Conformation-dependent partitioning of yeast nutrient transporters into starvation-protective membrane domains

Abstract

The eukaryotic plasma membrane is compartmentalized into domains enriched in specific lipids and proteins. However, our understanding of the molecular bases and biological roles of this partitioning remains incomplete. The best-studied domain in yeast is the membrane compartment containing the arginine permease Can1 (MCC) and later found to cluster additional transporters. MCCs correspond to static, furrow-like invaginations of the plasma membrane and associate with subcortical structures named "eisosomes" that include upstream regulators of the target of rapamycin complex 2 (TORC2) in the sensing of sphingolipids and membrane stress. However, how and why Can1 and other nutrient transporters preferentially segregate in MCCs remains unknown. In this study we report that the clustering of Can1 in MCCs is dictated by its conformation, requires proper sphingolipid biosynthesis, and controls its ubiquitin-dependent endocytosis. In the substrate-free outward-open conformation, Can1 accumulates in MCCs in a manner dependent on sustained biogenesis of complex sphingolipids. An arginine transport-elicited shift to an inward-facing conformation promotes its cell-surface dissipation and makes it accessible to the ubiquitylation machinery triggering its endocytosis. We further show that under starvation conditions MCCs increase in number and size, this being dependent on the BAR domain-containing Lsp1 eisosome component. This expansion of MCCs provides protection for nutrient transporters from bulk endocytosis occurring in parallel with autophagy upon TORC1 inhibition. Our study reveals nutrient-regulated protection from endocytosis as an important role for protein partitioning into membrane domains.

Keywords: endocytosis; membrane domain; transporter; ubiquitin; yeast.

Fig. 1.

During Arg transport, Can1 EMC clustering is abolished in a ubiquitylation-independent way. (A) Model of substrate transport-induced ubiquitylation and endocytosis of the Can1 permease. The binding site for Art1 (Art1BS) in the N-terminal tail is hidden (red hemicycle) when Can1 is in the substrate-free OF conformation. In the presence of Arg, the Art1BS is exposed (green hemicycle). A tripeptide sequence (87-ELK-89) in the N-terminal tail close to TMS1 (black rectangle) is required to mask the Art1BS when Can1 is in the OF conformation. (See text for details.) (B, Left) Shown are surface and middle section (also see Fig. S2B) confocal microscopy images of WT and art1Δ bul1/2Δ strains (gap1Δ can1Δ) expressing Pil1-mCherry and Can1-GFP or Can1(7KR)-GFP. Growth conditions are as described in Fig. S2A. (Right) Quantifications: Can1-GFP EMC/non-EMC fluorescence intensity ratios are plotted (n = 32–50 cells). The horizontal midline and the cross represent the median and average values, respectively. Each box is bounded by the upper and lower quartiles; the whiskers denote the maximal and minimal ratios. (C) Shown are surface section confocal images from representative FRAP experiments, of Can1(7KR)-GFP Pil1-mCherry–expressing cells, inside or outside the EMC in the absence of Arg (Upper Left and Upper Right, respectively) or inside the EMC in the presence of Arg (Lower Left). Specific regions (white arrows) were photobleached with or without the prior addition of 5 mM Arg for 30 min. The times stated refer to the time of the bleaching at t = 0 s. (Lower Right) Quantifications: Points and error bars respectively represent the mean and SD of the relative intensity for each time point (n = 8 per condition). FRAP curves are fitted models obtained by regression analysis. Dotted curves show 99% CI inside (black curve) and outside (blue curve) EMCs or inside EMCs in the presence of Arg (red curve). The probability that the calculated K values do not differ (F-pvalue of K): black and blue curves < 0.0001; black and red curves, 0.0023; blue and red curves, 0.7173. (D) Shown are surface section confocal microscopy images of an art1Δ bul1/2Δ gap1Δ can1Δ PIL1-mCherry strain expressing Can1-GFP, Can1(T180R)-GFP, or Can1(S176N,T456S)-GFP. Conditions and quantifications (n = 32–42) are as in B. ***P < 0.001; ns, nonsignificant, P > 0.05. (Scale bar: 2 μm.)

Fig. 2.

Substrate-triggered abolition of Can1 EMC clustering is induced by transition to the IF state. (A, Upper) Shown are surface section confocal microscopy images of an art1Δ bul1/2Δ gap1Δ PIL1-mCherry strain expressing Can1(T180R)-GFP or Can1(7KR)-GFP. For Arg washout, cells were washed and resuspended in Arg-free medium for 30 min. (Lower) Quantifications (n = 32–49) are as in Fig. 1B. (B) Shown are surface section confocal microscopy images (Left) and quantification (Right) of an art1Δ bul1/2Δ gap1Δ can1Δ PIL1-mCherry strain expressing Can1-GFP or Can1(S176N)-GFP. Conditions and quantifications (n = 34–42) are as in Fig. 1B. Representations are as in Fig. 1A. (C) Shown are surface section confocal microscopy images of an art1Δ bul1/2Δ gap1Δ can1Δ PIL1-mCherry strain expressing Can1-GFP or the indicated mutant. Conditions and quantifications (n = 22–38) as in Fig. 1B. The cartoons summarize previous knowledge (25, 32). The E184Q substitution stabilizes the transporter in an IF state, permanently exposing the Art1BS even in the absence of substrate. The S176N substitution is epistatic to E184Q and blocks Can1 in an OF state. Art1BS is no longer exposed. Can1(ELK89-AAA) is inactive and permanently exposes Art1BS. Representations are as in Fig. 1A. (D) Shown are surface section confocal images of one representative FRAP experiment in EMCs of a gap1Δ can1Δ PIL1-mCherry strain expressing Can1(7KR,E184Q)-GFP. Conditions (n = 9), analysis, and representations are as in Fig. 1C. ***P < 0.001; **0.001 < P < 0.01; ns, nonsignificant, P > 0.05. (Scale bar: 2 μm.)

Fig. 3.

Can1 EMC clustering requires SLs. (A, Left) Shown are surface and middle section (see also Fig. S4B) confocal microscopy images of gap1Δ can1Δ and gap1Δ can1Δ nce102Δ strains expressing Sur7-mRFP, Pil1(4A)-GFP as the sole Pil1, and Can1(7KR)-mCherry, grown in YNB Gal Am medium. Glu was added for 90 min and then 10 μM Μyr, 10 μM Myr + 10 μM PHS, 10 μM PHS, or 1 μg/mL ΑbA was added for 90 min. (Right) Quantifications (n = 41–67 for Sur7, 34–100 for Can1) are as in Fig. 1B. (B) The SL biosynthesis pathway of Saccharomyces cerevisiae. The metabolic intermediates and the genes encoding the enzymes involved are named. The steps inhibited by Myr and AbA are also highlighted. (C) FRAP experiments on EMCs carried out, analyzed, and represented as in Fig. 1C on a gap1Δ can1Δ strain expressing Can1(7KR)-mCherry and Pil1(4A)-GFP treated (red, n = 14) or not (black, n = 11) with 10-μΜ Myr for 90 min. F-pvalue of K < 0.0001. (D, Upper) Detergent resistance of Can1(7KR) and Can1(7KR,E184Q). can1Δ strains expressing either Can1(7KR)-GFP or Can1(7KR,E184Q)-GFP under the GAL1 promoter were grown in Gal Pro medium, and Glu was added for 1.5 h. Thirty-microgram aliquots of membrane-enriched protein extracts were treated with increasing concentrations of Triton X-100. Following centrifugation and washing, the detergent-resistant insoluble pellet was resuspended in sample buffer and immunoblotted. (Lower) Points and error bars respectively represent the mean and SD of the percentage relative intensity for each concentration (n = 3 biological replicates) of Can1-GFP in comparison with the extracts treated in the absence of Triton X-100. Statistically significant points are shown. See Fig. S4E for quantification of Gap1 and Pma1 signals. ***P < 0.001; **0.001 < P < 0.01; *P < 0.05; ns, nonsignificant, P > 0.05. (Scale bar: 2 μm.)

Fig. 4.

Can1 in EMCs is protected from ubiquitylation and endocytosis. (A, Left) Epifluorescence microscopy of Arg-induced endocytosis of Can1-GFP and Can1(T180R)-GFP in PIL1 CAN1 and pil1Δ CAN1 cells. (Right) Quantifications (n = 91–104) are as in Fig. S2A. (B) Concentration-dependent kinetics of [¹⁴C]Arg uptake into gap1Δ and gap1Δ pil1Δ cells. Error bars indicate the SD; n = 3. Curve fitting and calculation of the V_m and K_m values (in micromoles), 99% CI (dotted curves), and F-pvalues were carried out by Michaelis–Menten analysis. F-pvalue of V_m = 0.0003, F-pvalue of K_m = 0.9245. (C) gap1Δ can1Δ and gap1Δ can1Δ pil1Δ cells expressing Can1-GFP from the native or GAL1 promoter were grown in Glu Pro or Gal Pro medium, respectively. To the latter, Glu was added for 90 min before cells were collected. Total protein extracts were immunoblotted with anti-GFP and anti-Pgk1. Quantifications shown below the blots are the ratios of Can1-GFP/Pgk1 intensities. Ratios in the PIL1⁺ (WT) strain are set at 1. (D) Concentration-dependent kinetics of [¹⁴C]Arg uptake in gap1Δ can1Δ Can1-GFP cells expressing Sur7 or Sur7-GB and grown in Raf Pro medium. Gal was added for 1 h, and then Glu was added for 90 min. Error bars indicate SD; n = 2. Analysis and representations are as in B. F-pvalue of V_m = 0.7976; F-pvalue of K_m = 0.9492. (E, Left) Shown are surface section confocal images of SUR7-GB and SUR7 strains (gap1Δ can1Δ) expressing Pil1-mCherry and Can1(7KR)-GFP grown as in D. The cells were then treated or not with 5 mM Arg for 30 min. (Right) Quantifications (n = 22–37) are as in Fig. 1B. (F, Left) Epifluorescence microscopy of Arg-induced endocytosis of Can1-GFP in PIL1 and pil1Δ cells (gap1Δ can1Δ) expressing Sur7 or Sur7-GB, grown as in D. (Right) Quantifications (n = 74–106) are as in Fig. S2A. (G) Western blotting of total protein extracts of Can1-GFP–expressing strains (gap1Δ can1Δ) grown as in D and probed with anti-GFP. Asterisks indicate Ub-Can1-GFP conjugates. ***P < 0.001; ns, nonsignificant, P > 0.05. (Scale bar: 2 μm.)

Fig. 5.

The EMC as a reservoir of transporters for nutrient starvation. (A) Epifluorescence microscopy of PIL1 and pil1Δ cells (gap1Δ can1Δ) expressing Can1-GFP, Gap1-GFP, or Can1(7KR)-GFP, treated or not with 200 ng/mL Rap for 3 h. (B) [¹⁴C]-amino acid uptake measurements (Arg for Can1, Lys for Lyp1, Met for Mup1, uracil for Fur4, citrulline for Gap1) on PIL1 and pil1Δ cells (gap1Δ when not measuring citrulline) treated or not with 200 ng/mL Rap for 2 or 3 h. Error bars indicate SD; n = 3. (C) Growth curves of WT and pil1Δ cells starved for nitrogen for 4 h in minimal medium containing Arg or Am as the sole nitrogen source. Equal amounts of cells were seeded into 24-well plates. Error bars indicate the SD of two technical replicates. The experiment is representative of three biological replicates. (D, Left) Western blotting of total protein extracts (probed with anti-GFP, stripped and probed with anti-Pma1 and anti-Pgk1) of PIL1 and pil1Δ (gap1Δ can1Δ) cells expressing Can1-GFP and grown in glutamine plus Am at different ODs or having reached (E) or remained for 12 h in (L) the stationary phase. (Right) The Can1-GFP/Pgk1 intensity ratio, normalized to 1 for the PIL1⁺ in OD = 0.1 from two to three independent biological replicates shown in Fig. S6B. Quantifications are as in Fig. 3D. (E) Spot dilution tests of PIL1 and pil1Δ cells (± gap1Δ) that had remained in the stationary phase for 12 h. (F, Upper) Shown are surface section confocal images of gap1Δ can1Δ and gap1Δ can1Δ lsp1Δ strains expressing Pil1-mCherry and Can1(7KR)-GFP taken from early log-phase cultures on Gal Pro (N-poor) or Gal Am + Gln (N-rich) medium. Cells grown on N-rich medium and then shifted to medium lacking any nitrogen source (Shift to N−) for 2 h, were also observed at the beginning of the stationary phase (Early SP) and after 12 h in the stationary phase (Late SP). Pil1-mCherry images were differentially treated for brightness and contrast to improve the visibility of details and do not reflect differences in the intensity measured. (Lower) Quantifications (n = 33–79) are as in Fig. 1B (see also Materials and Methods and Fig. S6D). (G, Upper) FRAP experiments on EMCs of a gap1Δ can1Δ strain expressing Can1(7KR)-GFP and Pil1-mCherry from cells in the late stationary phase carried out, analyzed, and represented as in Fig. 1C. (Lower) Quantifications: the fitted curve (n = 11) is compared with the one of Can1 in EMCs (log phase, n = 8) from Fig. 1C. F-pvalue of K < 0.0001. ***P < 0.001; **0.001 < P < 0.01; *P < 0.05; ns, nonsignificant, P > 0.05. (Scale bar: 2 μm.)

Fig. 6.

Model of the mechanisms of Can1 lateral PM segregation and regulated protection from ubiquitylation by starvation-protective EMCs. (A) In the absence of substrate, Can1 in an OF conformation concentrates in EMCs (which associate subcortically with Pil1- and Lsp1-containing eisosomes), probably because it interacts with particular lipids (for instance SLs). (B) In the presence of Arg, a transient shift of the transporter to an IF conformation results in the loss of clustering in EMCs because its potential interaction with lipids (illustrated) or its partitioning in highly ordered membrane domains is reduced. Meanwhile, Arg activates Art1 via the TORC1-Npr1 pathway. Art1 recruits the Rsp5 Ub-ligase only to Can1 molecules that have adopted the IF conformation and are outside the EMCs. (C) Upon nutrient starvation, the eisosomes are induced at transcriptional and posttranslational levels; inhibition of TORC1 leads to Npr1 stimulation, which phosphorylates Lsp1 and also Orm1/2 at the Golgi, inducing the biogenesis of complex SLs. These events correlate with deeper eisosomal invaginations capable of hosting more proteins, protecting them from ubiquitylation and endocytosis promoted by certain uncharacterized α-arrestins (ARTs) that somehow are activated by nutrient starvation and/or TORC1 inhibition.