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
    • "Several potential novel targets of Rsp5 were also identified. Bmh2, a 14-3-3 protein, which has previously been shown to regulate the Rsp5 adaptor Bul1 [74], was isolated, and Ino1 (inositol 3-phosphate synthase) and Pdi1 (a protein disulfide isomerase) were also previously isolated in large-scale proteomic analyses of Rsp5- associated proteins [75,76]. Sul2 (a sulfur permease), two plasma membrane-associated t-SNAREs (Sso1 and Sso2), and Rvs161, a lipid raft protein, were also isolated, consistent with the role of Rsp5 in membrane protein trafficking. "
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    ABSTRACT: We describe a new class of reagents for identifying substrates, adaptors, and regulators of HECT and RING E3s. UBAITs (Ubiquitin-Activated Interaction Traps) are E3-ubiquitin fusion proteins and, in an E1- and E2-dependent manner, the C-terminal ubiquitin moiety forms an amide linkage to proteins that interact with the E3, enabling covalent co-purification of the E3 with partner proteins. We designed UBAITs for both HECT (Rsp5, Itch) and RING (Psh1, RNF126, RNF168) E3s. For HECT E3s, trapping of interacting proteins occurred in vitro either through an E3 thioester-linked lariat intermediate or through an E2 thioester intermediate, and both WT and active-site mutant UBAITs trapped known interacting proteins in yeast and human cells. Yeast Psh1 and human RNF126 and RNF168 UBAITs also trapped known interacting proteins when expressed in cells. Human RNF168 is a key mediator of ubiquitin signaling that promotes DNA double-strand break repair. Using the RNF168 UBAIT, we identify H2AZ-a histone protein involved in DNA repair-as a new target of this E3 ligase. These results demonstrate that UBAITs represent powerful tools for profiling a wide range of ubiquitin ligases.
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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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    • "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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