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. 2013 Mar;24(6):832-47.
doi: 10.1091/mbc.E12-10-0750. Epub 2013 Jan 23.

Energy metabolism regulates clathrin adaptors at the trans-Golgi network and endosomes

Quyen L Aoh  1 , Chao-wei Hung, Mara C Duncan
Affiliations

Energy metabolism regulates clathrin adaptors at the trans-Golgi network and endosomes

Quyen L Aoh et al. Mol Biol Cell. 2013 Mar.

Abstract

Glucose is a master regulator of cell behavior in the yeast Saccharomyces cerevisiae. It acts as both a metabolic substrate and a potent regulator of intracellular signaling cascades. Glucose starvation induces the transient delocalization and then partial relocalization of clathrin adaptors at the trans-Golgi network and endosomes. Although these localization responses are known to depend on the protein kinase A (PKA) signaling pathway, the molecular mechanism of this regulation is unknown. Here we demonstrate that PKA and the AMP-regulated kinase regulate adaptor localization through changes in energy metabolism. We show that genetic and chemical manipulation of intracellular ATP levels cause corresponding changes in adaptor localization. In permeabilized cells, exogenous ATP is sufficient to induce adaptor localization. Furthermore, we reveal distinct energy-dependent steps in adaptor localization: a step that requires the ADP-ribosylation factor ARF, an ATP-dependent step that requires the phosphatidyl-inositol-4 kinase Pik1, and third ATP-dependent step for which we provide evidence but for which the mechanism is unknown. We propose that these energy-dependent mechanisms precisely synchronize membrane traffic with overall proliferation rates and contribute a crucial aspect of energy conservation during acute glucose starvation.

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Figures

FIGURE 1:
FIGURE 1:
Snf1 is required for localization of the TGN–endosomal clathrin adaptors Gga2 and Ent5 during prolonged glucose starvation. (A) AMPK pathway proteins are required for Ent5 hyperphosphorylation. Cellular lysates were prepared from indicated cells before or 2 h after glucose starvation. The cell lysates were then probed with an antibody to Ent5. Arrowheads indicate hyperphosphorylated Ent5. (B) Snf1 is required for adaptor localization during prolonged glucose starvation. Wild-type (WT) and snf1∆ cells expressing Gga2-GFP (top) or Ent5-GFP (bottom) from their endogenous loci were imaged before (+glucose), within 10 min (acute starvation), and after 2 h of glucose starvation (prolonged starvation). Scale bar, 5 μm. (C) Quantification of the number of puncta per cell for cells in B. **p < 0.01; n.s., not significant. Charts show box plots of the data. The line indicates the median, the edges of the box indicate the 25 and 75% percentiles, and the whiskers extend to the extremes of the data not considered outliers. (D) Snf1 is activated within 5 min of glucose starvation. Cellular lysates were prepared from cells expressing Snf1-myc from its endogenous locus at the indicated time points before or after glucose starvation. Active Snf1 was detected with a phospho-AMPK, and total cellular Snf1 was detected with an anti-myc antibody.
FIGURE 2:
FIGURE 2:
Inhibition of cellular energy production induces adaptor redistribution. Wild-type cells expressing Gga2-GFP (A) or Ent5-GFP (B) from their endogenous loci were preadapted to different carbons sources by continuous logarithmic growth for 48 h. Cells were imaged during logarithmic phase growth (i), after treatment with 2 μg/ml antimycin (ii), immediately after carbon source withdrawal (iii), and immediately after carbon source withdrawal followed by treatment with 2 μg/ml antimycin (iv). Scale bar, 5 μm.
FIGURE 3:
FIGURE 3:
Glucose repression genes are required for glucose starvation–induced redistribution of Ent5. (A,C). Indicated cells expressing Ent5-GFP from the endogenous locus were imaged before or immediately after glucose starvation. (B,D). Quantification of the number of puncta per cell for cells in A and C. **p < 0.01; n.s., not significant. Chart shows box plot of data as described in Figure 1. Scale bar, 5 μm.
FIGURE 4:
FIGURE 4:
Cellular ATP concentration decrease significantly during glucose starvation and is regulated by glucose repression pathways. (A) Cellular ATP was measured in wild-type cells preadapted to different carbon sources (2% glucose, 2% galactose, 2% raffinose, 2% glycerol/3% ethanol, or 2% sucrose) before and after carbon source withdrawal. (B) TPK1-as tpk2∆ tpk3∆ cells were grown to mid–log phase, incubated with 2 μM 1NM-PP1 or DMSO for 1 h, and then starved. Cellular ATP was measured in cells before and after starvation. ATP concentrations were normalized to cells grown in glucose and treated with DMSO. (C) Cellular ATP was measured in reg1∆ cells before or after glucose starvation. The relative ATP concentrations were normalized to wild-type cells grown in glucose. (D) snf1Δ cells were processed as in C. **p < 0.01, *p < 0.05. Charts show average values and SD.
FIGURE 5:
FIGURE 5:
Cellular ATP concentrations correlate with the association of adaptors to membranes. (A) Cellular ATP measured in indicated cells before and after glucose starvation as described in Materials and Methods. (B) Wild-type cells were grown to mid–log phase in galactose and then starved. Various amounts of antimycin A were added to the cells. Cellular ATP measured as described. Charts show average values and SD. (C) Indicated cells were processed as in B and imaged. Scale bar, 5 μm. (D) Quantification of the number of puncta per cell for cells in C. Chart shows box plot of data as described in Figure 1.
FIGURE 6:
FIGURE 6:
ATP is sufficient to recruit adaptors in permeabilized cells. (A) Cells expressing Gga2-GFP or Ent5-GFP from the endogenous loci were permeabilized and incubated with or without indicated nucleotides for 5 min before imaging. (B) Ent5 is recruited to Sec7 containing organelles upon addition of ATP. Cells expressing Ent5-GFP and Sec7-mcherry were permeabilized and incubated with or without ATP. Cells were fixed, mounted for TIRF microscopy, and imaged. (C) Snf1 is not required for adaptor localization in the presence of exogenous ATP. Cells lacking Snf1 and expressing Gga2-GFP or Ent5-GFP from the endogenous loci were permeabilized and imaged with or without addition of exogenous ATP. Scale bar, 5 μm.
FIGURE 7:
FIGURE 7:
ATP and GTP both contribute to adaptor recruitment. (A, B) Right, addition of GTP reduces the amount of ATP required to induce adaptor localization in permeabilized cells. Cells were prepared as in Figure 6 and incubated with indicated amounts of nucleotides for 5 min before imaging. Left, quantification of adaptor localization in different conditions. Cells were classified as having no puncta, only small or dim puncta, or large puncta. Scale bar, 5 µm. Charts show representative data from one of three replicate experiments. n > 50 cells. (C) GTP-γ-S reduces the amount of ATP required to induce adaptor localization.
FIGURE 8:
FIGURE 8:
Glucose starvation alters the localization of Arf1, Pik1, and PI4p. (A) Arf relocalizes to dim puncta during acute and prolonged starvation. Diploid cells heterozygous for ARF1-GFP and homozygous for GGA2-mCherry were imaged before, within 15 min, or after 2 h of glucose starvation. (B) Pik1 redistributes to the cytosol upon glucose starvation. Haploid wild-type cells expressing GFP-Pik1 from a plasmid were imaged before, within 15 min, or after 2 h of glucose starvation. (C) GOLPH3 relocalizes to dim puncta during acute and prolonged starvation. Haploid wild-type cells expressing Gga2-mCherry from the endogenous locus and the PI4P probe GOLPH3-GFP from a plasmid were imaged before, within 15 min, or after 2 h of glucose starvation. (D) Quantification of puncta per cell for cells grown in or acutely starved for glucose as described in A–C. **p < 0 .01; n.s., not significant. Charts show box plot of data as described in Figure 1.
FIGURE 9:
FIGURE 9:
Arf1, Pik1, and PI4p show differential responses to exogenous nucleotides in permeabilized cells. (A) Diploid cells heterozygous for ARF1-GFP or haploid wild-type cells expressing GFP-Pik1 GOLPH3-GFP from plasmids were prepared as described in Figure 6A and imaged before or after addition of 15 mM indicated nucleotides. Scale bars, 5 μm. (B) Quantification of puncta per cell. **p < 0.01; *p < 0.05; n.s., not significant. Charts show box plot of data as described in Figure 1. (C) Quantification of adaptor localization in different conditions. Cells were classified as in Figure 7. Black portion of the bars indicates percentage of cells with large puncta; gray indicates small puncta; and white indicates no puncta.
FIGURE 10:
FIGURE 10:
Arf1, Pik1, and Sac1 modulate adaptor localization during glucose starvation. (A) Brefeldin A induces adaptor redistribution only during glucose starvation. erg6∆ cells expressing Gga2-GFP or Ent5-mCherry were imaged before or after 2 h of glucose starvation. Cells were treated with DMSO (control) or 150 μM brefeldin A for 5 min before imaging. (B) Adaptors show increased dependence on Pik1 during glucose starvation. pik1-83 cells expressing Gga2-mRFP or Ent5-GFP were imaged before or after 2 h of glucose starvation at the permissive temperature (25°C) or after a 30-min shift to nonpermissive (38°C) temperature. For temperature-shifted starved cells, cells were first starved for 1.5 h and then shifted to the nonpermissive temperature. (C) Gga2 but not Ent5 requires Sac1 for rapid redistribution during acute starvation. Wild-type and sac1∆ cells expressing Gga2-GFP or Ent5-GFP were imaged before or after 2 h of glucose starvation. Scale bar, 5 μm.
FIGURE 11:
FIGURE 11:
Model of energy-dependent steps in adaptor recruitment. The steps illustrated may occur concurrently or in a different sequence than illustrated. Before energy input, Arf is localized to membranes, but Pik1 and adaptors are cytosolic. 1) On availability of GTP, Pik1 becomes localized and activated. 2) On availability of ATP, Pik1 synthesizes PI4p; however, this is not sufficient for adaptor recruitment. 3) Adaptors are recruited only when a third, uncharacterized factor has access to sufficient ATP. All three activities—Arf1, Pik1 and this third factor—are required for localization of adaptors to membranes during glucose starvation.
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