AMP-activated kinase is required for adaptor localization in prolonged starvation
In response to glucose starvation, clathrin-dependent traffic at the TGN and endosomes is transiently blocked. All clathrin adaptors at the TGN and endosomes immediately redistribute to the cytosol. Only after 30 min or more of continuous starvation do the adapters partially regain their normal punctate localization, reflecting association with membranes of the TGN and endosomes (Aoh et al., 2011
; Supplemental Figure S1). We also demonstrated that these changes in localization reflect loss of adaptor association with membranes and not disassembly of the organelles (Aoh et al., 2011
). Delocalization of adaptors prevents clathrin-dependent traffic and thus prevents energy consumption by the clathrin-dependent cycling of proteins between the TGN and endosomes. However, our previous findings did not reveal the molecular mechanism by which glucose starvation regulates adaptor function.
We previously found that Ent5 becomes hyperphosphorylated at the same time that adaptors relocalize to membranes during prolonged starvation (Aoh et al., 2011
). This hyperphosphorylation is easily identified with immunoblot analysis (, arrowhead). Although the functional significance of this phosphorylation is unknown, mutations that prevented the immediate delocalization of adaptors during glucose starvation also prevented hyperphosphorylation (Aoh et al., 2011
). Therefore, to identify additional regulators of adaptors, we screened for gene deletions that prevented Ent5 hyperphosphorylation during glucose starvation using a focused library of strains lacking kinases or kinase cofactors. We performed immunoblot analysis of lysates from cells from this library to identify gene deletions that altered Ent5 hyperphosphorylation after prolonged starvation (unpublished data).
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 (more ...)
Using this approach, we found that mutation of several proteins in the AMPK pathway gave strong defects in Ent5 hyperphosphorylation (). AMPK consists of a core catalytic α subunit and one of three partially redundant β subunits, which contribute substrate specificity (reviewed in Hedbacker and Carlson, 2008
). AMPK also requires activating phosphorylation by one of three upstream kinases. In the screen, we found that deletion of GAL83
, a β subunit, blocked Ent5 hyperphosphorylation. We also found that deletion of the primary upstream activating kinase, SAK1
, caused partial defects (, arrowhead). In our initial screen, the core α catalytic subunit (SNF1
) was not identified as required for hyperphosphorylation. However, strains from deletion library collection that we used to make the focused library might contain errors or have second-site suppressor mutations. Therefore we generated a strain carrying a complete deletion of SNF1
. In this strain, Ent5 hyperphosphorylation was inhibited (). Thus the AMPK pathway is required for Ent5 hyperphosphorylation.
To determine whether Snf1 regulates adaptor localization responses, we investigated adaptor localization in snf1Δ
cells. We chose Gga2 and Ent5 as representatives of the two functional modules of clathrin-dependent TGN-endosome traffic: the Gga/Ent3 module and the AP-1/Ent5 module. These two modules have different requirements for localization and act at different stages in transport within the TGN and endosomes (Boman et al., 2000
; Costaguta et al., 2001
; Fernandez and Payne, 2006
; Daboussi et al., 2012
; Hung et al., 2012
). Furthermore, the Gga/Ent3 module promotes the recruitment of the Ent5/AP-1 module (Daboussi et al., 2012
; Hung et al., 2012
). Deletion of SNF1
did not dramatically alter adaptor localization in the presence of glucose or adaptor redistribution to the cytosol in the acute phase of glucose starvation (). However, deletion of SNF1
caused dramatic changes in adaptor localization after prolonged starvation. In wild-type cells, Gga2 and Ent5 accumulate in large puncta after prolonged starvation. In cells lacking Snf1, Gga2 accumulated in many very small puncta, and Ent5 was found in only a few small puncta (). These results indicate that Snf1 is required for proper localization of Gga2 and Ent5 during the prolonged phase of glucose starvation.
Snf1 is a major regulator of cell physiology during glucose starvation. It can phosphorylate several proteins involved in membrane traffic, raising the possibility that Snf1 may directly regulate adaptor localization (Ptacek et al., 2005
). However, Snf1 should be activated rapidly upon glucose starvation, but relocalization of Gga2 and Ent5 is delayed until 30 min after the onset of starvation (Aoh et al., 2011
; Mayer et al., 2011
; Ruiz et al., 2011
). A temporal difference in Snf1 activation and adaptor relocalization could mean that Snf1 acts indirectly in adaptor localization. To determine whether Snf1 activation coincided temporally with adaptor relocalization in our starvation conditions, we monitored Snf1 activation during glucose starvation. To monitor activation, we used an antibody that recognizes the active phosphorylated form of Snf1. Before glucose starvation, no active Snf1 was detected (). Within 5 min of starvation, Snf1 was phosphorylated and remained phosphorylated during the 2-h time course. Because Snf1 is activated substantially before adaptors relocalize to punctate structures, the effect of Snf1 on adaptors is likely indirect.
Glucose repression is required for adaptor relocalization in acute starvation
We previously identified another glucose-responsive kinase, PKA, as an indirect regulator of adaptor localization during glucose starvation (Aoh et al., 2011
). PKA and Snf1 converge on the regulation of several glucose-responsive pathways, including opposing regulation of glucose repression (Zaman et al., 2009
). Glucose repression is the suite of changes in transcription and cell physiology that optimizes the cell to exclusively derive energy from the glycolysis of glucose or fructose (Wilson and Roach, 2002
). Glucose repression is induced by the activity of PKA (Thevelein and de Winde, 1999
). During glucose starvation, glucose repression is inactivated by Snf1 (Carlson et al., 1981
). We previously discovered that PKA activity in the presence of glucose is required for adaptor delocalization during the acute phase of glucose starvation (Aoh et al., 2011
). Our finding that Snf1 is required for adaptor relocalization during the prolonged phase of glucose starvation suggested that the two pathways were playing opposing roles in adaptor localization. Because PKA and Snf1 play opposing roles in both adaptor localization and glucose repression, we hypothesized that adaptor localization was linked to the glucose repression program.
To investigate this hypothesis, we grew cells in carbon sources that activate glucose repression (repressing) or inactivate glucose repression (derepressing). In cells grown in the repressing carbon sources—glucose and sucrose—carbon starvation caused an immediate delocalization of Gga2 and Ent5 to the cytosol (). In contrast, in cells grown in the derepressing carbon sources—galactose, raffinose, and glycerol/ethanol—starvation did not dramatically alter the localization of Gga2 or Ent5 (). This result suggests that the starvation-induced redistribution of adaptors depends on glucose repression.
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 (more ...)
To further test the role of glucose repression in adaptor redistribution, we investigated adaptor redistribution in cells lacking proteins required to establish glucose repression. The phosphatase regulatory subunit, Reg1, and the 14-3-3 protein, Bmh1, are required to establish glucose repression (Dombek et al., 2004
). We found that cells in lacking Reg1 or Bmh1, the number of punctate structures containing adaptors was unchanged by glucose starvation (). This result further demonstrates that glucose repression is a necessary prerequisite for starvation-induced redistribution.
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 (more ...)
Energy metabolism is required for adaptor localization
One of the major effects of glucose repression upon glucose starvation is on ATP production. The glucose repression program maintains aerobic glycolysis as the only pathway for energy generation, inhibiting all other modes of energy generation (Johnston, 1999
). In the absence of parallel ATP generation pathways, ATP levels drop precipitously immediately after glucose starvation (Ashe et al., 2000
). Because of the link between glucose repression and adaptor localization, we hypothesized that adaptor redistribution was linked to energy generation. To test this hypothesis, we inhibited respiration with the cytochrome C reductase inhibitor antimycin A in cells grown under different conditions. In the absence of mitochondrial function, cells can produce ATP via glycolysis with six carbon sugars (glucose, galactose, or raffinose). In cells grown in glucose, galactose, or raffinose, antimycin A had no dramatic effect on adaptor localization (). This suggests that antimycin A does not influence adaptors in cells that can generate energy via glycolysis. In cells preadapted to glycerol/ethanol, addition of antimycin A did not cause redistribution of adaptors to the cytosol (). This result may reflect the ability of these cells to store and metabolize carbohydrates such as glycogen and trehalose (Shi et al., 2010
). Taken together, these results suggest that continued energy production is required for adaptor localization.
As a further test of this hypothesis, we treated cells that were preadapted and then starved for galactose or raffinose with antimycin A. Cells starved for galactose or raffinose generate ATP via respiration, and thus the addition of antimycin A reduces ATP levels in these cells. In cells that had been preadapted and then starved for either galactose or raffinose, adaptors redistributed to the cytosol immediately upon addition of antimycin A (). In cells preadapted to glycerol/ethanol and then starved and treated with antimycin A, we saw a reduction in the number of adaptor puncta; however, delocalization was not as complete as with other conditions. This may reflect the ability of these cells to generate ATP by glycolysis of glycogen and trehalose. These results suggest that adaptor localization is closely correlated to steady-state maintenance of energy levels.
To verify that changes in ATP concentration paralleled the changes in adaptor localization, we monitored ATP concentration in cells grown in different carbon sources and then starved. As expected, ATP concentration dropped >50% in cells grown in glucose or sucrose and then starved for the carbon source (). In contrast, ATP concentration remained high upon starvation of cells grown in galactose or raffinose. This observation is consistent with the known effects of carbon source on oxidative phosphorylation.
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, (more ...)
In cells grown in glycerol/ethanol, measured ATP concentrations were lower than in the other carbon sources. Furthermore, the levels after starvation were substantially lower than in unstarved cells. This was unexpected based on the continued localization of adaptors under these conditions. One possible explanation is that glycerol/ethanol-grown cells may have a lower volume of cytoplasm, and this causes an underestimation of actual ATP concentrations. Cytoplasmic volume depends on both the size of the cell and the volume of the cytoplasm occupied by the vacuole and other ATP-impenetrable organelles. We approximated total cell volume with optical density. Glycerol/ethanol-grown cells are smaller overall and have more mitochondria and larger vacuoles, and thus they are expected to have a lower cytoplasmic volume than glucose-grown cells (Jorgensen et al., 2002
; Hughes and Gottschling, 2012
). The ATP levels measured therefore likely underestimate ATP concentrations for the glycerol/ethanol-grown cells. However, even with this underestimation, the measured ATP concentration of glycerol/ethanol-starved cells were still 10% higher than those in cells starved for repressing sugars (; p
< 0.01). Thus, overall these results show that adaptor delocalization coincides with low ATP concentration in cells.
We next examined ATP concentrations in cells with characterized defects in adaptor localization during glucose starvation. Cells lacking functional PKA or Reg1 fail to induce adaptor delocalization in the acute phase of glucose starvation (Aoh et al., 2011
; ). To modulate PKA activity, we used a strain carrying Tpk1-as, an analogue-sensitive allele of the PKA catalytic subunit Tpk1, in which PKA activity can be inhibited by the kinase inhibitor analogue 1NM-PP1. In this strain two of the three alternate catalytic subunits (TPK2
) are deleted, and the third (TPK1
) is mutated such that it can be inhibited by the kinase inhibitor analogue 1NM-PP1. When Tpk1-as cells were treated with 1NM-PP1 for 1 h before starvation, the ATP concentration did not change upon starvation (). This is in contrast to control dimethyl sulfoxide (DMSO)–treated cells, in which the ATP concentration dropped upon glucose starvation. Similarly, in cells lacking Reg1, ATP concentration dropped only 30%. This change was significantly smaller than the 50% reduction of ATP concentration in wild-type cells (; p
< 0.01). These findings are consistent with the ability of 1NM-PP1 TPK1-as–treated cells and reg1Δ cells to generate ATP by mitochondrial respiration using nonglucose substrates. In contrast, in cells lacking Snf1, ATP concentration dropped 80% after glucose starvation, a level that was significantly lower than that of wild-type cells (; p
< 0.01). Taken together, these results show that low ATP concentrations parallel changes in adaptor delocalization and that mutations that prevent adaptor delocalization prevent low ATP concentrations in the acute phase of glucose starvation.
To further investigate the coincidence between ATP concentration and adaptor localization, we monitored ATP concentration during a time course of starvation. In the first 5 min of glucose starvation, ATP concentration dropped to 20% of the level before starvation. The concentration rose to 35% within 10 min and continued to rise over the next 60 min (). By 30 min—the time point when adaptors return to membranes—ATP concentration was up to 40% of the prestarved level. In contrast, in cells lacking Snf1, ATP concentration dropped to 10% of prestarved level and never rose above 30% in the 60-min time course (). Thus the adaptor recruitment is coincident with ATP concentrations >40% of prestarved cells.
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 (more ...)
Next we directly manipulated cellular ATP concentration and monitored adaptor localization. In derepressed starved cells, antimycin A caused a concentration-dependent reduction in ATP concentration (). Reduction of ATP concentration to <20% of replete concentration caused a nearly complete redistribution of adaptors to the cytosol (, second column). When ATP concentrations were at or >40% of replete, adaptors were recruited to membranes. Of interest, the effect was dose dependent. At lower concentrations of ATP, fewer punctate structures were observed than at higher concentrations of ATP (). Thus adaptor localization is closely correlated with cellular ATP concentration.
Addition of ATP is sufficient for adaptor localization
ATP is a ubiquitous molecule in cells. It is required for the proper functioning of motors, kinases, and other enzymes, for pH and ion homeostasis, and to maintain levels of GTP. Any one of these functions may directly or indirectly affect adaptor localization. To explore possible energy-dependent mechanisms, we developed a permeabilized cell assay of adaptor localization. To prevent ATP generation, we treated glucose-starved cells with antimycin A before permeabilizing the cells. After permeabilization, Gga2–green fluorescent protein (GFP) could be seen as many small puncta, similar to its appearance in glucose-starved snf1Δ cells. Ent5 was diffusely localized. Addition of ATP to permeabilized cells caused both adaptors to rapidly redistribute into a few large puncta that resembled their localization on organelles in intact cells (). Thus ATP can induce adaptor redistribution Furthermore, because permeabilized cells cannot maintain pH or ion gradients, cytosolic pH or ion concentrations can be excluded as the mechanism by which ATP regulates adaptors.
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 (more ...)
To determine whether adaptors were recruited to organelles, we performed colocalization analysis. Owing to difficulties in visualizing the weak mCherry fluorescence of mCherry-tagged proteins in permeabilized cells using a confocal microscope, we used total internal reflection microscopy (TIRF) for this analysis. Although TIRF only allows visualization of structures within close proximity of the cell surface, some Ent5-positive organelles are close enough to the cell surface to be captured via this method. Organelles were marked with Sec7-mCherry. Sec7 shows partial colocalization with Ent5 in intact cells in the presence of glucose and during prolonged starvation (Daboussi et al., 2012
; Supplemental Figure S1). Of importance, Sec7 does not delocalize upon glucose starvation; therefore it is a reliable marker of organelles even when adaptors are delocalized (Aoh et al., 2011
). When observed with TIRF microscopy, Ent5 appeared diffuse in starved, permeabilized cells. In contrast, Sec7 was apparent in bright puncta in the permeabilized cells (). On addition of ATP, Ent5 appeared in punctate structures. As in intact cells, some of the Ent5 punctate structures colocalized with Sec7. These results indicate that ATP directs adaptor recruitment to organelles in permeabilized cells.
The analysis of adaptor redistribution in intact cells suggested that Snf1 acts indirectly to regulate adaptor localization via energy metabolism. To further test this model, we performed ATP add-back experiments in permeabilized cells lacking Snf1. We found that ATP induced adaptor puncta even in permeabilized cells lacking Snf1 (). Because ATP can bypass the requirement for Snf1, this finding further supports our earlier conclusion that Snf1 regulates adaptor localization indirectly via its roles in energy metabolism.
We next examined the ability of other nucleotides to induce adaptor localization in permeabilized cells (). Addition of GTP caused minor changes in Gga2 and Ent5 localization. However, ATP-γ-S and GTP-γ-S caused little change in either Gga2 or Ent5 localization. This suggests that full adaptor localization requires ATP hydrolysis and suggests a possible role for GTP in adaptor localization.
To further explore the role of nucleotides on adaptor localization, we examined adaptor localization in cells treated with different concentrations of ATP. For this analysis, we classified cells as having no puncta, small puncta, or large puncta. In cells expressing Gga2-GFP without exogenous ATP, no cells contained large puncta, 75% of cells had small puncta, and 25% had no puncta (). Addition of 5 or 10 mM ATP caused an increase in the number of cells with large puncta and in the number of cells with small puncta and a decrease of the number of cells with no puncta of Gga2. Increasing ATP from 10 to 15 mM caused a dramatic increase in the number of cells containing large puncta of Gga2. Thus, Gga2 shows a dose-dependent response to ATP.
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 and incubated with indicated amounts of nucleotides (more ...)
Ent5 also shows a dose-dependent response to ATP. In cells expressing Ent5-GFP without exogenous ATP, no cells contained large puncta, 38% of cells had small puncta, and 62% had no puncta (). Addition of 5 mM ATP caused an increase in the number of cells with large puncta and in the number of cells with small puncta and a corresponding decrease in the number of cells with no puncta. In the presence of 10 mM ATP, 65% of cells contained large puncta and only 4% contained small puncta. Increasing ATP from 10 to 15 mM ATP further increased the number of cells containing large puncta to 95%. Thus, similar to Gga2, Ent5 shows a dose-dependent response to ATP. Furthermore, ATP induces more cells to contain large puncta of Ent5 than large puncta of Gga2 at all concentrations of ATP tested. This difference suggests that, similar to intact cells, Gga2 and Ent5 have different requirements for localization in permeabilized cells.
We next examined the effects of different concentrations of GTP on adaptor localization. We found that addition of 5, 10, or 15 mM GTP caused 10–16% of cells to contain large puncta of Gga2 (). This increase in large puncta compared with cells with no nucleotide was coincident with a slight decrease in the number of cells with small puncta and in the number of cells with no puncta. In contrast, for Ent5, addition of 5 or 10 mM GTP only increased the number of cells with small puncta; it had no effect on the number of cells with large puncta of Ent5 (). However, increasing GTP from 10 to 15 mM caused 10% of the cells to contain large puncta of Ent5. This increase was coincident with a decrease in the number of cells with small puncta. Thus, although Gga2 and Ent5 show dose-dependent responses to GTP, the effect of GTP is weaker than the effect of ATP on both adaptors.
The finding that GTP can induce weak adaptor localization could be explained by a role of GTP in adaptor localization or secondary production of ATP by the transfer of the γ phosphate from GTP to ADP in the permeabilized cells. To distinguish between these two possibilities, we examined whether GTP and ATP acted synergistically. We treated cells with combinations of 5 or 10 mM ATP and 5 or 10 mM GTP. Gga2 localization in large puncta was increased by the addition of both GTP and ATP (). This effect was most pronounced for cells treated with 10 mM GTP and 10 mM ATP. This combination increased the number of cells containing large puncta of Gga2 from 16% in the presence of 10 mM ATP alone to 47% in the presence of 10 mM of ATP and GTP. GTP-γ-S was more effective than GTP in inducing large puncta of Gga2. In cells treated with 10 mM ATP and 10 mM GTP-γ-S, 75% of cells had large puncta of Gga2 (, A, chart, and C). Because GTP-γ-S is a poor substrate for γ transfer reactions, this finding argues that the generation of ATP does not contribute to the synergy of GTP and ATP on Gga2 localization (Lacombe et al., 1990
). Taken together, these results suggest that ATP and GTP act together to promote Gga2 localization.
The effect of GTP on Ent5 localization in ATP-treated cells was even more substantial. Addition of 10 mM GTP to cells treated with 5 mM ATP increased the number of cells containing large puncta of Ent5 from 34% to >80% (). Furthermore, 10 mM GTP-γ-S was as effective as 10 mM GTP on the formation of large Ent5 puncta in cells treated with 5 mM ATP (, B, chart, and C). Taken together, these results suggest that as with Gga2, ATP and GTP act synergistically to promote Ent5 localization. Thus GTP and ATP both contribute to adaptor localization in permeabilized cells.
Although the concentration of nucleotide required for these effects was relatively high, the maximal ATP concentration used is only five times higher than present estimates of physiological ATP concentrations in yeast (Ozalp et al., 2011
). Furthermore, without an ATP/GTP-regenerating system, nucleotides may be rapidly consumed. Thus the ATP responses observed may be induced by ATP concentrations equal to endogenous ATP concentrations. Alternatively, high nucleotide levels may be required because key factors such as GTP exchange factors are more dilute in the permeabilized cells. If the substrates or cofactors are more dilute, this would require higher concentrations of nucleotide for the same effect.
Upstream regulators of adaptors are differentially regulated by energy metabolism
Neither Gga2 nor Ent5 has any obvious GTP- or ATP-sensing domains. Therefore the regulation of adaptor localization is likely mediated by one or more upstream factors. To begin to characterize these factors, we first determined whether known regulators of Gga2 and Ent5 respond to ATP concentration. Several nucleotide-binding proteins are known to act upstream of adaptors, including the functionally interchangeable small GTPases Arf1 and Arf2, the PI4 kinase Pik1, and its product PI4p. Although Pik1 is known to redistribute in cells starved for glucose, the kinetics of this delocalization were unknown (Faulhammer et al., 2007
; Demmel et al., 2008
). We investigated the changes in localization of the major Arf isoform (Arf1), Pik1, and the PI4p-binding domain from the human GOLPH3 proteins during glucose starvation. In the presence of glucose, Arf1-GFP, GFP-Pik1, and GFP-GOLPH3 all localized to many bright punctate structures in cells (). Immediately upon glucose starvation, all three lost bright punctate staining. GFP-Pik1 became completely diffuse, whereas both Arf1 and GOLPH3 retained localization to dim punctate structures. These results show that the majority of Arf1 and Pik1 are rapidly delocalized during glucose starvation and that the levels of PI4p at the TGN and endosomes drop dramatically.
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 (more ...)
On prolonged glucose starvation, when Gga2 relocalized to several bright punctate structures, Pik1 was not found in bright punctate structures. Arf1 and GOLPH3 punctate structures remained dim. Of note, the number of ARF structures went up only slightly, and GOLPH3 puncta did not change upon prolonged starvation (). These results show that unlike the adaptors, the localization of Arf1, Pik1, and GOLPH3 do not show adaptation in the form of increased membrane localization during prolonged glucose starvation.
We next investigated the colocalization of Arf1 and PI4p with Gga2. In the presence of glucose, Arf1 and GOLPH3 show partial colocalization with Gga2. During prolonged starvation, Arf1 and GOLPH3 also partially colocalized with Gga2. We observed three types of structures in ARF-expressing cells: those that contained both Arf1 and Gga2, those that contained only Arf1, and those that contained only Gga2 (). This partial colocalization is consistent with partial colocalization seen in glucose-replete cells. In contrast, for GOLPH3, we saw only two types of structures: those that contained both GOLPH3 and Gga2, and those that contained only Gga2 (). Gga2 is believed to activate the formation of PI4p in glucose-replete cells (Daboussi et al., 2012
). Thus the partial colocalization of GOLPH3 and Gga2 in glucose-starved cells may indicate that Gga2 plays a similar role in influencing PI4p levels in glucose-starved cells. Taken together, these results show that glucose starvation clearly reduces the levels of Arf1 and PI4p in the endosomal system; however, their ability to localize to organelles that recruit Gga2 is unchanged.
We next investigated whether ATP induces changes in the localization of Arf1, Pik1, or PI4p in permeabilized cells. In glucose-starved permeabilized cells, Arf1 localized to a few dim puncta, whereas GOLPH3 and Pik1 were diffuse (). Addition of ATP caused a slightly higher number of dim Arf1 puncta. In contrast, ATP immediately induced formation of many bright GOLPH3 or Pik1 puncta. Thus ATP addition does not have a prominent effect on Arf1 localization, but it can direct dramatic changes in PI4p and Pik1 localization.
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 (more ...)
We next investigated the effects of GTP and GTP-γ-S on Arf1, Pik1, and PI4p (). As with ATP, the localization of Arf1 was not substantially increased by either GTP or GTP-γ-S. In contrast, both Pik1 and GOLPH3 localized to puncta after the addition of GTP. However, the number of puncta per cell induced by GTP was less than the number induced by ATP (). GTP-γ-S was even less effective at recruiting Pik1 and GOLPH3 than was GTP. Both Pik1 and GOLPH3 formed fewer puncta in the presence of GTP-γ-S than in GTP. Furthermore, the number of cells with large puncta was lower in the presence of GTP-γ-S than in GTP (). Taken together, these results show that Arf1 localization is largely nonresponsive to nucleotides. In contrast, Pik1 and GOLPH3 localization is induced by ATP, partially by GTP, and weakly by GTP-γ-S.
Arf1 and PI4p are required for adaptor localization during glucose starvation
To investigate whether adaptor localization depends on Arf, Pik1, and PI4p during glucose starvation, we first tested whether Arf is required for adaptor localization in glucose starvation. To rapidly inhibit Arf function, we used the lactone antibiotic brefeldin A. Brefeldin A inhibits several Arf guanine nucleotide exchange factors (GEFs). Inhibition of these GEFs leads to rapid loss of active Arf at select locations (reviewed in Jackson and Casanova, 2000
). In cells treated with brefeldin A in the presence of glucose, both Gga2 and Ent5 localized to a few large puncta per cell ( and Supplemental Figure S2). This suggests that some Gga2 and Ent5 structures are independent of a brefeldin A–sensitive GEF in the presence of glucose. In contrast, during prolonged starvation both Gga2 and Ent5 became diffuse within minutes of brefeldin A treatment (). Thus the localization of both Gga2 and Ent5 depends on Arf during glucose starvation.
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 (more ...)
We next investigated whether Pik1 is required for adaptor localization during glucose starvation. To modulate Pik1 activity, we used a previously described temperature-sensitive pik1-83
allele (Hendricks et al., 1999
). As previously described, in the presence of glucose, the pik1-83
allele did not alter Gga2 localization ( and Supplemental Figure S2; Daboussi et al., 2012
). In contrast, Gga2 became diffuse when cells were shifted to the nonpermissive temperature during prolonged starvation. These results suggest that Gga2 localization requires Pik1 activity during starvation.
Ent5 localization was highly dependent on Pik1 activity under all conditions tested ( and Supplemental Figure S2). In the presence of glucose, Ent5 became diffuse upon shift to the nonpermissive temperature, confirming previous results that Ent5 requires Pik1 for localization (Daboussi et al., 2012
). Of note, even at permissive temperature, Ent5 did not localize to bright puncta in the pik1-83
cells during prolonged glucose starvation. This result suggests that pik1-83
is not fully functional in glucose-starved cells and that Ent5 requires full Pik1 activity for localization during glucose starvation. Taken together, these results suggest that Ent5 localization depends on Pik1 activity both in the presence and in the absence of glucose.
PI4p levels are also regulated by the lipid phosphatase Sac1, which redistributes to the TGN during glucose starvation (Faulhammer et al., 2005
). To investigate the role of Sac1 relocalization in adaptor localization during glucose starvation, we monitored Gga2 and Ent5 localization in cells lacking Sac1 ( and Supplemental Figure S2). In these cells, Gga2 mostly became diffuse after glucose starvation, although some dim Gga2 puncta persisted. Such dim puncta were not seen in wild-type cells. In contrast, Ent5 became diffuse immediately after glucose starvation and then relocalized to bright puncta during prolonged starvation. These results suggest that Sac1 has a minor role in Gga2 relocalization but not Ent5 during glucose starvation.