Substrate-induced ubiquitylation and endocytosis of yeast amino acid permeases

Abstract

Many plasma membrane transporters are downregulated by ubiquitylation, endocytosis, and delivery to the lysosome in response to various stimuli. We report here that two amino acid transporters of Saccharomyces cerevisiae, the general amino acid permease (Gap1) and the arginine-specific permease (Can1), undergo ubiquitin-dependent downregulation in response to their substrates and that this downregulation is not due to intracellular accumulation of the transported amino acids but to transport catalysis itself. Following an approach based on permease structural modeling, mutagenesis, and kinetic parameter analysis, we obtained evidence that substrate-induced endocytosis requires transition of the permease to a conformational state preceding substrate release into the cell. Furthermore, this transient conformation must be stable enough, and thus sufficiently populated, for the permease to undergo efficient downregulation. Additional observations, including the constitutive downregulation of two active Gap1 mutants altered in cytosolic regions, support the model that the substrate-induced conformational transition inducing endocytosis involves remodeling of cytosolic regions of the permeases, thereby promoting their recognition by arrestin-like adaptors of the Rsp5 ubiquitin ligase. Similar mechanisms might control many other plasma membrane transporters according to the external concentrations of their substrates.

FIG 1

The Gap1-112 mutant resists downregulation via the TORC1/Npr1 pathway. (A) Model of the regulation of Gap1 ubiquitylation by internal amino acids (aai) via the TORC1/Npr1 pathway (see the text). (B) (Inset) General topology of Gap1. The region colored blue has been enlarged to show the Gap1 residues replaced with alanines in the mutants used in this study. (C) gap1Δ (EK008) and gap1Δ npr1Δ (30794d) cells expressing Gap1-GFP (pJOD010) or Gap1-112–GFP (pMA074) were grown on Gal Pro medium. Glucose was added for 1.5 h before analysis by fluorescence microscopy. w-t, wild type (corresponding to strain EK008); DIC, differential interference contrast. (D) gap1Δ (EK008) and gap1Δ npr1(ts) (MA003) cells expressing Gap1-GFP (pJOD010) or Gap1-112–GFP (pMA074) were grown at 25°C on Gal Pro medium. The cells were transferred to 37°C for 2 h before analysis by fluorescence microscopy. (E) The Gap1-126 mutant is inactive. (Left) gap1Δ (EK008) and gap1Δ ssy1Δ (32501d) strains expressing Gap1-GFP (pJOD010), Gap1-126–GFP (pMA065), or no Gap1 protein (pFL38) (−) were grown for 4 days on solid medium containing citrulline (1 mM), proline (10 mM) plus d-histidine (0.05%), or phenylalanine (1 mM) as the sole nitrogen source. Citrulline and the toxic d-isomer of histidine enter cells only via Gap1. Phenylalanine (1 mM) enters cells via Gap1 and permeases (mainly Agp1) under the positive control of the Ssy1 sensor of amino acids. The results show that wild-type Gap1 complements the phenotypes due to the gap1Δ mutation, whereas the Gap1-126 variant does not. (Right) gap1Δ cells (EK008) were grown on Gal Pro medium, glucose was added for 2 h, and [¹⁴C]-citrulline (20 μM) was added to the medium before measurement of the incorporated radioactivity at various times. prot, protein. (F) (Right) Diagram of the experiment. Phe added to cells expressing the inactive Gap1-126 mutant is detected by the Ssy1 sensor. This leads to induction of permeases, such as Agp1, which incorporate Phe into the cells, thus causing endocytosis of Gap1-126. (Left) gap1Δ (EK008), gap1Δ ssy1Δ (32501d), and gap1Δ agp1Δ (30633c) cells expressing Gap1-126–GFP (pMA065) or Gap1-126-112–GFP (pES009) were grown on Gal Pro medium, glucose was added for 1.5 h, and Am (20 mM) or Phe (5 mM) was then added for 2 h or 3 h before analysis by fluorescence microscopy.

FIG 2

The Gap1-112 mutant is ubiquitylated and downregulated in response to substrate transport. (A) Fluorescence microscopy analysis of gap1Δ cells (EK008) expressing Gap1-GFP (pJOD010) or Gap1-112–GFP (pMA074) under the same conditions as for Fig. 1F. (B) gap1Δ ssy1Δ (32501d) and gap1Δ ssy5Δ (FB092) cells expressing Gap1-GFP (pJOD010) or Gap1-112–GFP (pMA074) were grown on Gal Pro medium. Glucose was added for 1.5 h, and Phe (5 mM) was then added for 1.5 h before analysis by fluorescence microscopy. (C) (Top) gap1Δ cells (EK008) expressing Gap1-112 (pMA074), Gap1-112-K9R-K16R (pKG057), or Gap1-126-112 (pMA108) fused to GFP were grown on Gal Pro medium and treated with Phe (5 mM) for 15 min. Crude cell extracts were prepared and immunoblotted with anti-GFP antibodies. (Middle) Conditions as for panel A, with gap1Δ cells (EK008) expressing Gap1 (pJOD010) or Gap1-112 (pMA074). (Bottom) Conditions as for panel A, except that cells were examined at several time intervals. (D) Fluorescence microscopy analysis of gap1Δ cells (EK008) expressing Gap1-112–GFP (pMA074) or Gap1-112-K9R-K16R–GFP (pKG057). Conditions were as in panel B, except that the cells were examined 3 h after Phe addition. (E) Diagram illustrating Gap1 ubiquitylation and endocytosis by internal and external amino acids. Internal amino acids (AA_i) act via the TORC1/Npr1 pathway and derive from Am uptake or from direct uptake of external amino acids (AA_e) via other permeases and via Gap1 itself. AA_e transport by Gap1 itself also promotes ubiquitylation and endocytosis of the permease.

FIG 3

A single substitution in the substrate-binding site abolishes the activity and substrate-induced endocytosis of Gap1. (A) Side (left) and top (right) views of the best energy-scored 3D model of Gap1 in the OF occluded state. The protein is depicted as a ribbon diagram, with the five TMs shaping the substrate-binding site highlighted in color (TM1, yellow; TM3, orange; TM6, purple; TM8, blue; and TM10, green). TM11 and 12 are not part of the 5 plus 5 fold. The phenylalanine substrate is shown as spheres colored according to the following scheme: cyan, carbon; red, oxygen; blue, nitrogen. (B) Close view of the binding pocket bound to phenylalanine. The substrate Phe and Gap1 residues within 4 Å of the substrate are shown, respectively, as ball-and-stick and stick models. The residues are colored as in panel A. (C) gap1Δ (EK008) and gap1Δ ssy1Δ (32501d) cells expressing Gap1-GFP (pEL003) or Gap1(G107N)-GFP (pMS018) were grown on glucose Pro medium; then, Phe (5 mM) or Am (20 mM) was added for 2 h before analysis by fluorescence microscopy. (D) Strain MS001, lacking eight amino acid permease genes and the SSY1 amino acid sensor gene, was transformed with a plasmid containing the wild-type GAP1 gene (pEL003), the GAP1(G107N) gene (pMS018), or no GAP1 gene (pFL38) (−). The strains were grown for 4 days on solid medium containing the indicated nitrogenous compounds as sole nitrogen sources. (E) Time course of accumulation of [¹⁴C]arginine (top) or [¹⁴C]phenylalanine (bottom) (initial concentration, 10 μM) measured in cells of strain MS001 grown on glucose-urea medium and transformed with a plasmid containing the wild-type GAP1 gene (pEL003), the GAP1(G107N) mutant gene (pMS018), or no GAP1 gene (pFL38).

FIG 4

Substrate-induced endocytosis of the Can1 permease. (A) Wild-type cells (23344c) expressing Can1-GFP (pKG036) were grown on glucose Pro medium. Am (20 mM) or Arg (5 mM) was added for 3 h before analysis by fluorescence microscopy. (B) Wild-type (23344c) and gap1Δ npr1Δ (30794d) cells expressing Can1-GFP (pKG036) were grown on glucose Pro medium and examined by fluorescence microscopy. (C) rsp5(npi1) cells (27038a) expressing Can1-GFP (pKG036) were grown on glucose Pro medium. Arg (5 mM) was added to the medium, and the cells were examined after 3 h by fluorescence microscopy. (D) (Top) Two close views of the binding pocket of wild-type Can1 and Can1(T180R) bound to arginine (purple). F295 and W177 are shown as they sandwich the arginine side chain. The residue at position 180 is colored green. (Bottom) Wild-type cells (23344c) expressing Can1-GFP (pKG036) or Can1(T180R)-GFP (pKG013) were grown on glucose Pro medium before addition of Arg (5 mM) for 3 h. (E) (Top) Wild-type (23344c) and gap1-1 can1-1 (21983c) cells expressing the indicated Can1 variants or no Can1 were grown for 2 days on solid medium containing Arg or Am as the sole nitrogen source and on Am medium supplemented with canavanine (10 μg/ml). (Bottom) Time course of [¹⁴C]arginine accumulation (initial concentration, 10 μM) measured in 22Δ8AA mutant cells (lacking Gap1, Can1, Lyp1, and five other yAAPs) expressing Can1 (pKG036), Can1(T180R) (pKG013), or no Can1 (pFL38) and grown on glucose Am medium. (F) Wild-type cells (23344c) expressing Can1-GFP (pKG036) or Can1(S176N-T456S)-GFP (pKG066) were grown on glucose Pro medium before addition of Arg or Lys (5 mM) for 3 h.

FIG 5

Substrate-induced endocytosis of Can1 can occur without transport. (A) Wild-type cells expressing Can1 (pCJ563), Can1(E184A) (pCJ573), or Can1(E184Q) (pCJ559) fused to GFP were grown on Gal Pro medium. Glucose was added to the cultures for 0.5 h before addition of Arg (5 mM) for 2 h. (B) Mutant strain 22Δ8AA, lacking Gap1, Can1, Lyp1, and five other yAAPs and expressing the indicated permeases, was grown for 3 days on solid glucose Am medium with or without thialysine (0.1 mM). (C) Conditions as in Fig. 4F, except that thialysine (5 mM) was added instead of Arg or Lys.

FIG 6

The Gap1(W179L) and Can1(E184D) mutants display reduced V_max values of transport and are largely resistant to substrate-mediated downregulation. (A) Concentration-dependent uptake rates of [¹⁴C]Phe in the MS001 strain grown on glucose urea medium and expressing Gap1-GFP (●) (pEL003) or Gap1(W179L)-GFP (▲) (pMS023). The error bars indicate standard deviations. (B) (Top) gap1Δ cells (EK008) expressing Gap1-GFP (pEL003) or Gap1(W179L)-GFP (pMS023) under their natural promoter were grown on glucose Pro medium before analysis by fluorescence microscopy. (Bottom) gap1Δ ssy1Δ cells (32501d) expressing Gap1-GFP (pJOD010) or Gap1(W179L)-GFP (pKG078) under the GAL promoter were grown on Gal Pro medium before addition of glucose for 1.5 h. Crude cell extracts were prepared and immunoblotted with anti-GFP antibodies. The indicated values correspond to relative quantification of the signal intensities. (C) gap1Δ cells (EK008) expressing Gap1-112 (pMA074) or Gap1-112-W179L (pKG068) fused to GFP were grown on Gal Pro medium before addition of glucose for 1.5 h. Phe (5 mM) was then added for 3 h. (D) Concentration-dependent uptake rates of [¹⁴C]arginine in 22Δ8AA cells grown on glucose Am medium and expressing Can1(E184D)-GFP (pES003). (E) Wild-type cells (23344c) expressing the GFP-fused Can1(E184D) variant (pES002) were grown on glucose Pro medium before addition of Arg (5 mM) for 3 h.

FIG 7

The Can1(T456S) mutant displaying higher V_max values of arginine and lysine transport is not downregulated by these amino acids. (A) Close view of three residues (456 from TM10 and 176 and 180 from TM3, depicted as balls) of the binding pocket of wild-type Can1 (left) and of Can1(T456S) (right). Replacement of T456 with serine introduces a void between the two TMs that could facilitate the conformational transition required for transport (Table 2). (B) Wild-type cells (23334c) expressing the GFP-fused Can1 (pKG036) or Can1(T456S) (pKG046) were grown on glucose Am medium before addition of Arg (5 mM) for 3 h and subsequent analysis by fluorescence microscopy. Vm, V_max. (C) Same as panel B, except that cells expressing the GFP-fused Can1(T456S) (pKG046) or Can1(S176N-T456S) (pKG026) were examined after addition of Lys (5 mM) to the medium.

FIG 8

Arrestin-like adaptors of Rsp5 promote substrate-induced downregulation of Gap1 and Can1. (A) (Top) gap1Δ (EK008) and gap1Δ bul1Δ bul2Δ (JA493) cells expressing Gap1-112–GFP (pMA074) were grown on Gal Pro medium before addition of glucose for 0.5 h. Phe (5 mM) was then added for 2 h. (Bottom) The same cells and growth conditions, except that Phe was added for 0.5 h. Crude cell extracts were then prepared and immunoblotted with anti-GFP antibodies. (B) Wild-type (23344c) and gap1Δ art1Δ (JA937) cells expressing GFP-fused Can1 (pKG036) or Can1(S176N-T456S) (pKG066) were grown on glucose Am medium (hence, Gap1 synthesis is repressed in the wild type). Arg or Lys was then added for 3 h before analysis by fluorescence microscopy. (C) (Top) gap1Δ can1 (ES006) and gap1Δ can1 rsp5(npi1) (35237c) cells expressing Art1-HA under the natural promoter of the ART1 gene were grown on glucose Pro medium. Crude cell extracts were then prepared and immunoblotted with anti-HA antibodies. (Bottom) gap1Δ can1 ART1-HA cells (ES006) transformed with a CAN1 plasmid (pKG036) were grown on glucose Pro medium. Arg (5 mM) or Am (20 mM) was added for 20 min, and crude cell extracts were prepared and immunoblotted with anti-HA antibodies. (D) (Top) gap1Δ cells (EK008) expressing Gap1-112–GFP (pMA074) were grown on Gal Pro medium before addition of glucose for 0.5 h (left), Am (20 mM) for 2 h (middle), and Phe (5 mM) for 2 h (right) to half of the culture. (Bottom) Cells expressing Bul2-HA under the natural promoter of the BUL2 gene (MA032) and Gap1-112–GFP (pMA074) were grown on Gal Pro medium and treated as described above for the indicated time intervals. Crude cell extracts were prepared after the indicated times and immunoblotted with anti-HA antibodies.

FIG 9

Two Gap1 mutants undergo constitutive Bul-dependent endocytosis. (A) gap1Δ (EK008) and gap1Δ bul1Δ bul2Δ (JA479) cells expressing the GFP-fused Gap1 (pJOD010), Gap1-124 (pMA019), Gap1-167 (pNG047), Gap1-124-(K9R-K16R) (pMA142), or Gap1-167-(K9R-K16R) (pMA148) were grown on Gal Pro medium, glucose was added for 1.5 h, and the cells were examined by fluorescence microscopy. (B) Concentration-dependent rates of [¹⁴C]Phe uptake in gap1Δ ssy1Δ cells (32501d) expressing Gap1(K9R-K16R)–GFP (pCJ038) (●), Gap1-124-(K9R-K16R)–GFP (pMA142) (○), or Gap1-167-(K9R-K16R)–GFP (pMA148) (▽). (C) The reduced V_max values for Gap1-124 and Gap1-167 stabilized at the plasma membrane are not due to reduced amounts of the permeases. gap1Δ ssy1Δ cells (32501d) expressing GFP-fused Gap1(K9R-K16R), Gap1-124-(K9R-K16R), or Gap1-167-(K9R-K16R) (plasmids as in panel B) and gap1Δ bul1Δ bul2Δ (JA493) expressing GFP-fused Gap1, Gap1-124, or Gap1-167 (plasmids as in panel A) were grown on Gal Pro medium, and glucose was added for 2 h. Crude cell extracts were prepared and immunoblotted with anti-GFP antibodies.

FIG 10

Hypothetical model of substrate-induced ubiquitylation and endocytosis of yeast amino acid permeases. (Top) Upon amino acid binding (a and b), the permease shifts to the outward-open-occluded (c) and then the inward-open-occluded (d) state. An inward open conformation preceding substrate release into the cytosol is prone to ubiquitylation (e and f). Substitutions in Can1 or Gap1 impeding shifts to different states (arrows) of the transport cycle are indicated. In the case of Can1(T456S), the state favoring ubiquitylation is not stable enough to promote efficient endocytosis (d to g direct shift). In the cases of Can1(E184Q) in the presence of Arg and Can1(S176N-T456S) and in the presence of thialysine, the permeases shift to the state favoring ubiquitylation without releasing the substrate into the cell. The conformational change favoring ubiquitylation involves the N-terminal tail, possibly interacting with an internal loop of the permease. The permease in the state labeled e or f can shift to the states leading to substrate release (g and a). (Bottom) Substitutions in cytosol-facing regions (e.g., Gap1-124 and -167) can mimic the conformation prone to ubiquitylation.