Yeast α-arrestin Art2 is the key regulator of ubiquitylation-dependent endocytosis of plasma membrane vitamin B1 transporters

Abstract

Endocytosis of membrane proteins in yeast requires α-arrestin-mediated ubiquitylation by the ubiquitin ligase Rsp5. Yet, the diversity of α-arrestin targets studied is restricted to a small subset of plasma membrane (PM) proteins. Here, we performed quantitative proteomics to identify new targets of 12 α-arrestins and gained insight into the diversity of pathways affected by α-arrestins, including the cell wall integrity pathway and PM-endoplasmic reticulum contact sites. We found that Art2 is the main regulator of substrate- and stress-induced ubiquitylation and endocytosis of the thiamine (vitamin B1) transporters: Thi7, nicotinamide riboside transporter 1 (Nrt1), and Thi72. Genetic screening allowed for the isolation of transport-defective Thi7 mutants, which impaired thiamine-induced endocytosis. Coexpression of inactive mutants with wild-type Thi7 revealed that both transporter conformation and transport activity are important to induce endocytosis. Finally, we provide evidence that Art2 mediated Thi7 endocytosis is regulated by the target of rapamycin complex 1 (TORC1) and requires the Sit4 phosphatase but is not inhibited by the Npr1 kinase.

Fig 1. Art2 and Rsp5 are required for CHX- and thiamine-induced Thi7 endocytosis.

(A) The WT, art2Δ, art9Δ, and rsp5 strains expressing THI7-GFP under their endogenous promoter were grown in thiamine-free selective medium up to early-log phase and harvested after 120 min with CHX (final concentration; 50 μg/ml) or EtOH (top panel) and with thiamine (“Th.,” final concentration: 100 μM) or water (H₂O) (bottom panel). Total protein extracts were immunoblotted with anti-GFP and anti-Pma1 as a loading control. Free GFP is the results of the degradation of Thi7-GFP in the vacuole. Thi7-GFP band intensity is normalized by the intensity of the Pma1-corresponding band. Representative of 3 experiments. (B) Localization of Thi7-GFP in the WT, art2Δ, art9Δ, rsp5, and art2Δart9Δ strains after addition of thiamine or CHX or mock-treated with water. The vacuolar membrane is stained with FM4–64. Quantification shows the ratio of internal-over-PM fluorescence intensity as described in Materials and methods section (n > 30 cells) (****p < 0.0001; ***p < 0.001; **p < 0.01; *p < 0.05). (C) Expression of 3xHA-ART2 restores thiamine-induced endocytosis of Thi7-GFP in the art2Δ background. THI7-GFP and either 3xHA-ART2 or its corresponding e.v. are expressed in the art2Δ strain, grown in thiamine-free selective medium, and complemented with thiamine (final concentration: 100 μM). Scale bar represents 5 μm. The numerical data are included in S1 Data. CHX, cycloheximide; EtOH, ethanol; e.v., empty vector; GFP, green fluorescent protein; ns, nonsignificant; PM, plasma membrane; Pma1, plasma membrane ATPase 1; WT, wild type.

Fig 2. The endocytosis of the low-affinity thiamine transporters Nrt1 and Thi72 is also mediated by Art2.

(A) Strains expressing NRT1-GFP (top panel) and THI72-GFP (bottom panel) under THI7 endogenous promoter were grown in thiamine-free medium up to early-log phase and harvested after 120-min incubation with thiamine (“Th.”) at a concentration of 100 μM for total protein extractions. Extracts were immunoblotted with anti-GFP and anti-Pma1 as a loading control. Values are quantification of band intensity. (B) Localization of Nrt1-GFP or Thi72-GFP in WT, art2Δ, art9Δ, rsp5, and art2Δart9Δ strains after addition of an excess of thiamine into culture grown in thiamine-free medium. The vacuolar membrane is stained with FM4–64. Scale bar represents 5 μm. Quantification shows the ratio of internal-over-PM fluorescence intensity as described in Materials and methods section (n > 50 cells). (****p < 0.0001; ***p < 0.001; **p < 0.01; *p < 0.05) The numerical data are included in S2 Data. GFP, green fluorescent protein; Nrt1, nicotinamide riboside transporter 1; ns, nonsignificant; PM, plasma membrane; Pma1, plasma membrane ATPase 1; WT, wild type.

Fig 3. The Art2-dependent ubiquitylation of Thi7-GFP C terminus is required for its endocytosis.

(A) GFP-tagged KR-mutated THI7 was expressed in a thi7Δnrt1Δthi72Δ strain and grown in a thiamine-free medium. Serial 10-fold dilutions of yeast cells in early log phase were spotted on thiamine-free oxythiamine-containing medium. The expression of pRS416 (empty vector) and the nontagged version were used as controls. Oxythiamine was transported by all the KR Thi7 mutants (B) Localization of the different KR mutant versions of Thi7-GFP in the WT strain after thiamine treatment (final concentration: 100 μM): Thi7^KR-GFP, Thi7^NterKR-GFP, Thi7^CterKR-GFP, and Thi7^Cter6KR-GFP. (C) Top: THI7-GFP and THI7^KR-GFP were expressed in end3Δ and end3Δart2Δ strains and grown in thiamine-free medium to early log phase before a 30-min incubation with 100 μM thiamine. Bottom: THI7-GFP, Thi7^KR-GFP, Thi7^NterKR-GFP, Thi7^CterKR-GFP, or Thi7^Cter6KR-GFP were expressed in art2Δ strain (complemented or not with 3xHA-ART2) and grown in thiamine-free medium to early log phase before a 30-min incubation with 100 μM thiamine. After solubilization, IP of transporters was performed using the GFP-Trap kit. Samples were immunoblotted with anti-GFP and anti-Ub. Quantification is based on the intensity of the Ub band and normalized to the intensity of the corresponding GFP signal. GFP, green fluorescent protein; IB, immunoblotting; IP, immunoprecipitation; KR, lysine-to-arginine; Ub, ubiquitin; WT, wild type.

Fig 4. Selection of 14 single-point Thi7 mutants with reduced transport activity and structural modelling of Thi7.

(A) Scheme of Thi7 topology generated by Protter software [43], representing the 14 single-point Thi7 mutants isolated by oxythiamine-based screening (red) or by side-directed mutagenesis (blue). The lysine residues mutated to generate Thi7^KR mutants are shown in purple. (B) Phenotypic growth test of thi7Δnrt1Δthi72Δ strain expressing single-point THI7-GFP mutants, WT THI7-GFP, or an e.v. on thiamine-free selective medium (SC-U-B1) supplemented or not with oxythiamine (final concentrations; 3, 6, and 15 μM). Representative of 5 experiments. (C) Phenotypic growth test of thi7Δnrt1Δthi72Δthi4Δ strain expressing single-point THI7-GFP mutants, WT THI7-GFP, or an e.v. on thiamine-free selective medium (SC-U-B1) or supplemented with thiamine (final concentrations; 10 nM, 1 μM, and 100 μM). Representative of 4 experiments. (D) A 3D model of Thi7 in an occluded state. (E) Proposed thiamine binding site in Thi7. Residues predicted to be at a distance equal to or less than 6 Å from the thiamine molecule are shown. Docking experiments predict that thiamine is stabilized by two hydrogen bridges with S55 and N137 (in purple) and one π-π interaction with Y346 (in green). Eight Thi7 mutants obtained in the oxythiamine-based screening result from mutation of specific residues surrounding the binding site (encircled in black: G59, D85, N133, M247, P286, N350, V398, M399). (F) Superimposition of the 3D model of WT Thi7 (residue N133 in yellow) in a thiamine-bound occluded state with the 3D model of Thi7^N133K (residue K133 in red) in an occluded state displays how the N133K mutation could affect the binding site and the stabilization of thiamine. (G) Superimposition of the 3D model of WT Thi7 (residue M399 in yellow) in a thiamine-bound occluded state with the 3D model of Thi7^M399R (residue R399 in green) in an occluded state displays how the M399R mutation could disturb the binding site and stabilization of thiamine. 3D, three-dimensional; C-ter, C terminus; e.v., empty vector; GFP, green fluorescent protein; KR, lysine-to-arginine; N-ter, N terminus; TM, transmembrane span; WT, wild type.

Fig 5. Single-point Thi7 mutants display thiamine-induced endocytic defects compared to WT Thi7.

Localization of Thi7 mutants in a thi7Δnrt1Δthi72Δ strain after thiamine addition (final concentration: 100 μM) for 3 h into culture grown in thiamine-free selective medium. Scale bar represents 5 μm. Quantification shows the ratio of internal-over-PM fluorescence intensity of all single-point Thi7 mutants before and after thiamine addition (100 μM) as described in the Materials and methods section (n > 69 cells per condition in 3 replicates). The upper graph is data not normalized, whereas the bottom graph is data normalized for each mutant by the ratio at time 0. (****p < 0.0001; ***p < 0.001; **p < 0.01; *p < 0.05) The numerical data are included in S3 Data. GFP, green fluorescent protein; ns, nonsignificant; PM, plasma membrane; WT, wild type.

Fig 6. Thiamine uptake through Thi7^6KR leads to endocytosis of the inactive Thi7 transporters.

(A) Localization of Thi7^M399R-GFP and Thi7^6KR-mDsRed after incubation with thiamine (final concentration: 100 μM) in a thi7Δnrt1Δthi72Δ strain coexpressing THI7^M399R-GFP and THI7^6KR-mDsRed, or THI7^M399R-GFP with an empty vector (pRS313; Φ) or THI7^6KR-mDsRed with an empty vector (pRS316; Φ). Relative quantification of the internal-over-PM fluorescence intensity ratio of Thi7^M399R-GFP before and after incubation with thiamine (100 μM) when coexpressed with Thi7^6KR-mDsRed or an empty vector (pRS313; Φ). (B) Localization of Thi7-GFP and Thi7^6KR-mDsRed after incubation with thiamine (final concentration: 100 μM) in a thi7Δnrt1Δthi72Δ strain coexpressing THI7-GFP with an empty vector (pRS313; Φ) or THI7-GFP with THI7^6KR-mDsRed. Relative quantification of the internal-over-PM fluorescence intensity ratio of Thi7^M399R-GFP before and after incubation with thiamine (100 μM) when coexpressed with Thi7^6KR-mDsRed or an empty vector (pRS313; Φ). (C) Localization of Thi7^N350K-GFP and Thi7^6KR-mDsRed after incubation with thiamine at a final concentration of 100 μM in a thi7Δnrt1Δthi72Δ strain coexpressing THI7^N350K-GFP and either THI7^6KR-mDsRed or an empty vector (pRS313; Φ). Relative quantification of the internal-over-PM fluorescence intensity of Thi7^N350K-GFP before and after incubation with thiamine (100 μM) when coexpressed with either Thi7^6KR-mDsRed or an empty vector (pRS313; Φ). For quantification, the median with 95% confidence interval is presented (n > 94 cells per condition in 3 biological replicates) (****p < 0.0001, Wilcoxon test). Scale bar represents 5 μm. The numerical data are included in S4 Data. GFP, green fluorescent protein; ns, nonsignificant; PM, plasma membrane.

Fig 7. Uptake of thiamine through Thi7^6KR is not sufficient to induce endocytosis of Thi7^D85G and Thi7^P291Q.

(A) Localization of Thi7^D85G-GFP and Thi7^6KR-mDsRed after incubation with thiamine (final concentration: 100 μM) in a thi7Δnrt1Δthi72Δ strain coexpressing THI7^D85G-GFP and either THI7^6KR-mDsRed or an empty vector (pRS313; Φ). Relative quantification of the internal-over-PM fluorescence intensity of Thi7^D85G-GFP before and after incubation with thiamine (100 μM) when coexpressed with either Thi7^6KR-mDsRed or an empty vector (pRS313; Φ). (B) Localization of Thi7^P291Q-GFP and Thi7^6KR-mDsRed after incubation with thiamine (final concentration: 100 μM) in a thi7Δnrt1Δthi72Δ strain coexpressing THI7^P291Q-GFP and either THI7^6KR-mDsRed or an empty vector (pRS313; Φ). Relative quantification of the internal-over-PM fluorescence intensity of Thi7^P291Q-GFP before and after incubation with thiamine (100 μM) when coexpressed with either Thi7^6KR-mDsRed or an empty vector (pRS313; Φ). (****p < 0.0001; ***p < 0.001; **p < 0.01; *p < 0.05) (C) Localization of Thi7^M399R-GFP, Thi7^N350K-GFP, Thi7^D85G-GFP, Thi7^P291Q-GFP, and Thi7-GFP after incubation with CHX 50 μg/ml for 3 h or EtOH in a thi7Δnrt1Δthi72Δ strain grown in thiamine-free medium. For quantification, the median with 95% confidence interval is presented (n > 101 cells per condition in 3 biological replicates) (*p < 0.05; Wilcoxon test). Scale bar represents 5 μm. The numerical data are included in S5 Data. CHX, cycloheximide; EtOH, ethanol; GFP, green fluorescent protein; KR, lysine-to-arginine; ns, nonsignificant; PM, plasma membrane.

Fig 8. Thiamine-induced endocytosis of Thi7 appears to require TORC1 and Sit4 but is not inhibited by Npr1.

(A) Localization of Thi7-GFP in WT, sit4Δ, and npr1Δ strains complemented with the pFL36-HIS3-LYS2 or pFL36-HIS3-MET15 plasmid grown in thiamine-free medium containing proline (10 mM) as the sole nitrogen source (“Pro–Thiamine”) or on ammonium-(38 mM) containing thiamine-free complete medium (“Am + a.a.–Thiamine”). Cells were incubated for 3 h with or without thiamine (100 μM) or CHX (50 μg/ml). Relative quantification of the internal-over-PM fluorescence intensity of Thi7-GFP in the different conditions (n > 52 cells). (B) Addition of thiamine or oxythiamine leads to de-phosphorylation of HA-Npr1. A WT strain expressing HA-NPR1 and complemented with the pFL36 plasmid was grown up to early log phase in thiamine-free medium containing proline (10 mM) as the sole nitrogen source and incubated or not for 3 h with thiamine (100 μM), on thiamine-free medium containing ammonium (38 mM) as sole nitrogen source and incubated for 3 h with or without thiamine (100 μM) and ammonium-containing thiamine-free complete medium incubated for 3 h with thiamine (100 μM), oxythiamine (100 μM), CHX (50 μg/ml), or EtOH before being harvested. Cell extracts were immunoblotted with anti-HA and anti-Pma1 antibodies. Normalized intensities; distribution of HA-Npr1 in ammonium-containing thiamine-free complete medium with or without incubation with thiamine 100 μM, oxythiamine 100 μM, CHX 50 μg/ml, or EtOH. (C) Localization of Thi7-GFP in WT and art2Δ strains grown in ammonium-containing thiamine-free complete medium (“Am + a.a.–Thiamine”) and incubated for 3 h with or without DMSO, rapamycin 100 ng/ml, rapamycin 200 ng/ml, rapamycin 200 ng/ml + thiamine 100 μM, or thiamine 100 μM. Relative quantification of the internal-over-PM fluorescence intensity of Thi7-GFP in the different conditions. For quantification in (A) and (C), the median with 95% confidence interval is presented (n > 32 cells). Scale bar represents 5 μm (****p < 0.0001; ***p < 0.001; **p < 0.01; *p < 0.05). The numerical data are included in S6 Data. CHX, cycloheximide; EtOH, ethanol; GFP, green fluorescent protein; HA, hemagglutinin; ns, nonsignificant; PM, plasma membrane; Pma1, plasma membrane ATPase1; TORC1, target of rapamycin complex 1; WT, wild type.

Fig 9. Model of Art2-dependent ubiquitylation and endocytosis of Thi7, Nrt1, and Thi72.

(Top panel) In the absence of thiamine, Art2 is inactive. Thiamine uptake through Thi7, Nrt1, or Thi72 induces conformational changes, leading to the exposure of an Art2-binding domain. Thiamine accumulation within the cytosol leads to Art2-mediated ubiquitylation of thiamine transporters. This process requires the Sit4 phosphatase, and our results also suggest an active TORC1 and a poorly phosphorylated version of Npr1. (Bottom panel) Alternatively, CHX diffuses through the cell membrane and stimulates TORC1. CHX-activated TORC1 leads to hyperphosphorylation and inactivation of Npr1. CHX treatment induces Art2-dependent endocytosis of thiamine transporters, which also depends on the Sit4 phosphatase. In the absence of thiamine transport, no conformational change occurs on the transporter. CHX, cycloheximide; K, Rsp5-targeted lysine residue(s); Nrt1, nicotinamide riboside transporter 1; PM, plasma membrane; TORC1, target of rapamycin complex 1; Ub, ubiquitin.