Activity of a ubiquitin ligase adaptor is regulated by disordered insertions in its arrestin domain

Abstract

The protein composition of the plasma membrane is rapidly remodeled in response to changes in nutrient availability or cellular stress. This occurs, in part, through the selective ubiquitylation and endocytosis of plasma membrane proteins, which in the yeast Saccharomyces cerevisiae is mediated by the HECT E3 ubiquitin ligase Rsp5 and arrestin-­related trafficking (ART) adaptors. Here, we provide evidence that the ART protein family members are composed of an arrestin fold with interspersed disordered loops. Using Art1 as a model, we show that these loop and tail regions, while not strictly required for function, regulate its activity through two separate mechanisms. Disruption of one loop mediates Art1 substrate specificity. Other loops are subjected to phosphorylation in a manner dependent on the Pho85 cyclins Clg1 and Pho80. Phosphorylation of the loops controls Art1’s localization to the plasma membrane, which promotes cargo ubiquitylation and endocytosis, demonstrating a mechanism through which Art1 activity is regulated.

Complementary α-arrestin-ubiquitin ligase complexes control nutrient transporter endocytosis in response to amino acids

“…The localization of 35 GFP-tagged proteins to the PM remained largely unchanged, although in some instances the vacuolar GFP fluorescence was increased ( Next, we examined how amino acid availability could regulate AAT endocytosis. Therefore, we used the high affinity methionine transporter Mup1 as a model cargo because its regulation in response to nutrient excess is well characterized (Busto et al, 2018, Lee et al, 2019, Gournas et al, 2018, Guiney et al, 2016, Baile et al, 2019. Moreover, Mup1 is one of the most abundant PM proteins and it is easy to follow its endocytosis and subsequent transport into the vacuole (Busto et al, 2018).…”

“…These domains have large, disordered loop-and tail-like insertions that contribute to the function of ARTs (Baile et al, 2019).…”

Complementary α-arrestin - Rsp5 ubiquitin ligase complexes control selective nutrient transporter endocytosis in response to amino acid availability

“…F) MUP1-HA-GFP (wild type (WT)), MUP1 141 T552,560A-HA-GFP or MUP1 S568,573A-HA-GFP cells were starved (-N) for 3h and analyzed as in 142 D). G) Live-cell fluorescence microscopy analysis of cells expressing MUP1 143 T6,25,26,34A,S9,22,23,24,31,33,42A,K16,27, and interspersed extended loops (Baile et al, 2019) in light grey. The basic amino acid residues shown 182 in blue mediate the starvation-induced endocytosis of Mup1, Can1 and Lyp1 (numbers correspond to 183 amino acid positions in the Art2 sequence).…”

“…the high affinity methionine transporter Mup1 as a model cargo because its regulation in response to 140 nutrient excess is well characterized (Busto et al, 2018, Lee et al, 2019, Gournas et al, 2018, Guiney 141 et al, 2016, Baile et al, 2019. Moreover, Mup1 is one of the most abundant PM proteins and it is easy 142 to follow its endocytosis and subsequent transport into the vacuole (Busto et al, 2018).…”

“…Perhaps a similar concept also 539 holds true for α-arrestins. This is not unlikely given that their arrestin fold appears to be interspersed 540 with disordered loops and very long, probably unstructured N-and/or C-terminal tails, some of which 541 participate in cargo recognition or membrane interactions (Baile et al, 2019). 542 543 Despite the possible plasticity in substrate interactions, the selectivity of Art1-Rsp5 and Art2-Rsp5 544 complexes in ubiquitinating lysine residues proximal to the acidic patches of Mup1 is remarkable.…”