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

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 downregulated 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 downregulation. 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 downregulation. 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.

Aneuploidy shortens replicative lifespan in Saccharomyces cerevisiae

“…3B). As the Q146K mutation in Bul1 is just 11 residues upstream of the PPXY motif necessary for interaction with its E3 ubiquitin ligase partner, Rsp5 (Merhi & André, 2012), we created a bul1Δ in the chr5 disome and found that complete deletion of BUL1 reproduces the lifespan extension (Fig. 3B).…”

“…Instead, damaged membrane proteins are targeted for endocytosis and pass through the multivesicular body (MVB) and to the lysosome (in yeast, the vacuole), where they are eventually degraded (Piper & Luzio, 2007; Zhao et al ., 2013). Bul1 plays an important role in this plasma membrane quality control system by facilitating the Rsp5 ‐ dependent polyubiquitination of multiple protein targets which directs their sorting to the vacuole for degradation (Yashiroda et al ., 1996; De Craene et al ., 2001; Helliwell et al ., 2001; Crespo et al ., 2004; Merhi & André, 2012; O'Donnell, 2012; Zhao et al ., 2013). Disruption of this membrane protein quality control pathway has been previously linked to accelerated aging (Fabrizio et al ., 2010), while the Rsp5/Bul1 node, in particular, has been shown to ameliorate the effects of protein aggregation in a model of neurodegenerative diseases (Tardiff et al ., 2013).…”

“…In response to poor nutrient conditions, such as growth on a nonpreferred nitrogen source, Bul1 and Bul2 are phosphorylated and inactivated. This allows the general amino acid transporter Gap1 to remain at the plasma membrane and scavenge for additional sources of nitrogen (Merhi & André, 2012; O'Donnell, 2012). Under nutrient‐replete conditions, signaling through TOR results in Bul1/Bul2 derepression, association with Rsp5, and resulting ubiquitination and endocytosis of Gap1 (Merhi & André, 2012; O'Donnell, 2012).…”

“…This allows the general amino acid transporter Gap1 to remain at the plasma membrane and scavenge for additional sources of nitrogen (Merhi & André, 2012; O'Donnell, 2012). Under nutrient‐replete conditions, signaling through TOR results in Bul1/Bul2 derepression, association with Rsp5, and resulting ubiquitination and endocytosis of Gap1 (Merhi & André, 2012; O'Donnell, 2012). Finally, additional support connecting Bul1/Bul2 function to aging via effects on nutrient signaling comes from a study where a natural variant of BUL2 prolonged chronological aging in yeast via Gln3‐dependent effects on telomere length (Kwan et al ., 2011).…”

Ubiquitin-Dependent Sorting in Endocytosis

“…In mammalian cells, ARRDC3 controls ubiquitination and down-regulation of b2 and b3 adrenergic receptors as well as the b4 integrin (Draheim et al 2010;Nabhan et al 2010;Patwari et al 2011;Shea et al 2012). Recent studies have shown that some a-arrestin proteins are regulated by phosphorylation, in particular kinases downstream from Tor1, thus allowing cell global regulation of plasma membrane protein levels in response to nutrient status (MacGurn et al 2011;Merhi and Andre 2012). As a consequence of directly binding Nedd4 Ub ligases, all of the a-arrestin-family proteins also undergo ubiquitination.…”