EmH-3 expression were reversible when sponges were no longer subjected to RA. After RA removal, EmH-3 expression returned to the high values found in untreated sponges, archaeocytes differentiated into choanocytes and sponges achieved a normal development. These results clearly show that, in freshwater sponges, the most primitive metazoan, RA may also act as a morphogen, regulating the expression of a homeobox-containing gene. They demonstrate that the expression of EmH-3 is necessary for the differentiation of archaeocytes into choanocytes and hence for the formation of a complete functional aquiferous system. © 2001 Elsevier Science Ireland Ltd.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Evidence for a direct role of the Doa4 deubiquitinating enzyme in protein sorting into the MVB pathway.

Degradation of various membrane proteins in the lumen of the vacuole/lysosome requires their prior sorting into the multivesicular body (MVB) pathway. In this process, ubiquitin serves as a sorting signal for most cargoes. The yeast ubiquitin hydrolase Doa4 acts late in the MVB pathway. It's role is to catalyze deubiquitination of cargo proteins prior to their sorting into the endosomal vesicles. This step rescues ubiquitin from degradation in the vacuole/lysosome, enabling it to be recycled. Accordingly, the level of monomeric ubiquitin is typically reduced in doa4 mutants. Although MVB sorting of cargo proteins is also impaired in doa4 mutants, the question of whether this defect is due solely to Doa4's role in maintaining a normal pool of ubiquitin in the cell remains open. We here show that the requirement of Doa4 for correct MVB sorting of the endocytic cargo general amino acid permease and of the biosynthetic cargo carboxypeptidase S are not because of the role of Doa4 in ubiquitin recycling. This suggests a direct role of Doa4 in MVB sorting and we show that this role depends on Doa4's catalytic activity. We propose that deubiquitination by Doa4 of cargo proteins and/or some components of the MVB sorting machinery is essential to correct sorting of cargoes into the MVB pathway.Journal ArticleResearch Support, Non-U.S. Gov'tFLWINSCOPUS: ar.jinfo:eu-repo/semantics/publishe