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. 2011 Oct;22(19):3671-83.
doi: 10.1091/mbc.E11-04-0309. Epub 2011 Aug 10.

Glucose regulates clathrin adaptors at the trans-Golgi network and endosomes

Quyen L Aoh  1 , Lee M Graves, Mara C Duncan
Affiliations

Glucose regulates clathrin adaptors at the trans-Golgi network and endosomes

Quyen L Aoh et al. Mol Biol Cell. 2011 Oct.

Abstract

Glucose is a rich source of energy and the raw material for biomass increase. Many eukaryotic cells remodel their physiology in the presence and absence of glucose. The yeast Saccharomyces cerevisiae undergoes changes in transcription, translation, metabolism, and cell polarity in response to glucose availability. Upon glucose starvation, translation initiation and cell polarity are immediately inhibited, and then gradually recover. In this paper, we provide evidence that, as in cell polarity and translation, traffic at the trans-Golgi network (TGN) and endosomes is regulated by glucose via an unknown mechanism that depends on protein kinase A (PKA). Upon glucose withdrawal, clathrin adaptors exhibit a biphasic change in localization: they initially delocalize from the membrane within minutes and later partially recover onto membranes. Additionally, the removal of glucose induces changes in posttranslational modifications of adaptors. Ras and Gpr1 signaling pathways, which converge on PKA, are required for changes in adaptor localization and changes in posttranslational modifications. Acute inhibition of PKA demonstrates that inhibition of PKA prior to glucose withdrawal prevents several adaptor responses to starvation. This study demonstrates that PKA activity prior to glucose starvation primes membrane traffic at the TGN and endosomes in response to glucose starvation.

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Figures

FIGURE 1:
FIGURE 1:
Adaptors relocalize in response to glucose withdrawal. (A) Fluorescence microscopy of adaptors in cells expressing Ent5-GFP (DLY3), Gga2-GFP (DLY4), the AP-1 subunit Apl4-GFP (DLY36), or Ent3-GFP (DLY6). Adaptors localize to puncta in the presence of glucose. Immediately after glucose washout, adaptors are localized to cytoplasm. After prolonged starvation, adaptors are both diffusely localized and found in puncta. Five minutes after glucose is reintroduced following prolonged starvation, Ent5, Gga2, and Ent3 are found predominately in puncta, while AP-1 is found both diffusely localized and in puncta. (B) The number of puncta per cell was quantitated during growth in glucose, acute glucose starvation, or prolonged glucose starvation, or 5 min after the addition of 2% glucose to starved cells. (C) Glucose alone can regulate adaptor localization. Cells expressing Ent5-GFP were grown in the presence of glucose for 2 h then transferred to water with 2% glucose (left) or into water (Center), or first washed into water for 15 min and then transferred into 2% glucose (right). (D) Cells expressing Ent5-GFP were grown in the presence of glucose for 2 h, then transferred to water with 2% glucose (left) or into 2% sorbitol (center), or first washed into 2% sorbitol for 15 min and then transferred into 2% glucose (right).
FIGURE 2:
FIGURE 2:
Glucose starvation does not delocalize other markers of the TGN and endosomes, but affects clathrin localization. (A) Fluorescence microscopy of cells expressing Sec7-GFP (DLY35) or Snf7-GFP (DLY22). Both Sec7 and Snf7 are predominantly localized to puncta during growth in glucose, immediately following or after prolonged glucose washout, and following reintroduction of glucose to starved cells. (B) The number of Sec7 puncta per cell was quantitated during growth in glucose, acute glucose starvation, or prolonged glucose starvation, or 5 min after the addition of 2% glucose to starved cells. (C) Clathrin colocalizes with Ent5 during glucose starvation. Fluorescence microscopy of cells coexpressing clathrin heavy chain (Chc1-red fluorescent protein [RFP]) and Ent5-GFP (DLY7). Cells were imaged during growth in glucose, immediately following or after prolonged glucose washout. Glucose was then added to the cells, which were imaged 5 min later. Arrowheads indicate examples of colocalization.
FIGURE 3:
FIGURE 3:
Adaptors are released from membranes during glucose starvation. Wild-type or Sec7-GFP-expressing cells (DLY35) were grown to mid-log phase and then incubated in the presence (left) or absence (right) of glucose for 15 min. The cells were then lysed by mechanical disruption, and the lysate was centrifuged at 200,000 × g to give the S200 supernatant and P200 membrane pellet. (A) Lysates were subject to immunoblot analysis and probed with antibodies to clathrin, Ent5, Gga2, Ent3, or GFP to detect Sec7. (B) Quantitative analysis of the fraction of adaptors found in P200 pellet to total from samples treated +/− glucose. Error bars represent SD (n = 3 for Ent5, Gga2, and Ent3; n = 2 for Sec7-GFP).
FIGURE 4:
FIGURE 4:
Posttranslational modifications of adaptors are regulated by glucose. (A) Glucose depletion causes hyperphosphorylation of Ent5, dephosphorylation of Gga2, and a change in Ent3 posttranslational modification. Wild-type cells (BY4742) were grown to mid-log phase, then washed with and resuspended in media without glucose for 2 h. Cell lysates were then probed with antibodies to adaptors. (B) Phosphorylation of adaptors. Adaptors were immunoprecipitated from wild-type cells grown to stationary phase (Ent5) or mid-log phase (Gga2 and Ent3). Immunoprecipitated samples were then treated +/− calf intestinal alkaline phosphatase (CIP) and subjected to immunoblot analysis. (C) Time course showing changes in adaptor modification in response to glucose. Wild-type cells were grown to mid-log phase and then washed with and incubated in media without glucose for 4 h. Following starvation, glucose was added to the culture media for 30 min. Samples were taken at indicated time points, lysed, and subjected to immunoblot analysis using antibodies to Ent5, Gga2, Ent3, or Adh1 as a loading control. The fraction of the highest-molecular-weight band (arrowheads) to total was quantitated and is presented graphically in (D). (E) Ent5 phosphorylation does not induce protein turnover during glucose starvation. Cells were grown in the absence (lanes 1–3) or presence (lanes 4–6) of 35 μg/ml cycloheximide to mid-log phase in glucose for 2 h (lanes 1 and 4) and then washed and starved for glucose for an additional 3 h (lanes 2 and 5). Glucose (2%) was then added back to cells for 30 min (lanes 3 and 6).
FIGURE 5:
FIGURE 5:
PKA is required for Ent5 relocalization and hyperphosphorylation. (A) Prolonged inhibition of PKA is required to prevent Ent5 relocalization during glucose starvation. ENT5-GFP TPK1-as tpk2Δ tpk3Δ cells (DLY13) were incubated with 2 μM 1NM-PP1 for indicated times, and then imaged immediately after glucose removal. (B) PKA is required for starvation-induced changes in Ent5 localization. Cells were grown in glucose in the presence of DMSO (top) or 2 μM 1NM-PP1 (bottom) for 2 h. Cells were imaged just before starvation or at indicated times following glucose washout. (C) When PKA is inhibited, Ent5 is not hyperphosphorylated during glucose starvation. Cells were treated with DMSO or 2 μM 1NM-PP1 for 2 h during growth in glucose and then starved for glucose. Samples were taken from cells just before DMSO or 1NM-PP1 treatment (lane 1) or just prior to glucose starvation (lanes 2 and 6) or at indicated time-points after starvation. Cell lysates were subject to SDS–PAGE and Western blot analysis with antibodies to Ent5 or Adh1.
FIGURE 6:
FIGURE 6:
PKA is required for starvation-induced relocalization of all TGN–endosome adaptors. TPK1-as tpk2Δ tpk3Δ cells expressing GGA2-GFP (DLY14), ENT3-GFP (DLY17), and the AP-1 subunit APL4-GFP (DLY16) were imaged in the presence of 2 μM 1NM-PP1 during growth in glucose or at indicated times following glucose washout.
FIGURE 7:
FIGURE 7:
Glucose-stimulated PKA activity is a prerequisite for Ent5 relocalization and hyperphosphorylation during starvation. (A) Glucose-stimulated PKA activity permits relocalization of Ent5 during starvation. ENT5-GFP TPK1-as tpk2Δ tpk3Δ cells (DLY13) were treated with 2 μM 1NM-PP1 or DMSO for 2 h prior to glucose starvation (top) or coincident with glucose starvation (bottom). Cells were imaged in the presence of glucose or at indicated times following glucose washout. (B) PKA must be active prior to glucose removal to allow hyperphosphorylation of Ent5. Cell lysates from TPK1-as tpk2Δ tpk3Δ cells treated with 2 μM 1NM-PP1 prior to (lanes 2–5) or coincident with (lanes 6–9) glucose starvation. Samples were taken from cells grown prior to treatment with DMSO or 1NM-PP1 (lane 1), 2 h after growth in glucose in the presence of DMSO or 1NM-PP1 (lanes 2 and 6), or at indicated times after glucose removal, or 30 min after glucose readdition (lanes 5 and 9). Cell lysates were subject to SDS–PAGE and Western blot analysis with antibodies to Ent5 or Adh1.
FIGURE 8:
FIGURE 8:
Gpr1 and Ras2 regulate adaptor modification and localization. (A) Gpr1 and Ras2, but not Ras1, affect changes in adaptors' phosphorylation. Wt, gpr1Δ, ras1Δ, and ras2Δ cells were grown to mid-log phase in the presence of glucose or subsequently washed and incubated with glucose-free media for 2 h. Cells were lysed, and cell lysates were analyzed by immunoblotting with antibodies to Ent5, Gga2, Ent3, or Adh1 as a loading control. (B) Gpr1 and Ras2 are required for starvation-induced relocalization of Ent5. Fluorescence microscopy of ENT5-GFP in wild-type (DLY3), gpr1Δ (DLY11), or ras2Δ cells (DLY12). Cells were imaged during growth in glucose, or at indicated times following glucose washout. (C) Hyperactivation of PKA does not inhibit relocalization of Ent5 during glucose starvation. Cells expressing ENT5-GFP (DLY3) were transformed with a plasmid containing the dominant active Ras2Val19 allele under the control MET3 promoter. Ras2Val19 expression was induced by growth in media without methionine (−met; bottom). In cells with hyperactive PKA activity, Ent5 localization is not significantly affected during growth in glucose. Following acute glucose starvation, Ent5 is predominantly diffusely localized and found in some punctate structures in cells with hyperactive PKA activity. During prolonged starvation, and following reintroduction of glucose, localization of Ent5 in hyperactive cells is similar to that in uninduced cells.
FIGURE 9:
FIGURE 9:
Possible model of the role of PKA in the acute phase of glucose starvation and the effect on adaptor localization. (A) In the presence of glucose, PKA directs the activation of an unknown factor (X). This factor is kept inactive by the presence of glucose. (B) On glucose starvation, the unknown factor becomes active and reduces levels of PI4P at the TGN and endosomes. (C) When PKA is inhibited, the unknown factor is not active, and the immediate response does not occur upon glucose starvation. Depletion of activity of the unknown factor levels after prolonged starvation also allows recovery of PI4P levels and adaptor re-recruitment.
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