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    ABSTRACT: Many plasma membrane transporters are downregulated by ubiquitylation, endocytosis, and delivery to the lysosome, in response to various stimuli. We report here that two amino acid transporters of Saccharomyces cerevisiae, the general amino-acid permease (Gap1) and the arginine-specific permease (Can1), undergo ubiquitin-dependent downregulation in response to their substrates, and that this downregulation is not due to intracellular accumulation of the transported amino acids but to transport catalysis itself. Following an approach based on permease structural modeling, mutagenesis, and kinetic parameter analysis, we obtained evidence that substrate-induced endocytosis requires transition of the permease to a conformational state preceding substrate release into the cell. Furthermore, this transient conformation must be stable enough and thus sufficiently populated for the permease to undergo efficient downregulation. Additional observations including the constitutive downregulation of two active Gap1 mutants altered in cytosolic regions support the model that the substrate-induced conformational transition inducing endocytosis involves remodeling of cytosolic regions of the permeases, thereby promoting their recognition by arrestin-like adaptors of the Rsp5 ubiquitin ligase. Similar mechanisms might control many other plasma membrane transporters according to the external concentrations of their substrates.
    Molecular and Cellular Biology 10/2014; · 5.04 Impact Factor
  • Myriam Crapeau, Ahmad Merhi, Bruno Andre
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    ABSTRACT: Gap1, the yeast general amino acid permease, is a convenient model for studying how the intracellular traffic of membrane transporters is regulated. Present at the plasma membrane under poor nitrogen supply conditions, it undergoes ubiquitylation, endocytosis, and degradation upon activation of the TORC1 kinase complex in response to an increase in internal amino acids. This downregulation is stimulated by TORC1-dependent phosphoinhibition of the Npr1 kinase, resulting in activation by dephosphorylation of the arrestin-like Bul1 and Bul2 adaptors recruiting the Rsp5 ubiquitin ligase to Gap1. We here report that Gap1 is also downregulated when cells are treated with the TORC1 inhibitor rapamycin or subjected to various stresses, and that a lack of the Tco89 subunit of TORC1 causes constitutive Gap1 downregulation. Both the Bul1 and Bul2 and the Aly1 and Aly2 arrestin-like adaptors of Rsp5 promote this downregulation without undergoing dephosphorylation. Furthermore, they act via C-terminal regions of Gap1 not involved in ubiquitylation in response to internal amino acids, whereas a Gap1 mutant altered in the N-terminal tail and resistant to ubiquitylation by internal amino acids is efficiently downregulated under stress via the Bul and Aly adaptors. While the Bul proteins mediate Gap1 ubiquitylation of two possible lysines, K9 and K16, the Aly proteins promote ubiquitylation of the K16 residue only. This stress-induced pathway of Gap1 downregulation targets other permeases as well, and likely allows cells facing adverse conditions to retrieve amino acids from permease degradation.
    The Journal of biological chemistry. 06/2014;
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    ABSTRACT: Amino-acid uptake in yeast cells is mediated by about sixteen plasma membrane permeases, most of which belong to the APC (amino acid, polyamine, organocation) transporter family. These proteins display various substrate specificity ranges. For instance, the general amino-acid permease Gap1 transports all amino acids, whereas Can1 and Lyp1 catalyze specific uptake of arginine and lysine, respectively. Though Can1 and Lyp1 have different narrow substrate specificities, they are close homologs. Here we investigated the molecular rules determining the substrate specificity of the H+-driven arginine-specific permease Can1. Using a Can1-Lyp1 sequence alignment as a guideline and a 3D Can1 structural model based on the crystal structure of the bacterial APC-family arginine-agmatine antiporter, we introduced amino acid substitutions liable to alter Can1 substrate specificity. We show that the single substitution T456S results in a Can1 variant transporting lysine in addition to arginine, and that the combined substitutions T456S and S176N convert Can1 to a Lyp1-like permease. Replacement of a highly conserved glutamate in the Can1 binding site leads to variants (E184Q, E184A) incapable of any amino acid transport, pointing to a potential role for this glutamate in H+ coupling. Measurements of the kinetic parameters of arginine and lysine uptake by the wild-type and mutant Can1 permeases, together with docking calculations for each amino acid in their binding site, suggest a model in which residues at positions 176 and 456 confer substrate selectivity at the ligand-binding stage and/or in the course of conformational changes required for transport.
    Journal of Biological Chemistry 01/2014; 289:7232-7246. · 4.65 Impact Factor
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    ABSTRACT: Decades of work requiring heterologous expression of eukaryotic proteins have shown that no expression system can be considered as the panacea and the appropriate expression strategy is often protein-dependent. In a large number of cases, yeasts have proven to be reliable organisms for heterologous protein expression by combining eukaryotic cellular organization with the ease of use of simpler microorganisms. During this work, a novel promoter system based on the nitrogen catabolite regulation has been developed to produce the general amino acid permease (Gap1) in its natural host, the yeast Saccharomyces cerevisiae. A simple purification protocol was also established that allows to purify milligrams of Gap1 from cells cultivated in a five liters bio-reactor. In order to test the ability of the system to be used for expression of other proteins, the yeast specific transporter of gamma-aminobutyric acid (Uga4), a human vesicular transporter of glutamate (Vglut1) and a small secreted glycoprotein (MD-2) were also expressed using the nitrogen catabolite regulation. All proteins were fused to GFP and their presence and localization were confirmed by western blot analysis and fluorescence microscopy. Our work shows that the nitrogen catabolite repressible GAP1 promoter can be used to obtain high levels of recombinant protein while allowing for large biomass production in S. cerevisiae. This approach can be used to express membrane and soluble proteins from higher eukaryotes (from yeast to human). Therefore, this system stands as a promising alternative to commonly used expression procedure in yeasts.
    Microbial Cell Factories 12/2013; 12(1):129. · 3.31 Impact Factor
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    ABSTRACT: Cystinosin, the lysosomal cystine exporter defective in cystinosis, is the founding member of a family of heptahelical membrane proteins related to bacteriorhodopsin and characterized by a duplicated motif termed the PQ loop. PQ-loop proteins are more frequent in eukaryotes than in prokaryotes; except for cystinosin, their molecular function remains elusive. In this study, we report that three yeast PQ-loop proteins of unknown function, Ypq1, Ypq2, and Ypq3, localize to the vacuolar membrane and are involved in homeostasis of cationic amino acids (CAAs). We also show that PQLC2, a mammalian PQ-loop protein closely related to yeast Ypq proteins, localizes to lysosomes and catalyzes a robust, electrogenic transport that is selective for CAAs and strongly activated at low extracytosolic pH. Heterologous expression of PQLC2 at the yeast vacuole rescues the resistance phenotype of an ypq2 mutant to canavanine, a toxic analog of arginine efficiently transported by PQLC2. Finally, PQLC2 transports a lysine-like mixed disulfide that serves as a chemical intermediate in cysteamine therapy of cystinosis, and PQLC2 gene silencing trapped this intermediate in cystinotic cells. We conclude that PQLC2 and Ypq1-3 proteins are lysosomal/vacuolar exporters of CAAs and suggest that small-molecule transport is a conserved feature of the PQ-loop protein family, in agreement with its distant similarity to SWEET sugar transporters and to the mitochondrial pyruvate carrier. The elucidation of PQLC2 function may help improve cysteamine therapy. It may also clarify the origin of CAA abnormalities in Batten disease.
    Proceedings of the National Academy of Sciences 11/2012; · 9.81 Impact Factor
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    Ahmad Merhi, Bruno André
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    ABSTRACT: Ubiquitylation of many plasma membrane proteins promotes their endocytosis followed by degradation in the lysosome. The yeast general amino acid permease, Gap1, is ubiquitylated and down-regulated when a good nitrogen source like ammonium is provided to cells growing on a poor nitrogen source. This ubiquitylation requires the Rsp5 ubiquitin ligase and the redundant arrestin-like Bul1 and Bul2 adaptors. Previous studies have shown that Gap1 ubiquitylation involves the TORC1 kinase complex, which inhibits the Sit4 phosphatase. This causes inactivation of the protein kinase Npr1, which protects Gap1 against ubiquitylation. However, the mechanisms inducing Gap1 ubiquitylation after Npr1 inactivation remain unknown. We here show that on a poor nitrogen source, the Bul adaptors are phosphorylated in an Npr1-dependent manner and bound to 14-3-3 proteins that protect Gap1 against down-regulation. After ammonium is added and converted to amino acids, the Bul proteins are dephosphorylated, dissociate from the 14-3-3 proteins, and undergo ubiquitylation. Furthermore, dephosphorylation of Bul requires the Sit4 phosphatase, which is essential to Gap1 down-regulation. The data support the emerging concept that permease ubiquitylation results from activation of the arrestin-like adaptors of the Rsp5 ubiquitin ligase, this coinciding with their dephosphorylation, dissociation from the inhibitory 14-3-3 proteins, and ubiquitylation.
    Molecular and Cellular Biology 09/2012; · 5.04 Impact Factor
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    ABSTRACT: With the growing number of available microbial genome sequences, regulatory signals can now be revealed as conserved motifs in promoters of orthologous genes (phylogenetic footprints). A next challenge is to unravel genome-scale regulatory networks. Using as sole input genome sequences, we predicted cis-regulatory elements for each gene of the yeast Saccharomyces cerevisiae by discovering over-represented motifs in the promoters of their orthologs in 19 Saccharomycetes species. We then linked all genes displaying similar motifs in their promoter regions and inferred a co-regulation network including 56,919 links between 3171 genes. Comparison with annotated regulons highlights the high predictive value of the method: a majority of the top-scoring predictions correspond to already known co-regulations. We also show that this inferred network is as accurate as a co-expression network built from hundreds of transcriptome microarray experiments. Furthermore, we experimentally validated 14 among 16 new functional links between orphan genes and known regulons. This approach can be readily applied to unravel gene regulatory networks from hundreds of microbial genomes for which no other information is available except the sequence. Long-term benefits can easily be perceived when considering the exponential increase of new genome sequences.
    Nucleic Acids Research 05/2011; 39(15):6340-58. · 8.81 Impact Factor
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    ABSTRACT: The yeast Ssy5 protein is a serine-type endoprotease autoprocessed into a catalytic domain and a large inhibitory prodomain. When external amino acids are detected by the plasma membrane Ssy1 sensor, Ssy5 is activated and catalyzes endoproteolytic processing of the Stp1 and Stp2 transcription factors. These Stp proteins then migrate into the nucleus and activate transcription of several amino acid permease genes. Previous studies showed that Ssy5 activation involves the SCFGrr1 ubiquitin ligase complex, but the molecular mechanisms of this activation remain unclear. We here report that the prodomain of Ssy5 is phosphorylated in a casein kinase I-dependent manner in response to amino acid detection. We describe a mutant form of Ssy5 whose prodomain is not phosphorylated and show that it is nonfunctional. Amino acid detection also induces ubiquitylation of the Ssy5 prodomain. This prodomain ubiquitylation requires its prior phosphorylation and the SCFGrr1 complex. When this ubiquitylation is defective, Ssy5 accumulates as a phosphorylated form but remains inactive. A constitutive Ssy5 form in which the prodomain fails to inhibit the catalytic domain does not need to be phosphorylated or ubiquitylated to be active. Finally, we provide evidence that ubiquitylation of the inhibitory prodomain rather than its subsequent degradation is the key step in the Ssy5 activation mechanism. We propose that the Ssy5 protease is activated by phosphorylation-induced ubiquitylation, the effect of which is relief from inhibition by its prodomain.
    Journal of Biological Chemistry 02/2011; 286(14):12006-15. · 4.65 Impact Factor
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    ABSTRACT: The yeast general amino acid permease Gap1 is a convenient model for studying the intracellular trafficking of membrane proteins. Present at the plasma membrane when the nitrogen source is poor, it undergoes ubiquitin-dependent endocytosis and degradation upon addition of a good nitrogen source, e.g., ammonium. It comprises 12 transmembrane domains (TM) flanked by cytosol-facing N- and C-terminal tails (NT, CT). The NT of Gap1 contains the acceptor lysines for ubiquitylation and its CT includes a sequence essential to exit from the endoplasmic reticulum (ER). We used alanine-scanning mutagenesis to isolate 64 mutant Gap1 proteins altered in the NT, the CT, or one of the five TM-connecting intracellular loops (L2, -4, -6, -8 and -10). We found 17 mutations (in L2, L8, L10 and CT) impairing Gap1 exit from the ER. Of the 47 mutant proteins reaching the plasma membrane normally, two are unstable and rapidly down-regulated even when the nitrogen source is poor. Six others are totally inactive and another four, altered in a 16-amino-acid sequence in the NT, are resistant to ammonium-induced down-regulation. Finally, a mutation in L6 causes missorting of Gap1 from the secretory pathway to the vacuole. Interestingly, this direct vacuolar sorting seems to be independent of Gap1 ubiquitylation. This study illustrates the importance of multiple intracellular regions of Gap1 in its secretion, transport activity, and down-regulation.
    PLoS ONE 01/2011; 6(4):e18457. · 3.53 Impact Factor
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    ABSTRACT: Membrane transporters constitute one of the largest functional categories of proteins in all organisms. In the yeast Saccharomyces cerevisiae, this represents about 300 proteins ( approximately 5% of the proteome). We here present the Yeast Transport Protein database (YTPdb), a user-friendly collaborative resource dedicated to the precise classification and annotation of yeast transporters. YTPdb exploits an evolution of the MediaWiki web engine used for popular collaborative databases like Wikipedia, allowing every registered user to edit the data in a user-friendly manner. Proteins in YTPdb are classified on the basis of functional criteria such as subcellular location or their substrate compounds. These classifications are hierarchical, allowing queries to be performed at various levels, from highly specific (e.g. ammonium as a substrate or the vacuole as a location) to broader (e.g. cation as a substrate or inner membranes as location). Other resources accessible for each transporter via YTPdb include post-translational modifications, K(m) values, a permanently updated bibliography, and a hierarchical classification into families. The YTPdb concept can be extrapolated to other organisms and could even be applied for other functional categories of proteins. YTPdb is accessible at http://homes.esat.kuleuven.be/ytpdb/.
    Biochimica et Biophysica Acta 10/2010; 1798(10):1908-12. · 4.66 Impact Factor
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    ABSTRACT: Yeast permeases, that act as transporters for nutrients including amino acids, nucleobases and metals, provide a powerful model system for dissecting the physiological control of membrane protein trafficking. Modification of these transporters by ubiquitin is known to target them for degradation in the vacuole, the degradation organelle of fungi. Recent studies have uncovered the role of specific adaptors for recruiting the Rsp5 ubiquitin ligase to these proteins. In addition, the role of ubiquitin at different trafficking steps including early endocytosis, sorting into the multivesicular body (MVB) pathway and Golgi-to-endosome transit is now becoming clear. In particular, K63-linked ubiquitin chains now emerge as a specific signal for protein sorting into the MVB pathway. A complete view of the ubiquitin code governing yeast permease trafficking might not be far off.
    Trends in cell biology 02/2010; 20(4):196-204. · 12.12 Impact Factor
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    ABSTRACT: When yeast cells detect external amino acids via their permease-like Ssy1 sensor, the cytosolic precursor forms of Stp1 and Stp2 transcription factors are activated by endoproteolytic removal of their N-terminal domains, a reaction catalyzed by the Ssy5 endoprotease. The processed Stp factors then migrate into the nucleus, where they activate transcription of several amino acid permease genes including AGP1. We report here that the STP1 and STP2 genes most likely derive from the whole genome duplication that occurred in a yeast ancestor. Although Stp1 and Stp2 have been considered redundant, we provide evidence that they functionally diverged during evolution. Stp2 is the only factor processed when amino acids are present at low concentration, and the transcriptional activation of AGP1 promoted by Stp2 is moderate. Furthermore, only Stp2 can sustain Agp1-dependent utilization of amino acids at low concentration. In contrast, Stp1 is only processed when amino acids are present at high concentration, and it promotes higher level transcriptional activation of AGP1. Domain swapping experiments show that the N-terminal domains of Stp1 and Stp2 are responsible for these proteins being cleaved at different amino acid concentrations. Last, induction of the DIP5 permease gene by amino acids depends on Stp2 but not Stp1. We propose that post-whole genome duplication co-conservation of the STP1 and STP2 genes was favored by functional divergence of their products, likely conferring to cells an increased ability to adapt to various amino acid supply conditions.
    Journal of Biological Chemistry 11/2009; 285(2):855-65. · 4.65 Impact Factor
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    ABSTRACT: A growing number of yeast and mammalian plasma membrane proteins are reported to be modified with K63-linked ubiquitin (Ub) chains. However, the relative importance of this modification versus monoubiquitylation in endocytosis, Golgi to endosome traffic, and sorting into the multivesicular body (MVB) pathway remains unclear. In this study, we show that K63-linked ubiquitylation of the Gap1 permease is essential for its entry into the MVB pathway. Carboxypeptidase S also requires modification with a K63-Ub chain for correct MVB sorting. In contrast, monoubiquitylation of a single target lysine of Gap1 is a sufficient signal for its internalization from the cell surface, and Golgi to endosome transport of the permease requires neither its ubiquitylation nor the Ub-binding GAT (Gga and Tom1) domain of Gga (Golgi localizing, gamma-ear containing, ARF binding) adapter proteins, the latter being crucial for subsequent MVB sorting of the permease. Our data reveal that K63-linked Ub chains act as a specific signal for MVB sorting, providing further insight into the Ub code of membrane protein trafficking.
    The Journal of Cell Biology 05/2009; 185(3):493-502. · 10.82 Impact Factor
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    ABSTRACT: Nitrogen is an essential nutrient for all life forms. Like most unicellular organisms, the yeast Saccharomyces cerevisiae transports and catabolizes good nitrogen sources in preference to poor ones. Nitrogen catabolite repression (NCR) refers to this selection mechanism. We propose an approach based on Gaussian graphical models (GGMs), which enable to distinguish direct from indirect interactions between genes, to identify putative NCR genes from putative NCR regulatory motifs and over-represented motifs in the upstream noncoding sequences of annotated NCR genes. Because of the high-dimensionality of the data, we use a shrinkage estimator of the covariance matrix to infer the GGMs. We show that our approach makes significant and biologically valid predictions. We also show that GGMs are more effective than models that rely on measures of direct interactions between genes.
    Evolutionary Computation, Machine Learning and Data Mining in Bioinformatics, 7th European Conference, EvoBIO 2009, Tübingen, Germany, April 15-17, 2009, Proceedings; 01/2009
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    ABSTRACT: Ammonium transport proteins of the Mep/Amt/Rh family include microbial and plant Mep/Amt members, crucial for ammonium scavenging, and animal Rhesus factors likely involved in ammonium disposal. Recent structural information on two bacterial Mep/Amt proteins has revealed the presence, in the hydrophobic conducting pore, of a pair of preserved histidines proposed to play an important role in substrate conductance, by participating either in NH(4)(+) deprotonation or in shaping the pore. Here we highlight the existence of two functional Mep/Amt subfamilies distinguishable according to whether the first of these histidines is conserved, as in yeast ScMep2, or replaced by glutamate, as in ScMep1. Replacement of the native histidine of ScMep2 with glutamate leads to conversion from ScMep2 to ScMep1-like properties. This includes a two-unit upshift of the optimal pH for transport and an increase of the transport rate, consistent with alleviation of an energy-limiting step. Similar effects are observed when the same substitution is introduced into the Escherichia coli AmtB protein. In contrast to ScMep1, ScMep2 is proposed to play an additional signaling role in the induction of filamentous growth, a dimorphic change often associated with virulence in pathogenic fungi. We show here that the histidine to glutamate substitution in ScMep2 leads to uncoupling of the transport and sensor functions, suggesting that a ScMep2-specific transport mechanism might be responsible for filamentation. Our overall data suggest the existence of two functional groups of Mep/Amt-type proteins with different transport mechanisms and distinct impacts on cell physiology and signaling.
    Journal of Biological Chemistry 06/2008; 283(31):21362-70. · 4.65 Impact Factor
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    ABSTRACT: Nitrogen is an essential nutrient for all life forms. Like most unicellular organisms, the yeast Saccharomyces cerevisiae transports and catabolizes good nitrogen sources in preference to poor ones. Nitrogen catabolite repression (NCR) refers to this selection mechanism. All known nitrogen catabolite pathways are regulated by four regulators. The ultimate goal is to infer the complete nitrogen catabolite pathways. Bioinformatics approaches offer the possibility to identify putative NCR genes and to discard uninteresting genes. We present a machine learning approach where the identification of putative NCR genes in the yeast Saccharomyces cerevisiae is formulated as a supervised two-class classification problem. Classifiers predict whether genes are NCR-sensitive or not from a large number of variables related to the GATA motif in the upstream non-coding sequences of the genes. The positive and negative training sets are composed of annotated NCR genes and manually-selected genes known to be insensitive to NCR, respectively. Different classifiers and variable selection methods are compared. We show that all classifiers make significant and biologically valid predictions by comparing these predictions to annotated and putative NCR genes, and by performing several negative controls. In particular, the inferred NCR genes significantly overlap with putative NCR genes identified in three genome-wide experimental and bioinformatics studies. These results suggest that our approach can successfully identify potential NCR genes. Hence, the dimensionality of the problem of identifying all genes involved in NCR is drastically reduced.
    BMC proceedings 02/2008; 2 Suppl 4:S5.
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    Elsa Lauwers, Guido Grossmann, Bruno André
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    ABSTRACT: Current models for plasma membrane organization integrate the emerging concepts that membrane proteins tightly associate with surrounding lipids and that biogenesis of surface proteins and lipids may be coupled. We show here that the yeast general amino acid permease Gap1 synthesized in the absence of sphingolipid (SL) biosynthesis is delivered to the cell surface but undergoes rapid and unregulated down-regulation. Furthermore, the permease produced under these conditions but blocked at the cell surface is inactive, soluble in detergent, and more sensitive to proteases. We also show that SL biogenesis is crucial during Gap1 production and secretion but that it is dispensable once Gap1 has reached the plasma membrane. Moreover, the defects displayed by cell surface Gap1 neosynthesized in the absence of SL biosynthesis are not compensated by subsequent restoration of SL production. Finally, we show that down-regulation of Gap1 caused by lack of SL biogenesis involves the ubiquitination of the protein on lysines normally not accessible to ubiquitination and close to the membrane. We propose that coupled biogenesis of Gap1 and SLs would create an SL microenvironment essential to the normal conformation, function, and control of ubiquitination of the permease.
    Molecular Biology of the Cell 09/2007; 18(8):3068-80. · 4.60 Impact Factor
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    Elina Nikko, Bruno André
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    ABSTRACT: 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.
    Eukaryotic Cell 09/2007; 6(8):1266-77. · 3.59 Impact Factor
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    Elina Nikko, Bruno André
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    ABSTRACT: 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.
    Traffic 06/2007; 8(5):566-81. · 4.65 Impact Factor

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