Transition of yeast Can1 transporter to the inward-facing state unveils an α-arrestin target sequence promoting its ubiquitylation and endocytosis

Abstract

Substrate-transport-elicited endocytosis is a common control mechanism of membrane transporters avoiding excess uptake of external compounds, though poorly understood at the molecular level. In yeast, endocytosis of transporters is triggered by their ubiquitylation mediated by the Rsp5 ubiquitin-ligase, recruited by α-arrestin-family adaptors. We here report that transport-elicited ubiquitylation of the arginine transporter Can1 is promoted by transition to an inward-facing state. This conformational change unveils a region of the N-terminal cytosolic tail targeted by the Art1 α-arrestin, which is activated via the TORC1 kinase complex upon arginine uptake. Can1 mutants altered in the arginine-binding site or a cytosolic tripeptide sequence permanently expose the α-arrestin-targeted region so that Art1 activation via TORC1 is sufficient to trigger their endocytosis. We also provide evidence that substrate-transport elicited endocytosis of other amino acid permeases similarly involves unmasking of a cytosolic Art1-target region coupled to activation of Art1 via TORC1. Our results unravel a mechanism likely involved in regulation of many other transporters by their own substrates. They also support the emerging view that transporter ubiquitylation relies on combinatorial interaction rules such that α-arrestins, stimulated via signaling cascades or in their basal state, recognize transporter regions permanently facing the cytosol or unveiled during transport.

FIGURE 1:

The Art1 and Bul α-arrestins promote Can1 ubiquitylation in response to arginine. (A) Strains (with gap1Δ can1Δ mutations) expressing Can1-GFP were grown on Gal Pro medium. GAL-promoter–driven expression of CAN1-GFP was repressed by addition of 3% Glu for 1.5 h. Arg (5 mM) was then added for 3 h. After CMAC staining, the cells were observed by epifluorescence microscopy. Scale bar here and below is 2 μm. (B) Plasma membrane (PM) to intracellular GFP fluorescence intensity ratios, for the experiment of A, are plotted (n = 90 cells). The horizontal midline represents the median, the box is bounded by the upper and lower quartiles, and the whiskers denote the maximal and minimal ratios. ***, P < 0.001; ns, nonsignificant, P > 0.05. Lower middle. (C) The strains of A were grown on Gal Pro; Glu was added for 0.5 h and then Arg for 30 min. Total protein extracts were probed with antibodies against GFP and Pma1. (D) Immunoprecipitation (IP), via GFP, of Can1-GFP or Can1(7KR)-GFP. Cells were grown in Gal Pro media, and glucose was added for 90 min, followed by 5 mM Arg for 25 min. After immunoprecipitation using GFP, extracts were probed with antibodies against GFP or Ub. (E, F) The effects of Arg or Lys addition to Gal Pro grown gap1Δ can1Δ lyp1Δ cells expressing Can1-GFP, Can1(S176N,T456S)-GFP, or Can1(T180R)-GFP were examined by epifluorescence microscopy (E) or by immunoblotting total protein extracts (F); conditions as in A and C, respectively. (G) Strains (with a gap1Δ mutation) expressing Can1-GFP were grown on Gal Pro or Gal Am medium. Glu was added for 30 min and then Arg for 15 min. Protein extracts were probed as in C. (H) Epifluorescence microscopy analysis of strains in G. Glu was added for 90 min and then Arg for 3 h. Quantification of the fluorescence signals is presented in Supplemental Figure S1.

FIGURE 2:

Can1 is internalized and recycles to the plasma membrane via the Golgi upon Arg addition to the art1 mutant. (A) Scheme illustrating the role of Ypt6 Rab family small GTPase in recycling of membrane proteins from endosomes to the late Golgi complex. (B) Strains (all with gap1Δ can1Δ mutations) carrying or not a deletion of the YTP6 gene, and expressing Can1-GFP, were grown in Gal Pro medium. Glu was added for 1.5 h and then Arg for the time indicated, before imaging by epifluorescence microscopy. (C) Strains expressing Can1-GFP and Sec7-mCherry were grown in Gal Pro medium. Glu was added for 1.5 h and then Arg for 15 min, before imaging by confocal microscopy. Arrows indicate sites of colocalization between Can1 and Sec7. Right: The Pearson’s correlation coefficient for Can1-GFP and Sec7-mCherry are plotted (n = 40 cells). Representations as in Figure 1B.

FIGURE 3:

Lys-42 and Lys-45 are the main Ub-acceptors and required for efficient vacuolar sorting of Can1. (A) Schematic representation of the 23 mutants obtained by Ala-scanning mutagenesis of the N-tail of Can1. Mutants resistant to Art1-dependent endocytosis are shown in red, and those resistant to Bul1/2-dependent ubiquitylation in blue. The two main Ub-acceptor Lys residues required for efficient vacuolar sorting are also highlighted. See also Supplemental Figure S3 and Figure 4. (B) Strains (with gap1Δ can1Δ mutations) expressing Can1-GFP or Can1-GFP mutants carrying Ala-substitutions of the indicated residues of the N-tail were grown on Gal Am. Glu was added for 90 min and then Arg for 3 h before imaging as in Figure 1A. Quantifications are shown in Supplemental Figure S3D. (C, D, E) Epifluorescence microscopy analysis (C, as in Figure 1A) and immunoblots of total protein extracts (D, as in Figure 1G) of Gal Am grown strains (with gap1Δ can1Δ mutations) expressing Can1-GFP or the indicated Lys-to-Arg substitution alleles. For the immunoblot, cells were first grown in Raf Am and Gal was added for 1 h, Glu for 1.5 h and then Arg for 15 min. (E) C¹⁴-Arg uptake measurements in a gap1Δ can1Δ bul1/2Δ strain expressing Can1-GFP, the corresponding mutants, or no Can1 protein. (F, G, H) Strains (with gap1Δ can1Δ mutations) expressing Can1-GFP or Can1-(7KR)-GFP were grown in Gal Am; experiments and conditions as in C, D, and E, respectively.

FIGURE 4:

Art1 and Bul1/2 act via distinct N-terminal regions of Can1 to promote its ubiquitylation. (A) Strains (all with gap1Δ can1Δ mutations) expressing Can1-GFP or Can1-GFP mutants carrying Ala-substitutions of the indicated residues of the N-tail 70–81 region were grown on Gal Am. Glu was added for 0.5 h and then Arg for 15 min. Protein extracts were probed as in Figure1C. (B) Strains (with gap1Δ can1Δ mutations) expressing Can1-GFP or Can1-GFP mutants carrying Ala-substitutions of the indicated residues of the N-tail 62–69 region were examined as in A. See also Supplemental Figure S3F. (C, D, E) Epifluorescence microscopy analysis (C), immunoblotting (D), and C¹⁴-Arg uptake measurements (E) of Can1-GFP and a mutant carrying Ala substitutions of 66–69 and 74–77 residues, conditions as in Figure 3, F, G, and H, respectively.

FIGURE 5:

The inactive Can1(E184Q) mutant is down-regulated upon Arg uptake. (A) Schematic representation of the regulation of the Art1 and Bul α-arrestins by nitrogen availability via the TORC1 pathway. (B) Immunoblots of total protein extracts of a wild-type strain expressing HA-Npr1. Samples with or without rapamycin addition 30 min before Am or Arg addition were collected and probed with anti-HA antibodies. (C) Epifluorescence microscopy analysis of Gal Pro grown gap1Δ can1Δ cells expressing Can1-GFP or Gap1-GFP. Glu was added for 1.5 h and then Arg or Am for 3 h. (D) Strains (with gap1Δ can1Δ mutations) expressing Can1-GFP were grown on Gal Pro. Glu was added for 30 min and then Arg or Am for 15 min. Total protein extracts were probed as in Figure 1C. (E) Epifluorescence microscopy analysis (as in Figure 1A) and (F) immunoblotting (as in Figure 1C) of Can1-GFP and the indicated mutants in Gal Pro grown strains (with a gap1Δ mutation). (G) Epifluorescence microscopy analysis (as in Figure 1A) and (H) immunoblotting of Can1-GFP and the indicated mutants; conditions as in E and F, respectively. (I) Epifluorescence microscopy analysis (as Figure 1A) of the corresponding strains expressing Can1-GFP or Can1(E184Q)-GFP; conditions as in E. (J) Epifluorescence microscopy analysis (as in Figure 1A) of a gap1Δ can1Δ strain expressing Can1(7KR)-mCherry and Can1(E184Q)-GFP.

FIGURE 6:

TORC1-mediated activation of Art1 is sufficient to down-regulate the inactive Can1(E184Q) and Can1(87-ELK-89>AAA) mutants. (A) Hypothetical model for the mechanism of Art1-mediated down-regulation of Can1(E184Q) following Gap1-mediated Arg uptake. (B) Strains (with gap1Δ can1Δ mutations) expressing Can1(E184Q) and the gap1Δ can1Δ bul1/2Δ strain expressing GFP-fused wild-type or mutant Can1 (as indicated) were grown on Gal Pro. Glu was added for 1.5 h and then Am for 3 h, before imaging. The PM-to-intracellular-GFP fluorescence intensity ratios (as in Figure 1B) are plotted for the main conditions (n = 60 cells). Quantifications for other control strains are shown in Supplemental Figure S4A. (C) The same strains were grown on Gal Pro, Glu was added for 0.5 h and then Am for 20 min. Protein extracts were probed as in Figure 1C. (D) Strains expressing Can1-GFP or the indicated mutants were grown in Gal Pro. Glu was added for 1.5 h before imaging as in Figure 1A. See also Supplemental Figure S4B. (E) Strains expressing Can1-GFP or the mutant carrying Ala substitutions of residues 87–89 were examined as in Figure 5C. See also Supplemental Figure S4D.

FIGURE 7:

The E184Q substitution is predicted to stabilize Can1 in an IF conformation. (A) Left, 3D models of Can1 in the OF open, OF occluded, and IF open states, highlighting a shift (white arrow) of TM1 in the IF open conformation. Right, Close-up of TM1 colored in blue, purple, and green, respectively, in the OF open, OF occluded, and IF open conformations. The location of the 87–89 sequence is shown as red balls marking the Cα position of the residues. (B) Top, view of the surroundings of Gln-184 in two 3D models of substrate-free IF open Can1(E184Q). Gln-184 is shown as balls and sticks and the residue hydrogen-bonding to it is shown as sticks. A ribbon diagram depicting neighboring residues of TM1, TM3, and TM10 is also shown. Hydrogen bonds formed by Gln-184 are shown as blue broken lines. Bottom, summary table of the analysis of H-bonds formed by residue at position 184 in structural models of Can1(E184Q) and wild-type Can1, with Glu-184 in the protonated (Glu-184h) or charged (Glu-184-) form, in the OF open, OF occluded and IF open conformations. 0H, 1H, 2H: number of Can1 models (out of 10) with no, one, or two H-bonds established by the side chain of residue 184 (in TM3) with residues of other TMs. totH: total number of H-bonds established by the side chain of residue 184 with other TMs in the 10 analyzed Can1 models. (C) Close-up view of the region encompassing residue 176 in representative OF occluded models of Can1, Can1(S176N,T456S), and Can1(S176N). In two Can1(S176N) models, pink broken lines show steric hindrance between the N176 side chain, depicted as balls and sticks, and neighboring residues, including those of the middle (W177) and distal (Y173, E301, W464) gates. Portions of TM1, TM3, and TM10 are depicted as ribbons. (D) Epifluorescence microscopy analysis of a gap1Δ can1Δ bul1/2Δ strain expressing Can1-GFP or the indicated mutant grown on Gal Pro. Glu was added for 1.5 h and then Am for 3 h. (E) immunoblots of cell extracts from the strains of D grown on Gal Pro. Glu was added for 0.5 h and then Am for 0.5 h. (F) ¹⁴C-Arg uptake measurements in a gap1Δ can1Δ strain expressing the indicated Can1 mutant. See also Supplemental Figure S5.

FIGURE 8:

Substrate-transport–elicited unveiling of permease cytosolic regions brings specificity to Art1-mediated ubiquitylation. (A) Epifluorescence microscopy analysis of a strain expressing Mup1-GFP and of a gap1Δ strain expressing Can1-GFP or Lyp1-GFP. For Can1-GFP, Gal Pro was used and Glu was added for 1.5 h. For Lyp1-GFP and Mup1-GFP, Glu Pro was used. Arg, Lys, or Met was added for 3 h before observation. (B) Immunoblotting of total protein extracts as in Figure 5B. Arg, Lys, or Met was added in rapamycin-treated and untreated wild-type cells. (C) Epifluorescence microscopy analysis and (D) ¹⁴C-amino acid uptake measurements on the indicated strains expressing Can1(E184Q) and grown in Gal Pro. For microscopy, Glu was added for 1.5 h and then Arg, Lys, or Met for 3 h.

FIGURE 9:

Model of the mechanisms governing ubiquitylation and endocytosis of yeast amino acid permeases. (A) Art1- and Rsp5-mediated ubiquitylation and down-regulation of Can1 permease in response to substrate transport and TORC1 activation, as in the mutant lacking the Bul α-arrestins, for example. In the absence of substrate, the short ELK sequence (black) close to TM1 is oriented toward the core of the transporter and interacts with cytoplasmic loops, structuring the remaining tail so that the binding site for Art1 (aa 70–81, red hemicycle) is masked. In the presence of Arg, a transient switch of Can1 to an IF conformation, via repositioning of TM1 and the ELK tripeptide, causes a structural rearrangement unmasking the binding site for Art1 (now green). Arg uptake also stimulates TORC1, which activates Art1. Once activated, Art1 can recognize the unveiled binding site. This results in Rsp5-mediated Can1 ubiquitylation, mainly on Lys-42 or Lys-45. (B) Ubiquitylation and endocytosis of Gap1 and Can1 in response to TORC1 stimulation. Activation of TORC1 stimulates the Art1 and Bul α-arrestins. Once activated, the Buls can act through a permanently exposed region of the Gap1 N-tail (residues 20–35), causing its ubiquitylation on Lys-9 or Lys-16. This modification efficiently targets Gap1 to the vacuole. The activated Buls also recognize a permanently exposed binding site in the Can1 N-tail (residues 62–69), causing its ubiquitylation. This modification does not efficiently target the internalized Can1 to the vacuole. The TORC1-activated Art1 does not efficiently recognize Can1 because its binding site remains masked.