Methionine triggers Ppz-mediated dephosphorylation of Art1 to promote cargo-specific endocytosis

Abstract

Regulation of plasma membrane (PM) protein abundance by selective endocytosis is critical for cellular adaptation to stress or changing nutrient availability. One example involves rapid endocytic turnover of Mup1, a yeast methionine transporter, in response to increased methionine availability. Here, we report that methionine triggers rapid translocation of the ubiquitin ligase adaptor Art1 to the PM and dephosphorylation of Art1 at specific threonine residues. This methionine-induced dephosphorylation of Art1 is mediated by Ppz phosphatases, and analysis of phosphomimetic and phosphorylation-defective variants of Art1 indicates that these events toggle Art1 recognition of Mup1 at the PM. Importantly, we find that Ppz phosphatases are dispensable for Art1 PM translocation, but are required for Art1 interaction with Mup1. Based on our findings, we propose that methionine influx triggers Art1 translocation to the PM, followed by Ppz-mediated dephosphorylation which promotes cargo recognition at the PM.

Figure 1.

Methionine stimulation induces dephosphorylation of Art1. (A) Yeast cells expressing Mup1-GFP (green) and Vph1-mCherry (red; localized to the limiting membrane of the vacuole) were grown to mid-log phase and imaged by fluorescence deconvolution microscopy (untreated) or stimulated with methionine for the indicated amount of time and then imaged. (B) Yeast cells (WT or Δart1) expressing Mup1-pHluorin were cultured to mid-log phase, and flow cytometry was used to measure pHluorin-positive cells at the indicated time points following methionine stimulation (+ met.) or an untreated control culture. Sample data of the Mup1-pHluorin–trafficking analysis by microscopy and flow cytometry are provided in Fig. S1 A. Error bars indicate standard deviation from multiple biological replicate experiments (n = 3). (C) Yeast cells expressing Art1-GFP (green) and Mup1-MARS (red) were grown to mid-log phase and imaged by fluorescence deconvolution microscopy (untreated) or stimulated with methionine for 3 min and then imaged. (D) Pearson’s correlation coefficients were measured for yeast cells (n > 25 cells each for untreated or methionine stimulation conditions) expressing Art1-GFP and Mup1-MARS-RFP (as shown in C). Error bars indicate standard deviation for the set, and significance was determined by an unpaired t test. (E) SILAC-based quantification of phosphorylation events was performed (according to the scheme shown in Fig. S1 B) using endogenous Art1-FLAG as bait. A schematic representation of Art1 (top) is shown with domains color-coded to indicate location of phosphopeptides detected and quantified in this analysis (bottom graph). Normalized H:L ratios were measured and averaged over multiple biological replicates (n = 3). Error bars indicate standard deviation of measurements on peptides from three experiments (n = 3), except for S104 and S113, which were only resolved and quantified in two replicate experiments. (F) Complementation analysis of Δart1 mutant yeast cells using the Mup1-pHluorin–trafficking assay. For all Mup1-pHluorin–trafficking assays plots represents average of multiple biological replicate experiments (n ≥ 4), and error bars indicate standard deviation. For the final five time points of each time course, statistical significance was calculated using an unpaired t test (P < 0.005).

Figure 2.

Ppz phosphatases are required for Mup1 endocytic trafficking in response to methionine. (A) WT (expressing Vph1-mCherry) or Δppz1Δppz2 mutant (lacking mCherry expression) yeast cells (SEY6210 strain background) expressing Mup1-GFP were cultured to mid-log phase, mixed, stimulated by addition of methionine for 30 min, and imaged by fluorescence deconvolution microscopy. (B) Mup1-GFP signal intensity was measured at the PM and the vacuole lumen, and a Vac:PM ratio was calculated for many individual yeast cells (n ≥ 40). Statistical significance was calculated using a two-way ANOVA test (P values indicated in red). (C) A methionine stimulation time course was performed on WT and ppz mutant yeast cells expressing Mup1-FLAG. At the indicated time points after stimulation, cell lysates were analyzed by SDS-PAGE and immunoblot. (D) Flow cytometry analysis of yeast cells expressing Mup1-pHluorin grown in the absence of methionine and then stimulated by addition of methionine. Fluorescence of pHluorin is pH sensitive and lost during endocytic trafficking when the cargo encounters an acidic environment. Plot represents average of multiple biological replicate experiments (n ≥ 3), and error bars indicate standard deviation. Statistical significance was calculated using a Student’s t test (P < 0.005). (E) WT (expressing Vph1-mCherry) or Δppz1Δppz2 mutant (lacking mCherry expression) yeast cells (SEY6210 strain background) expressing Fur4-GFP were cultured to mid-log phase, mixed, stimulated by addition of methionine for 30 min, and imaged by fluorescence deconvolution microscopy. (F) Fur4-GFP signal intensity was measured at the PM and the vacuole lumen, and a Vac:PM ratio was calculated for many individual yeast cells (n ≥ 30). Statistical significance was calculated using a two-way ANOVA test, but no statistically significant difference between WT and Δppz1Δppz2 mutant cells was observed. (G and H) Flow cytometry analysis of yeast cells expressing Mup1-pHluorin grown in the absence of methionine and then stimulated by addition of methionine. Fluorescence of pHluorin is pH sensitive and lost during endocytic trafficking when the cargo encounters an acidic environment. Plot represents average of multiple biological replicate experiments (n ≥ 3), and error bars indicate standard deviation. Statistical significance was calculated using a Student’s t test (P < 0.005). Vac, vacuole lumen.

Figure 3.

Ppz catalytic activity and PM localization are required for Mup1 endocytosis. (A) Schematic representation of functional features known in Ppz1 and Ppz2 phosphatases. (B) Complementation analysis of Δppz1Δppz2 mutant yeast cells using the Mup1-pHluorin–trafficking assay. Error bars indicate standard deviation from multiple biological replicate experiments (n = 3). Expression of WT and catalytic dead (R451L) Ppz1-FLAG was confirmed by immunoblot (inset). (C) Complementation analysis of Δppz1 mutant yeast cells using the Mup1-pHluorin–trafficking assay. Error bars indicate standard deviation from multiple biological replicate experiments (n = 4). (D) Yeast cells expression WT Ppz1-GFP or a ppz1-G2A-GFP mutant were analyzed by fluorescence microscopy. (E) WT (strain SEY6210) yeast cells expressing Art1-GFP and Ppz1-mCherry were stimulated with methionine for 3 min and imaged using fluorescence deconvolution microscopy. (F) Complementation analysis of Δppz1 mutant yeast cells using the Mup1-pHluorin–trafficking assay. Immunoblot (inset) shows expression of Ppz-FLAG from indicated vectors. For all Mup1-pHluorin–trafficking assays, plots represents average of multiple biological replicate experiments (n ≥ 3), and error bars indicate standard deviation. For the final three time points of each time course, statistical significance was calculated using a Student’s t test (P < 0.005). (G) WT or Δppz1Δppz2 mutant yeast cells (SEY6210 strain background) harboring the indicated Ppz1 expression plasmids were plated on minimal media in the presence of canavanine (toxic arginine analogue).

Figure 4.

Ppz function in ubiquitin homeostasis is separable from its endocytic function. (A) WT or Δppz1Δppz2 mutant yeast cells (SUB280 strain background) harboring the indicated ubiquitin expression plasmids were plated on minimal media in the presence of canavanine (toxic arginine analogue). (B) The indicated yeast cells (SEY6210 background) containing either empty vector or PPZ1-FLAG vectors were analyzed for total cellular ubiquitin levels by immunoblot analysis. (C) Quantification of ubiquitin expression yeast cells (SEY6210 background) expressing the indicated Ppz expression plasmids. Error bars indicate standard deviation from multiple biological replicate experiments (n = 4). Statistical significance was calculated using a Student’s t test (P < 0.005).

Figure 5.

Ppz phosphatases regulate the phosphorylation and activity of Art1. (A) Mup1-pHluorin–trafficking assays were performed to characterize endocytic trafficking in the indicated yeast strains. ART1 is expressed from its native promoter on a pRS416 centromere plasmid and contains a C-terminal 3× FLAG fusion. Error bars indicate standard deviation from multiple biological replicate experiments (n = 3). (B) SILAC-based quantification of phosphorylation events was performed (according to the scheme shown in Fig. S1 A) using endogenous Art1-FLAG as bait. Color coding of regions of Art1 corresponds to the schematic representation of Art1 (Fig. 1 E). Normalized H:L ratios were measured and averaged over multiple biological replicates (n = 3). Error bars indicate standard deviation of measurements on peptides from three experiments (n = 3), except for S85, S106, S722, and S798, which were only resolved and quantified in two replicate experiments. (C) The indicated yeast strains (SEY6210 background) expressing empty vector or the indicated Art1 expression plasmid were plated on minimal media (SCD-ura) containing the indicated concentration of canavanine. (D) Mup1-pHluorin–trafficking assays were performed to characterize endocytic trafficking in the indicated yeast strains with Art1 variants expressed from a pRS416 centromere plasmid (as in Fig. 4 A and Fig. S1 A). Error bars indicate standard deviation from multiple biological replicate experiments (n = 3). (E) The indicated yeast strains (SEY6210 background) expressing empty vector or the indicated Art1 expression plasmid were plated on minimal media (SCD-ura) containing the indicated concentration of canavanine.

Figure 6.

Ppz phosphatases regulate specific phosphorylation events within the Rsp5 adaptor network. (A) SILAC-based quantitation of the FLAG-Rsp5 interaction network in WT (H) and ppz mutant (L) cells. Experiment was performed as shown in Fig. S1 A using FLAG-Rsp5 as the bait. Normalized H:L ratios were measured and averaged over multiple biological replicates (n = 3). Error bars indicate standard deviation of measurements on peptides from at least two experiments. Immunoblot (inset) shows recovery (1%) of FLAG-Rsp5 as bait in these samples. Additional data for this experiment are shown in Fig. S4. (B) SILAC-based quantification was performed on Rsp5 phosphopeptides enriched by immobilized metal affinity chromatography. A schematic (top) illustrates the phosphorylation events detected and quantified relative to known domain organization. Color coding of domain organization applies to the graph depicting Rsp5 phosphopeptide quantification (bottom panel). (C–E) SILAC-based quantification of phosphorylation events resolved and quantified in the Rsp5 adaptor network, including Art3/Aly2 (C), Art4/Rod1 (D), and Art6/Aly1 (E). For all panels in Figure 6, normalized H:L ratios were measured and averaged over multiple biological replicates (n = 3). Error bars indicate standard deviation of measurements on peptides from at least two experiments (n ≥ 2). Schematic representations of each adaptor are shown at the left, and phosphopeptide quantification is shown on the right. Additional phosphoprofiling data from these experiments are shown in Fig. S4.

Figure 7.

Ppz phosphatases promote Art1 interaction with Mup1 at the PM. (A) WT yeast cells (expressing Vph1-mCherry) and Δppz1Δppz2 yeast cells (lacking mCherry expression) were grown to mid-log phase, mixed, stimulated with methionine for 3 min, and imaged by fluorescence deconvolution microscopy. (B and C) A split-ubiquitin two-hybrid system was used to probe for Art1-cargo interactions. Different cargo (Mup1, Can1, and Lyp1) were expressed as bait constructs fused to LexA and CUb. Art1 was expressed as a prey construct fused to NUb-HA. (D) β-Galactosidase activity assays were performed to quantify the Art1-Mup1 interaction in WT and Δppz1Δppz2 yeast cells. Error bars indicate standard deviation from multiple biological replicate experiments (n = 7). Expression of bait and prey constructs is shown in Fig. S5 (E and F). (E) An integrated model summarizing current understanding of Art1 phosphoinhibition at the PM. Ppz phosphatases promote dephosphorylation of Art1 at Thr245, Thr795, and Thr93 positions, which contributes to Art1 activation at the PM (I). Specifically, Ppz phosphatase activity promotes the interaction of Art1 with Mup1 at the PM. Activated Art1 targets Rsp5 activity to Mup1, which is ubiquitylated (II) and subsequently sorted for endocytosis. At the PM, Npr1 kinase antagonizes Art1 activity by phosphorylating its N terminus, resulting in Art1 departure from the PM (III); thus, both Ppz phosphatases and Npr1 kinase participate in a complex phosphorylation/dephosphorylation cycle that controls Art1 activity and cargo ubiquitylation.