Internal Amino Acids Promote Gap1 Permease Ubiquitylation via TORC1/Npr1/14-3-3-Dependent Control of the Bul Arrestin-Like Adaptors

Molecular Physiology of the Cell, Université Libre de Bruxelles, IBMM, Gosselies, Belgium.
Molecular and Cellular Biology (Impact Factor: 4.78). 09/2012; 32(22). DOI: 10.1128/MCB.00463-12
Source: PubMed


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.

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Available from: Bruno André, Jan 29, 2014
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    • "Previous studies suggest that -arrestins are activated by dephosphorylation (Macgurn et al., 2011; Becuwe et al., 2012b; Merhi and Andre, 2012; O'Donnell et al., 2013). Therefore, we proceeded to examine whether pH-dependent activation of the RIM pathway correlates with changes in the phosphorylation status of Rim8. "
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    ABSTRACT: α-arrestins play a key role as trafficking adaptors in both yeast and mammals. The yeast Rim8/Art9 α-arrestin mediates the recruitment of ESCRT (endosomal sorting complex required for transport) to the seven-transmembrane protein Rim21 in the ambient pH signaling RIM pathway. ESCRT is thought to function as a signaling platform that enables the proteolytic activation of the Rim101 transcription factor upon external alkalization. Here, we provide evidence that the pH signal promotes the stable association of Rim8 with Rim21 at the plasma membrane. We show that Rim8 is phosphorylated in a pH-independent but Rim21-dependent manner, by the plasma-membrane-associated casein kinase 1 (CK1). We further show that this process involves a cascade of phosphorylation events within the hinge region connecting the arrestin domains. Strikingly, loss of casein kinase 1 activity causes constitutive activation of the RIM pathway and, accordingly, pH signaling is activated in a phospho-deficient Rim8 mutant and impaired in the corresponding phosphomimetic mutant. Our results indicate that Rim8 phosphorylation prevents its accumulation at the plasma membrane at acidic pH and thereby inhibits RIM signaling. These findings support a model where CK1-mediated phosphorylation of Rim8 contributes to setting a signaling threshold required to inhibit the RIM pathway at acidic pH. © 2015 by The American Society for Cell Biology.
    Molecular biology of the cell 04/2015; 26(11). DOI:10.1091/mbc.E14-11-1552 · 4.47 Impact Factor
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    • "It has recently been demonstrated that arrestinrelated trafficking adaptors (ARTs) in S. cerevisiae containing structurally conserved features with the mammalian arrestin proteins interact with Rsp5 and act as adaptor molecules for the ubiquitination of the nutrient transporters by the ubiquitin ligase to regulate their intracellular trafficking. Moreover, the ARTs are themselves ubiquitinated by Rsp5 (Lin et al., 2008; Nikko et al., 2008; Nikko and Pelham, 2009; O'Donnell et al., 2010; Hatakeyama et al., 2010; Merhi and André, 2012). "
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    ABSTRACT: The Tsc1-Tsc2 complex homologous to human tuberous sclerosis complex proteins governs amino acid uptake by regulating the expression and intracellular distribution of amino acid transporters in Schizosaccharomyces pombe. Here, we performed a genetic screening for molecules that are involved in amino acid uptake and found Arn1 (also known as Any1). Arn1 is homologous to ART1, an arrestin-related trafficking adaptor (ART) in Saccharomyces cerevisiae, and contains a conserved arrestin motif, a ubiquitination site, and two PY motifs. Overexpression of arn1(+) confers canavanine resistance on cells, whereas its disruption causes hypersensitivity to canavanine. We also show that Arn1 regulates endocytosis of the Cat1 amino acid transporter. Furthermore, deletion of arn1(+) suppresses a defect of amino acid uptake and the aberrant Cat1 localization in tsc2Δ. Arn1 interacts with and is ubiquitinated by the Pub1 ubiquitin ligase, which is necessary to regulate Cat1 endocytosis. Cat1 undergoes ubiquitinations on lysine residues within the N-terminus, which are mediated, in part, by Arn1 to determine Cat1 localization. Correctively, Arn1 is an ART in S. pombe and contributes to amino acid uptake through regulating Cat1 endocytosis in which Tsc2 is involved.
    Biology Open 05/2014; 3(6). DOI:10.1242/bio.20148367 · 2.42 Impact Factor
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    • "It is known that inactivation of TORC1 leads to Tap42-Sit4 dependent dephosphorylation of Npr1 (Schmidt, et al., 1998, Jacinto, et al., 2001, Gander, et al., 2008). Most recent work links Tap42-Sit4 dependent Npr1 dephosphorylation to its activation and subsequent phosphorylation of arrestin-like Bul proteins, which in turn inhibits endocytosis of Gap1 under nitrogen-limitation (Merhi & Andre, 2012). Although being an attractive model fitting with several observations, this model still fails to explain how under similar conditions Bul1,2-dependent vacuolar sorting of Tat2 can take place (Abe & Iida, 2003). "
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    ABSTRACT: The yeast Saccharomyces cerevisiae has been a favorite organism for pioneering studies on nutrient-sensing and signaling mechanisms. Many specific nutrient responses have been elucidated in great detail. This has led to important new concepts and insight into nutrient-controlled cellular regulation. Major highlights include the central role of the Snf1 protein kinase in the glucose repression pathway, galactose induction, the discovery of a G-protein coupled receptor system and role of Ras in glucose-induced cAMP signaling, the role of the protein synthesis initiation machinery in general control of nitrogen metabolism, the cyclin-controlled protein kinase Pho85 in phosphate regulation, nitrogen catabolite repression and the nitrogen-sensing TOR pathway, and the discovery of transporter-like proteins acting as nutrient sensors. In addition, a number of cellular targets, like carbohydrate stores, stress tolerance and ribosomal gene expression, are controlled by the presence of multiple nutrients. The PKA signaling pathway plays a major role in this general nutrient response. It has led to the discovery of nutrient transceptors (transporter-receptors) as nutrient sensors. Major shortcomings in our knowledge are the relationship between rapid and steady-state nutrient signaling, the role of metabolic intermediates in intracellular nutrient sensing and the identity of the nutrient sensors controlling cellular growth. This article is protected by copyright. All rights reserved.
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