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Cited by 18Cellular transport and secretionAutophagy in Disease and TherapyUbiquitin and proteasome pathways

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Art2 mediates selective endocytosis of methionine transporters during adaptation to sphingolipid depletion

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Hepowit, Nathaniel L., Moon, Bradley, Ebert, Adam C., Dickson, Robert C., MacGurn, Jason A.·2023·Journal of Cell Science
DOI 10.1242/jcs.260675PMC PMC10399987

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Abstract

ABSTRACT2: Accumulating evidence in several model organisms indicates that reduced sphingolipid biosynthesis promotes longevity, although underlying mechanisms remain unclear. In yeast, sphingolipid depletion induces a state resembling amino acid restriction, which we hypothesized might be due to altered stability of amino acid transporters at the plasma membrane. To test this, we measured surface abundance for a diverse panel of membrane proteins in the presence of myriocin, a sphingolipid biosynthesis inhibitor, in Saccharomyces cerevisiae. Unexpectedly, we found that surface levels of most proteins examined were either unaffected or increased during myriocin treatment, consistent with an observed decrease in bulk endocytosis. In contrast, sphingolipid depletion triggered selective endocytosis of the methionine transporter Mup1. Unlike methionine-induced Mup1 endocytosis, myriocin triggered Mup1 endocytosis that required the Rsp5 adaptor Art2, C-terminal lysine residues of Mup1 and the formation of K63-linked ubiquitin polymers. These findings reveal cellular adaptation to sphingolipid depletion by ubiquitin-mediated remodeling of nutrient transporter composition at the cell surface.

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Figures

Fig. 1. Myr treatment selectively decreases plasma membrane abundance of the primary methionine permease Mup1. (A) Yeast cells expressing the indicated amino acid transporters (AATs) with a C-terminal mNG tag (green) were visualized under a fluorescence microscope after treatment with Myr (400 ng ml−1) or mock solution (95% ethanol). Myr-treated cells expressed MARS- or mCherry-tagged Vph1 (a vacuolar marker, red; indicated as Vph1–RFP) and mock-treated cells have unlabeled vacuoles. Note that Myr-treated cells expressing Mup1–mNG, Dip5–mNG and Lyp1–mNG also expressed Vph1–MARS, whereas Myr-treated cells expressing Can1–mNG and Bap3–mNG also expressed Vph1–mCherry. Images are representative of triplicate experiments. (B) Quantification of the mean fluorescence intensity of the indicated AAT–mNG at the PM, measured using Fiji (n=30–60 cells from triplicate experiments). Bars show the mean±s.d. ns, not significant (one-tailed paired t-test).
Fig. 1. Myr treatment selectively decreases plasma membrane abundance of the primary methionine permease Mup1. (A) Yeast cells expressing the indicated amino acid transporters (AATs) with a C-terminal mNG tag (green) were visualized under a fluorescence microscope after treatment with Myr (400 ng ml−1) or mock solution (95% ethanol). Myr-treated cells expressed MARS- or mCherry-tagged Vph1 (a vacuolar marker, red; indicated as Vph1–RFP) and mock-treated cells have unlabeled vacuoles. Note that Myr-treated cells expressing Mup1–mNG, Dip5–mNG and Lyp1–mNG also expressed Vph1–MARS, whereas Myr-treated cells expressing Can1–mNG and Bap3–mNG also expressed Vph1–mCherry. Images are representative of triplicate experiments. (B) Quantification of the mean fluorescence intensity of the indicated AAT–mNG at the PM, measured using Fiji (n=30–60 cells from triplicate experiments). Bars show the mean±s.d. ns, not significant (one-tailed paired t-test).CC BY · PubMed Central
Fig. 2. Myr decreases the cellular uptake of glucose despite increased PM abundance of the glucose transporters Hxt1 and Hxt2. (A) Time-course measurements of 3H-glucose uptake in mock-treated versus Myr-treated yeast cells (n=3). Bars show the mean±s.d. P-values were calculated using a one-tailed paired t-test. (B) Yeast cells expressing the indicated glucose transporters with a C-terminal mNG tag (green) were visualized under a fluorescence microscope after treatment with Myr (400 ng ml−1) or mock solution (95% ethanol). Myr-treated cells expressed MARS-tagged Vph1 (a vacuolar marker, red) and mock-treated cells have unlabeled vacuoles. Images are representative of triplicate experiments. (C) Quantification of the total fluorescence intensity of select glucose transporters (C-terminally tagged with mNG) at the PM, measured using Fiji (n=30–60 cells from triplicate experiments). Bars show the mean±s.d. ns, not significant (one-tailed paired t-test).
Fig. 2. Myr decreases the cellular uptake of glucose despite increased PM abundance of the glucose transporters Hxt1 and Hxt2. (A) Time-course measurements of 3H-glucose uptake in mock-treated versus Myr-treated yeast cells (n=3). Bars show the mean±s.d. P-values were calculated using a one-tailed paired t-test. (B) Yeast cells expressing the indicated glucose transporters with a C-terminal mNG tag (green) were visualized under a fluorescence microscope after treatment with Myr (400 ng ml−1) or mock solution (95% ethanol). Myr-treated cells expressed MARS-tagged Vph1 (a vacuolar marker, red) and mock-treated cells have unlabeled vacuoles. Images are representative of triplicate experiments. (C) Quantification of the total fluorescence intensity of select glucose transporters (C-terminally tagged with mNG) at the PM, measured using Fiji (n=30–60 cells from triplicate experiments). Bars show the mean±s.d. ns, not significant (one-tailed paired t-test).CC BY · PubMed Central
Fig. 3. Myr inhibits bulk endocytic trafficking of a lipophilic tracer dye. (A) Yeast cells expressing the vacuole membrane marker Vph1–mNG (green) were pulse-labeled with the lipophilic tracer dye FM 4-64 (red) and imaged by fluorescence microscopy. Cells were treated with 400 ng ml−1 Myr (for 0, 1, 2 or 4 h). FM 4-64 pulse-chase times and Myr-treatment times are indicated at the top. Images are representative of duplicate experiments. (B) Colocalization of FM 4-64 and Vph1–mNG measured as Pearson's correlation coefficient values using softWoRx (v7.0.0); n=60 cells from duplicate experiments. Bars show the mean±s.d. ns, not significant (one-tailed paired t-test).
Fig. 3. Myr inhibits bulk endocytic trafficking of a lipophilic tracer dye. (A) Yeast cells expressing the vacuole membrane marker Vph1–mNG (green) were pulse-labeled with the lipophilic tracer dye FM 4-64 (red) and imaged by fluorescence microscopy. Cells were treated with 400 ng ml−1 Myr (for 0, 1, 2 or 4 h). FM 4-64 pulse-chase times and Myr-treatment times are indicated at the top. Images are representative of duplicate experiments. (B) Colocalization of FM 4-64 and Vph1–mNG measured as Pearson's correlation coefficient values using softWoRx (v7.0.0); n=60 cells from duplicate experiments. Bars show the mean±s.d. ns, not significant (one-tailed paired t-test).CC BY · PubMed Central
Fig. 4. Sphingolipid depletion induces Art2-mediated endocytic trafficking of Mup1. (A) Myr treatment decreased the level of Mup1 (C-terminally tagged with either FLAG or GFP), as measured using western blot analysis of cell lysates. (B) Phytosphingosine (PHS) supplementation stabilized Mup1–pHluorin at the PM of Myr-treated cells but not aureobasidin A (AbA)-treated cells. Mup1–pHluorin fluorescence signal at the PM of cells was measured by a flow cytometer after 5 h of treatment (eight biological replicates; n=10,000 cells). The yeast sphingolipid biosynthesis pathway is shown on the right. Myr and AbA inhibit the serine palmitoyl transferase (Lcb1/Lcb2 complex) and phosphatidylinositol:ceramide phosphoinositol transferase (Aur1), respectively. (C) ART2 is required for Myr-induced endocytic trafficking of Mup1–pHluorin. Mup1–pHluorin fluorescence signal at the PM of cells was measured by a flow cytometer after 5 h of treatment (four biological replicates; n=10,000 cells). (D) Mup1–pHluorin-expressing yeast cells were either mock-treated (expressing Vph1–MARS, red) or Myr-treated (unlabeled vacuoles) were mixed prior to analysis by fluorescence microscopy. Genotypes of the yeast strains used for each comparison (WT, Δart1 and Δart2) are indicated on the top. Images are representative of triplicate experiments. (E) Quantification of Mup1–pHluorin fluorescence at the PM of cells shown in D. Values are ratios of mean fluorescence intensities between cell pairs in proximity to each other; n=30 randomly selected cell pairs from triplicate experiments. Bars show the mean±s.d. ns, not significant; *P<0.01; **P<0.001; ***P<0.0001.
Fig. 4. Sphingolipid depletion induces Art2-mediated endocytic trafficking of Mup1. (A) Myr treatment decreased the level of Mup1 (C-terminally tagged with either FLAG or GFP), as measured using western blot analysis of cell lysates. (B) Phytosphingosine (PHS) supplementation stabilized Mup1–pHluorin at the PM of Myr-treated cells but not aureobasidin A (AbA)-treated cells. Mup1–pHluorin fluorescence signal at the PM of cells was measured by a flow cytometer after 5 h of treatment (eight biological replicates; n=10,000 cells). The yeast sphingolipid biosynthesis pathway is shown on the right. Myr and AbA inhibit the serine palmitoyl transferase (Lcb1/Lcb2 complex) and phosphatidylinositol:ceramide phosphoinositol transferase (Aur1), respectively. (C) ART2 is required for Myr-induced endocytic trafficking of Mup1–pHluorin. Mup1–pHluorin fluorescence signal at the PM of cells was measured by a flow cytometer after 5 h of treatment (four biological replicates; n=10,000 cells). (D) Mup1–pHluorin-expressing yeast cells were either mock-treated (expressing Vph1–MARS, red) or Myr-treated (unlabeled vacuoles) were mixed prior to analysis by fluorescence microscopy. Genotypes of the yeast strains used for each comparison (WT, Δart1 and Δart2) are indicated on the top. Images are representative of triplicate experiments. (E) Quantification of Mup1–pHluorin fluorescence at the PM of cells shown in D. Values are ratios of mean fluorescence intensities between cell pairs in proximity to each other; n=30 randomly selected cell pairs from triplicate experiments. Bars show the mean±s.d. ns, not significant; *P<0.01; **P<0.001; ***P<0.0001.CC BY · PubMed Central
Fig. 5. Myr-induced trafficking of Mup1 requires K63-linked polyubiquitylation at the C-terminal lysines of Mup1. (A) Diagram showing the predicted protein structure of Mup1 (AlphaFold). Most of the N-terminal cytosolic tail (gray) lacks predictable structure, whereas the C-terminal cytosolic tail (green) contains secondary structure elements that are predicted with higher confidence. Functional lysine residues at the N- and C-terminal cytosolic tails of Mup1 are colored in red. (B) Yeast cells expressing WT Mup1–mNG (cells with Vph1–MARS, red) or variants with C-terminal truncations (Mup11–571–mNG and Mup11–566–mNG) were visualized by fluorescence microscopy after treatment with Myr (400 ng ml−1) for 5 h. Images are representative of triplicate experiments. (C) Quantification of Mup1–mNG expressed as mean fluorescence intensity at the PM of cells visualized in B (n=30 cells from triplicate experiments). (D) Mup1–GFP variants (Mup1WT, Mup1K567R, Mup1K572R and Mup1K567R,K572R) were expressed exogenously from a plasmid. Mean fluorescence intensity levels of Mup1–GFP at the PM (n=94–121 cells from duplicate experiments) were quantified following mock treatment or treatment with Myr (400 ng ml−1) for 5 h. (E) Anti-FLAG and anti-K63-linked polyubiquitin immunoblots of Mup1–FLAG enriched by anti-FLAG immunoprecipitation after 2 or 4 h of Myr treatment. Images are representative of duplicate experiments. MW, molecular weight; MWM, molecular weight marker. (F) Mup1–pHluorin trafficking was analyzed by flow cytometry of yeast cells expressing either WT or K63R ubiquitin from a single source. Mup1–pHluorin levels were measured after 5 h treatment with or without Myr (four biological replicates; n=10,000 cells). (G) Yeast cells expressing Mup1–mNG (green) and either WT ubiquitin (labeled with Vph1–mRuby in red) or K63R ubiquitin (unlabeled vacuoles) were mixed and imaged by fluorescence microscopy following mock or Myr treatment. Images are representative of triplicate experiments. (H) Quantification of Mup1–mNG mean fluorescence intensity at the PM of cells shown in G. n=30 cells. Bars show the mean±s.d. ns, not significant; **P<0.001; ***P<0.0001 (one-tailed paired t-test).
Fig. 5. Myr-induced trafficking of Mup1 requires K63-linked polyubiquitylation at the C-terminal lysines of Mup1. (A) Diagram showing the predicted protein structure of Mup1 (AlphaFold). Most of the N-terminal cytosolic tail (gray) lacks predictable structure, whereas the C-terminal cytosolic tail (green) contains secondary structure elements that are predicted with higher confidence. Functional lysine residues at the N- and C-terminal cytosolic tails of Mup1 are colored in red. (B) Yeast cells expressing WT Mup1–mNG (cells with Vph1–MARS, red) or variants with C-terminal truncations (Mup11–571–mNG and Mup11–566–mNG) were visualized by fluorescence microscopy after treatment with Myr (400 ng ml−1) for 5 h. Images are representative of triplicate experiments. (C) Quantification of Mup1–mNG expressed as mean fluorescence intensity at the PM of cells visualized in B (n=30 cells from triplicate experiments). (D) Mup1–GFP variants (Mup1WT, Mup1K567R, Mup1K572R and Mup1K567R,K572R) were expressed exogenously from a plasmid. Mean fluorescence intensity levels of Mup1–GFP at the PM (n=94–121 cells from duplicate experiments) were quantified following mock treatment or treatment with Myr (400 ng ml−1) for 5 h. (E) Anti-FLAG and anti-K63-linked polyubiquitin immunoblots of Mup1–FLAG enriched by anti-FLAG immunoprecipitation after 2 or 4 h of Myr treatment. Images are representative of duplicate experiments. MW, molecular weight; MWM, molecular weight marker. (F) Mup1–pHluorin trafficking was analyzed by flow cytometry of yeast cells expressing either WT or K63R ubiquitin from a single source. Mup1–pHluorin levels were measured after 5 h treatment with or without Myr (four biological replicates; n=10,000 cells). (G) Yeast cells expressing Mup1–mNG (green) and either WT ubiquitin (labeled with Vph1–mRuby in red) or K63R ubiquitin (unlabeled vacuoles) were mixed and imaged by fluorescence microscopy following mock or Myr treatment. Images are representative of triplicate experiments. (H) Quantification of Mup1–mNG mean fluorescence intensity at the PM of cells shown in G. n=30 cells. Bars show the mean±s.d. ns, not significant; **P<0.001; ***P<0.0001 (one-tailed paired t-test).CC BY · PubMed Central
Fig. 6. Myr-induced trafficking of Mup1 requires the endocytic adaptor proteins Ent1 and Ede1. (A) Yeast cells expressing the indicated fluorescent protein fusions were analyzed by fluorescence microscopy following 5 h of mock treatment or Myr treatment. Colocalization was measured using Pearson's correlation coefficient (n=30–60 cells from triplicate experiments). (B) Colocalization between Ede1–mCherry and Mup1–mNG was analyzed by measuring Pearson's correlation coefficients (n=30 cells from triplicate experiments) for the indicated conditions. (C) A split-Venus fluorescence complementation assay with the N-terminal fragment of Venus fused to Mup1 (Mup1–VN) and the C-terminal fragment of Venus fused to Ede1 (Ede1–VC) was used to analyze proximity between Mup1 and Ede1. Cells expressing either WT or K63R ubiquitin were analyzed by fluorescence microscopy after 5 h of mock treatment or Myr treatment, as indicated. Images are representative of triplicate experiments. (D) Total Venus fluorescence (Mup1–VN and Ede1–VC) of cells analyzed from the experiment shown in C (n=30 cells from triplicate experiments). (E) Myr-triggered Mup1–pHluorin trafficking was analyzed by flow cytometry in yeast cells with the indicated genotypes. Mup1–pHluorin fluorescence was measured in cells after 5 h with or without Myr treatment (four biological replicates; n=10,000 cells). (F) Fluorescence microscopy of WT and Δent1Δede1 yeast cells expressing Mup1–pHluorin (green) after 5 h with or without Myr treatment. Images are representative of triplicate experiments. Bars show the mean±s.d. ns, not significant (one-tailed paired t-test).
Fig. 6. Myr-induced trafficking of Mup1 requires the endocytic adaptor proteins Ent1 and Ede1. (A) Yeast cells expressing the indicated fluorescent protein fusions were analyzed by fluorescence microscopy following 5 h of mock treatment or Myr treatment. Colocalization was measured using Pearson's correlation coefficient (n=30–60 cells from triplicate experiments). (B) Colocalization between Ede1–mCherry and Mup1–mNG was analyzed by measuring Pearson's correlation coefficients (n=30 cells from triplicate experiments) for the indicated conditions. (C) A split-Venus fluorescence complementation assay with the N-terminal fragment of Venus fused to Mup1 (Mup1–VN) and the C-terminal fragment of Venus fused to Ede1 (Ede1–VC) was used to analyze proximity between Mup1 and Ede1. Cells expressing either WT or K63R ubiquitin were analyzed by fluorescence microscopy after 5 h of mock treatment or Myr treatment, as indicated. Images are representative of triplicate experiments. (D) Total Venus fluorescence (Mup1–VN and Ede1–VC) of cells analyzed from the experiment shown in C (n=30 cells from triplicate experiments). (E) Myr-triggered Mup1–pHluorin trafficking was analyzed by flow cytometry in yeast cells with the indicated genotypes. Mup1–pHluorin fluorescence was measured in cells after 5 h with or without Myr treatment (four biological replicates; n=10,000 cells). (F) Fluorescence microscopy of WT and Δent1Δede1 yeast cells expressing Mup1–pHluorin (green) after 5 h with or without Myr treatment. Images are representative of triplicate experiments. Bars show the mean±s.d. ns, not significant (one-tailed paired t-test).CC BY · PubMed Central
Fig. 7. Comparison of Mup1 endocytic trafficking as stimulated by excess methionine (left) or sphingolipid depletion (right). Methionine stimulates Art1-dependent ubiquitin modification of the N-terminus of Mup1, which then undergoes endocytosis mediated by the endocytic adaptor Ede1. In contrast, myriocin stimulates Art2-dependent ubiquitin modification of the C-terminus of Mup1, endocytosis of which requires K63-linked ubiquitin polymers and the endocytic adaptors Ent1 and Ede1.
Fig. 7. Comparison of Mup1 endocytic trafficking as stimulated by excess methionine (left) or sphingolipid depletion (right). Methionine stimulates Art1-dependent ubiquitin modification of the N-terminus of Mup1, which then undergoes endocytosis mediated by the endocytic adaptor Ede1. In contrast, myriocin stimulates Art2-dependent ubiquitin modification of the C-terminus of Mup1, endocytosis of which requires K63-linked ubiquitin polymers and the endocytic adaptors Ent1 and Ede1.CC BY · PubMed Central

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Metadata

Authors
Hepowit, N. L., Moon, B., Ebert, A. C., Dickson, R. C., & MacGurn, J. A.
Affiliations
  • Vanderbilt University (US)
  • University of Kentucky (US)
Corresponding author
  • Dickson, Robert C.
  • MacGurn, Jason A.
ORCID
Venue
Journal of Cell Science
Publisher
The Company of Biologists
Published
2023-07-15
Volume
136
Issue
14
ISSN
  • 0021-9533 (ISSN-L)
  • 1477-9137
DOI
10.1242/jcs.260675
PMC
PMC10399987
OpenAlex
W4381309905
Cited by
18
References
48
Citations by year
2026: 2 · 2025: 5 · 2024: 8 · 2023: 3
FWCI
2.59
Type
journal article
Language
en
Open access
Licence
Locations
  • Journal of Cell Science (publishedVersion, cc-by)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion, cc-by-nc)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
Funders
  • National Institutes of Health (R56 AG024377, R35 GM144112, T32 GM65086)
  • Vanderbilt University
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › Cellular transport and secretion
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › Autophagy in Disease and Therapy
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Ubiquitin and proteasome pathways
Keywords
ART2, myriocin, Rsp5 ubiquitin ligase, amino acid transporters, amino acid restriction, longevity, Saccharomyces cerevisiae
MeSH terms
Endocytosis, Methionine, Saccharomyces cerevisiae, Sphingolipids, Ubiquitin, Saccharomyces cerevisiae Proteins, Ubiquitination, Endosomal Sorting Complexes Required for Transport

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