BACKGROUND
in the budding yeast saccharomyces cerevisiae, glucose stimulates its transport across the plasma membrane by inducing expression of several hxt  <cit> .
under glucose-limited conditions, the transcriptional repressor rgt <dig> binds to the hxt promoters and recruits general corepressors ssn <dig> and tup <dig>  <cit> .
rgt <dig> does this in conjunction with its corepressor mth <dig>  which physically interacts with rgt <dig>  <cit> .
therefore, it has been proposed that rgt <dig> forms a repression complex with mth <dig>  ssn <dig>  and tup <dig> on the hxt promoters, inhibiting transcription  <cit> .
glucose appears to prevent formation of this protein complex by causing degradation of mth <dig>  resulting in release of rgt <dig> from hxt promoters, thereby inducing expression of hxt  <cit> .
the glucose signal that leads to degradation of mth <dig> is generated by the plasma-membrane spanning glucose sensors rgt <dig> and snf <dig>  signal generation is a receptor-mediated process and does not require glucose metabolism.
this idea is supported by evidence that dominant mutations exist in the glucose sensor genes that lock the sensor proteins in glucose-bound conformations, generating a constitutive signal  <cit> .
indeed, mth <dig> is constitutively degraded in cells expressing the active glucose sensor mutants  <cit> .
subsequent studies have shown that the plasma membrane-tethered casein kinases yck1/ <dig> phosphorylate mth <dig>  triggering its ubiquitination and subsequent degradation  <cit> .
it has also been shown by yeast-two-hybrid assay that mth <dig> interacts with the c-terminal tails of the glucose sensors, suggesting that mth <dig> is recruited to the plasma membrane  <cit> .
these observations have led to the current view of glucose-induced hxt expression.
upon glucose binding, the glucose sensors are converted from inactive to active forms through a conformational change, activating yck1/ <dig> in their vicinity.
mth <dig>  recruited by the glucose sensors to the plasma membrane, is phosphorylated by yck1/ <dig> and, subsequently, ubiquitinated by scfgrr <dig>  finally, the ubiquitinated mth <dig> is targeted for degradation by the 26s proteasome  <cit> .
however, this hypothesis is mainly based on the following assumptions:  mth <dig> is excluded from the nucleus upon glucose addition and recruited to the plasma membrane by any means, and  yck1/ <dig> are activated through a direct interaction with the glucose sensors.
in this study, we specifically tested these assumptions and provide evidence that yck1/ <dig> do not directly couple to the glucose sensors during transmission of the glucose signal from the plasma membrane to the nucleus.
a possible mechanism for how the glucose sensors and yck1/ <dig> collaborate to degrade mth <dig> is discussed.
RESULTS
subcellular localization of mth <dig> is not regulated
we have previously reported that gfp-mth <dig> is mainly nuclear in glucose-depleted medium and is rapidly degraded upon glucose addition  <cit> .
to gain more insights into glucose-dependent degradation of mth <dig>  we first examined subcellular localization of gfp-mth <dig> in cells grown under conditions where hxt expression is repressed  or induced  .
because glucose also regulates expression of mth <dig>  <cit> , gfp-mth <dig> was expressed from the met <dig> promoter, which is not regulated by glucose, in a low copy centromeric plasmid  <cit> .
fluorescence microscopy analysis demonstrates that gfp-mth <dig> is localized primarily to two specific foci within the nucleus and that ~50% of fluorescence is recovered within 30- <dig> seconds after photobleaching .
these results suggest that, under glucose-limited conditions, mth <dig> predominantly accumulates in the nucleus but does not shuttle between the nucleus and cytoplasm.
we also monitored glucose-induced degradation of mth <dig> in a time-lapse manner and observed that more than half of gfp-mth <dig> is degraded within 5- <dig> min after glucose addition , which is consistent with the western blot analysis obtained previously  <cit> .
to determine whether mth <dig> is excluded from the nucleus in response to glucose, we determined in which cellular compartment mth <dig> is localized when expressed under high glucose conditions.
toward this aim, we examined subcellular localization of gfp-mth <dig> in the rgt2snf <dig> and grr <dig> mutants, where glucose-dependent degradation of mth <dig> is severely impaired  <cit> .
as shown in figure 2a, the gfp-mth <dig> proteins accumulate in the nucleus of the mutants grown in high-glucose medium.
to further test a possibility that the glucose sensors regulate nuclear exclusion of mth <dig>  we determined subcellular localization of a nondegradable form of mth <dig> in wild-type cells, where the glucose sensors are active.
the dominant htr1- <dig> mutation in mth <dig>   <cit>  converts mth <dig> into a degradation-resistant form  <cit> .
our results indicate that the mutant mth <dig> proteins  are not degraded, as expected, but accumulate in the nucleus in high-glucose medium .
we also observed that subcellular localization and degradation of mth <dig> are not influenced by the absence of rgt <dig>  a downstream target of the rgt2/snf3-mediated signaling pathway  <cit> .
therefore, it is likely that neither glucose nor the glucose sensors control nuclear export of mth <dig>
glucose-dependent degradation of mth <dig> occurs in the nucleus
to more directly assess whether mth <dig> degradation occurs in the nucleus or cytoplasm, we examined degradation of gfp-mth <dig> proteins engineered to have cytoplasmic localization.
to do so, gfp-mth <dig> was tagged with the nuclear export signal  of the yeast pkiα   <cit> .
to test the functionality of the nes motif, we constructed plasmid expressing gfp-nes-mth <dig> containing a single amino acid substitution within the motif.
the levels and subcellular localization of the resulting fusion proteins were determined by western blot and fluorescence microscopy analysis, respectively .
we observed that gfp-nes-mth <dig> is excluded from the nucleus, as expected , but significantly degraded regardless of the presence of glucose or the glucose sensors .
in contrast, gfp-mth <dig> with a nonfunctional nes motif -mth1) behaves like gfp-mth1; it accumulates in the nucleus in the absence of glucose  but is degraded when glucose is present , providing evidence that the nes motif used is functional.
therefore, collectively, these results suggest that glucose-dependent degradation of mth <dig> occurs in the nucleus.
nuclear degradation of mth <dig> is grr1-dependent
grr <dig> appears to be present in both the nucleus and cytoplasm, but deletion of the putative nls in the amino-terminus of grr <dig>  causes it to be localized to the cytoplasm  <cit> .
previous studies have shown that expression of grr1Δn is sufficient to restore the morphology of, and to mediate degradation of gic <dig> in, the grr <dig> mutant  <cit> , suggesting that grr1Δn is fully functional in the cytoplasm.
to provide more compelling evidence that grr1-dependent degradation of mth <dig> takes place in the nucleus, we examined if gfp-grr1Δn  can mediate mth <dig> degradation .
our results show that gfp-grr1Δn is predominantly localized to the cytoplasm  and able to restore the morphology of the grr <dig> mutant , as reported previously  <cit> .
however, expression of grr1Δn in the grr <dig> mutant does not cause degradation of gfp-mth <dig> , probably due to translocation of grr1Δn from the nucleus to the cytoplasm .
these results suggest that scfgrr1-mediated degradation of mth <dig> occurs in the nucleus, not in the cytoplasm.
direct interaction between yck1/ <dig> and the glucose sensors is not required for glucose-dependent degradation of mth1
next, we tested whether the glucose sensors are coupled with yck1/ <dig> in order to transduce glucose binding into an intracellular signaling cascade that leads to mth <dig> degradation.
toward this aim, we examined whether mth <dig> is degraded when yck1/ <dig> are mislocalized from the plasma membrane.
it has been well described that yck <dig> is targeted to the plasma membrane through palmitoylation of the c-terminal cys-cys sequence by the palmitoyl transferase akr <dig>  <cit> .
confocal microscopy images show that, like gfp-yck <dig>  <cit> , gfp-yck <dig> is associated with the plasma membrane in wild-type cells, but diffused throughout the entire cell when akr <dig> is absent .
importantly, glucose-induced degradation of mth <dig> is not significantly impaired in the absence of akr <dig> .
these results suggest that neither localization of yck1/ <dig> to the plasma membrane nor a direct interaction between yck1/ <dig> and the glucose sensors is required for degradation of mth <dig>
four different regions of mth <dig> are required for glucose-induced degradation of mth1
to identify the regions of mth <dig> that are required for its glucose-induced degradation, we constructed a series of successive internal deletions of 10- <dig> residues covering the entire mth <dig> sequence .
the amino acid positions between  <dig> and  <dig> of mth <dig> were not deleted because this region is not well conserved in the mth <dig> orthologs from other yeast species.
most of the deletion mutants of mth <dig> protein were detectable by western blotting, except two mutants that contain Δ235- <dig>  and Δ298- <dig>  .
our results show that four internal deletions--Δ81- <dig> , Δ118- <dig> , 156- <dig> , and Δ326- <dig> --render mth <dig> resistant to degradation.
these mth <dig> mutant proteins are constitutively nuclear  and significantly inhibit glucose induction of hxt <dig> expression .
proteasomal degradation of proteins is a multi-step process, involving phosphorylation, ubiquitination, and degradation in the 26s proteasome.
thus, it is likely that these four regions are involved in at least one of these steps.
discussion
the yeast glucose sensors convert glucose binding events into an intracellular signal that leads to degradation of mth <dig>  which is known to require activity of the plasma membrane-tethered yck1/ <dig>  <cit> .
because both the glucose sensors and yck1/ <dig> are associated with the plasma membrane, it has been hypothesized that, upon glucose addition, yck1/ <dig> are activated through a direct interaction with the glucose sensors and phosphorylate mth <dig>  triggering its proteasomal degradation.
in this study, we provide several lines of evidence that mth <dig> does not shuttle between the nucleus and cytoplasm and is degraded in the nucleus when glucose is present: 1) mth <dig> is not excluded from the nucleus in response to glucose ; 2) when engineered to be localized to the cytoplasm, mth <dig> is degraded in the cytoplasm regardless of the presence of glucose and the glucose sensors ; 3) mth <dig> is not degraded when grr <dig> is removed from the nucleus .
the majority of yck1/ <dig> targets are plasma membrane proteins.
yck1/ <dig> are responsible for phosphorylating the pest-like ubiquitination-endocytosis signal of the mating pheromone receptors  <cit> , and the uracil permease fur <dig>  <cit> .
these kinases are also known to regulate the activity of the maltose permease mal <dig>  <cit> , the multidrug transporter pdr <dig>  <cit> , and plasma membrane h+-atpase  <cit> .
another important target of yck1/ <dig> is ptr <dig>  a component of the yeast amino-acid signaling pathway.
extracellular amino acids trigger activation of the ssy1-ptr3-ssy <dig> -amino acid signaling pathway that leads to induction of endoproteolytic processing of stp <dig> and stp <dig>  enabling them to enter the nucleus and induce expression of the amino acid permease genes  <cit> .
this processing requires yck1/ <dig>  the novel chymotrypsin like protease ssy <dig>  and scfgrr <dig>  <cit> .
yck1/ <dig> have been shown to phosphorylate the peripheral plasma membrane protein ptr <dig> at thr- <dig>  increasing ssy5c -dependent proteolytic processing of stp <dig> and stp <dig>  <cit> .
mth <dig> is required to form a repression complex with rgt <dig> on the hxt promoters  <cit>  and appears not to contain endogenous nes-like motifs in its sequence .
these observations support the idea that mth <dig> neither shuttles between the nucleus and cytoplasm nor is excluded from the nucleus.
therefore, it is unlikely that the plasma membrane-tethered yck1/ <dig> directly phosphorylate the nuclear-localized mth <dig>
we also provide evidence that mth <dig> degradation is not impaired by the mislocalization of yck1/ <dig> from the plasma membrane, suggesting that direct interaction between the glucose sensors and yck1/ <dig> is not required for mth <dig> degradation .
although yck1/ <dig> have been reported to be necessary for mth <dig> degradation  <cit> , our results suggest that they do not exert their function through the glucose sensors.
it is not known how the glucose binding to the glucose sensors is converted to an intracellular signal that leads to degradation of mth <dig>  in this regard, we surmise that there is a yet unidentified kinase that receives the glucose signal and converts it into an intracellular signal.
in this scenario, the kinase is recruited to the glucose sensors upon glucose addition and phosphorylated by yck1/ <dig> at the plasma membrane.
finally, the kinase is translocated from the cytoplasm into the nucleus and catalyzes phosphorylation of mth <dig>  triggering its degradation by the 26s proteasome.
CONCLUSIONS
glucose-induced, the glucose sensor-regulated mth <dig> degradation occurs in the nucleus and requires neither nuclear export of mth <dig> nor direct interaction between the glucose sensors and yck1/ <dig>  the glucose sensors transmit their signal across the plasma membrane through a yet unidentified signaling component, not through yck1/ <dig>

 in the budding yeast saccharomyces cerevisiae, glucose stimulates its transport across the plasma membrane by inducing expression of several hxt. under glucose-limited conditions, the transcriptional repressor rgt binds to the hxt promoters and recruits general corepressors ssn and tup. rgt does this in conjunction with its corepressor mth which physically interacts with rgt. therefore, it has been proposed that rgt forms a repression complex with mth ssn and tup on the hxt promoters, inhibiting transcription. glucose appears to prevent formation of this protein complex by causing degradation of mth resulting in release of rgt from hxt promoters, thereby inducing expression of hxt. the glucose signal that leads to degradation of mth is generated by the plasma-membrane spanning glucose sensors rgt and snf signal generation is a receptor-mediated process and does not require glucose metabolism. this idea is supported by evidence that dominant mutations exist in the glucose sensor genes that lock the sensor proteins in glucose-bound conformations, generating a constitutive signal. indeed, mth is constitutively degraded in cells expressing the active glucose sensor mutants. subsequent studies have shown that the plasma membrane-tethered casein kinases yck1/ phosphorylate mth triggering its ubiquitination and subsequent degradation. it has also been shown by yeast-two-hybrid assay that mth interacts with the c-terminal tails of the glucose sensors, suggesting that mth is recruited to the plasma membrane. these observations have led to the current view of glucose-induced hxt expression. upon glucose binding, the glucose sensors are converted from inactive to active forms through a conformational change, activating yck1/ in their vicinity. mth recruited by the glucose sensors to the plasma membrane, is phosphorylated by yck1/ and, subsequently, ubiquitinated by scfgrr finally, the ubiquitinated mth is targeted for degradation by the 26s proteasome. however, this hypothesis is mainly based on the following assumptions: mth is excluded from the nucleus upon glucose addition and recruited to the plasma membrane by any means, and yck1/ are activated through a direct interaction with the glucose sensors. in this study, we specifically tested these assumptions and provide evidence that yck1/ do not directly couple to the glucose sensors during transmission of the glucose signal from the plasma membrane to the nucleus. a possible mechanism for how the glucose sensors and yck1/ collaborate to degrade mth is discussed.  subcellular localization of mth is not regulated we have previously reported that gfp-mth is mainly nuclear in glucose-depleted medium and is rapidly degraded upon glucose addition. to gain more insights into glucose-dependent degradation of mth we first examined subcellular localization of gfp-mth in cells grown under conditions where hxt expression is repressed or induced. because glucose also regulates expression of mth, gfp-mth was expressed from the met promoter, which is not regulated by glucose, in a low copy centromeric plasmid. fluorescence microscopy analysis demonstrates that gfp-mth is localized primarily to two specific foci within the nucleus and that ~50% of fluorescence is recovered within 30- seconds after photobleaching. these results suggest that, under glucose-limited conditions, mth predominantly accumulates in the nucleus but does not shuttle between the nucleus and cytoplasm. we also monitored glucose-induced degradation of mth in a time-lapse manner and observed that more than half of gfp-mth is degraded within 5- min after glucose addition, which is consistent with the western blot analysis obtained previously. to determine whether mth is excluded from the nucleus in response to glucose, we determined in which cellular compartment mth is localized when expressed under high glucose conditions. toward this aim, we examined subcellular localization of gfp-mth in the rgt2snf and grr mutants, where glucose-dependent degradation of mth is severely impaired. as shown in figure 2a, the gfp-mth proteins accumulate in the nucleus of the mutants grown in high-glucose medium. to further test a possibility that the glucose sensors regulate nuclear exclusion of mth we determined subcellular localization of a nondegradable form of mth in wild-type cells, where the glucose sensors are active. the dominant htr1- mutation in mth converts mth into a degradation-resistant form. our results indicate that the mutant mth proteins are not degraded, as expected, but accumulate in the nucleus in high-glucose medium. we also observed that subcellular localization and degradation of mth are not influenced by the absence of rgt a downstream target of the rgt2/snf3-mediated signaling pathway. therefore, it is likely that neither glucose nor the glucose sensors control nuclear export of mth glucose-dependent degradation of mth occurs in the nucleus to more directly assess whether mth degradation occurs in the nucleus or cytoplasm, we examined degradation of gfp-mth proteins engineered to have cytoplasmic localization. to do so, gfp-mth was tagged with the nuclear export signal of the yeast pkiα. to test the functionality of the nes motif, we constructed plasmid expressing gfp-nes-mth containing a single amino acid substitution within the motif. the levels and subcellular localization of the resulting fusion proteins were determined by western blot and fluorescence microscopy analysis, respectively. we observed that gfp-nes-mth is excluded from the nucleus, as expected, but significantly degraded regardless of the presence of glucose or the glucose sensors. in contrast, gfp-mth with a nonfunctional nes motif -mth1) behaves like gfp-mth1; it accumulates in the nucleus in the absence of glucose but is degraded when glucose is present, providing evidence that the nes motif used is functional. therefore, collectively, these results suggest that glucose-dependent degradation of mth occurs in the nucleus. nuclear degradation of mth is grr1-dependent grr appears to be present in both the nucleus and cytoplasm, but deletion of the putative nls in the amino-terminus of grr causes it to be localized to the cytoplasm. previous studies have shown that expression of grr1Δn is sufficient to restore the morphology of, and to mediate degradation of gic in, the grr mutant, suggesting that grr1Δn is fully functional in the cytoplasm. to provide more compelling evidence that grr1-dependent degradation of mth takes place in the nucleus, we examined if gfp-grr1Δn can mediate mth degradation. our results show that gfp-grr1Δn is predominantly localized to the cytoplasm and able to restore the morphology of the grr mutant, as reported previously. however, expression of grr1Δn in the grr mutant does not cause degradation of gfp-mth, probably due to translocation of grr1Δn from the nucleus to the cytoplasm. these results suggest that scfgrr1-mediated degradation of mth occurs in the nucleus, not in the cytoplasm. direct interaction between yck1/ and the glucose sensors is not required for glucose-dependent degradation of mth1 next, we tested whether the glucose sensors are coupled with yck1/ in order to transduce glucose binding into an intracellular signaling cascade that leads to mth degradation. toward this aim, we examined whether mth is degraded when yck1/ are mislocalized from the plasma membrane. it has been well described that yck is targeted to the plasma membrane through palmitoylation of the c-terminal cys-cys sequence by the palmitoyl transferase akr. confocal microscopy images show that, like gfp-yck, gfp-yck is associated with the plasma membrane in wild-type cells, but diffused throughout the entire cell when akr is absent. importantly, glucose-induced degradation of mth is not significantly impaired in the absence of akr. these results suggest that neither localization of yck1/ to the plasma membrane nor a direct interaction between yck1/ and the glucose sensors is required for degradation of mth four different regions of mth are required for glucose-induced degradation of mth1 to identify the regions of mth that are required for its glucose-induced degradation, we constructed a series of successive internal deletions of 10- residues covering the entire mth sequence. the amino acid positions between and of mth were not deleted because this region is not well conserved in the mth orthologs from other yeast species. most of the deletion mutants of mth protein were detectable by western blotting, except two mutants that contain Δ235- and Δ298-. our results show that four internal deletions--Δ81-, Δ118-, 156-, and Δ326- --render mth resistant to degradation. these mth mutant proteins are constitutively nuclear and significantly inhibit glucose induction of hxt expression. proteasomal degradation of proteins is a multi-step process, involving phosphorylation, ubiquitination, and degradation in the 26s proteasome. thus, it is likely that these four regions are involved in at least one of these steps. discussion the yeast glucose sensors convert glucose binding events into an intracellular signal that leads to degradation of mth which is known to require activity of the plasma membrane-tethered yck1/. because both the glucose sensors and yck1/ are associated with the plasma membrane, it has been hypothesized that, upon glucose addition, yck1/ are activated through a direct interaction with the glucose sensors and phosphorylate mth triggering its proteasomal degradation. in this study, we provide several lines of evidence that mth does not shuttle between the nucleus and cytoplasm and is degraded in the nucleus when glucose is present: 1) mth is not excluded from the nucleus in response to glucose; 2) when engineered to be localized to the cytoplasm, mth is degraded in the cytoplasm regardless of the presence of glucose and the glucose sensors; 3) mth is not degraded when grr is removed from the nucleus. the majority of yck1/ targets are plasma membrane proteins. yck1/ are responsible for phosphorylating the pest-like ubiquitination-endocytosis signal of the mating pheromone receptors, and the uracil permease fur. these kinases are also known to regulate the activity of the maltose permease mal, the multidrug transporter pdr, and plasma membrane h+-atpase. another important target of yck1/ is ptr a component of the yeast amino-acid signaling pathway. extracellular amino acids trigger activation of the ssy1-ptr3-ssy -amino acid signaling pathway that leads to induction of endoproteolytic processing of stp and stp enabling them to enter the nucleus and induce expression of the amino acid permease genes. this processing requires yck1/ the novel chymotrypsin like protease ssy and scfgrr. yck1/ have been shown to phosphorylate the peripheral plasma membrane protein ptr at thr- increasing ssy5c -dependent proteolytic processing of stp and stp. mth is required to form a repression complex with rgt on the hxt promoters and appears not to contain endogenous nes-like motifs in its sequence. these observations support the idea that mth neither shuttles between the nucleus and cytoplasm nor is excluded from the nucleus. therefore, it is unlikely that the plasma membrane-tethered yck1/ directly phosphorylate the nuclear-localized mth we also provide evidence that mth degradation is not impaired by the mislocalization of yck1/ from the plasma membrane, suggesting that direct interaction between the glucose sensors and yck1/ is not required for mth degradation. although yck1/ have been reported to be necessary for mth degradation, our results suggest that they do not exert their function through the glucose sensors. it is not known how the glucose binding to the glucose sensors is converted to an intracellular signal that leads to degradation of mth in this regard, we surmise that there is a yet unidentified kinase that receives the glucose signal and converts it into an intracellular signal. in this scenario, the kinase is recruited to the glucose sensors upon glucose addition and phosphorylated by yck1/ at the plasma membrane. finally, the kinase is translocated from the cytoplasm into the nucleus and catalyzes phosphorylation of mth triggering its degradation by the 26s proteasome.  glucose-induced, the glucose sensor-regulated mth degradation occurs in the nucleus and requires neither nuclear export of mth nor direct interaction between the glucose sensors and yck1/ the glucose sensors transmit their signal across the plasma membrane through a yet unidentified signaling component, not through yck1/ 
we also show that, like yck yck is localized to the plasma membrane via c-terminal palmitoylation mediated by the palmitoyl transferase akr however, glucose-dependent degradation of mth is not impaired in the absence of akr suggesting that a direct interaction between the glucose sensors and yck1/ is not required for mth degradation. glucose-induced, glucose sensor-regulated degradation of mth occurs in the nucleus and does not require direct interaction of the glucose sensors with yck1/ in yeast, glucose-dependent degradation of the mth protein, a corepressor of the glucose transporter gene repressor rgt is a crucial event enabling expression of several hxt. in addition, removal of grr from the nucleus prevents degradation of gfp-mth these results suggest that glucose-induced, glucose sensor-dependent mth degradation occurs in the nucleus. in this study, we examined whether the glucose sensors directly couple with yck1/ to convert glucose binding into an intracellular signal that leads to the degradation of mth fluorescence microscopy analysis indicates that, under glucose-limited conditions, gfp-mth is localized in the nucleus and does not shuttle between the nucleus and cytoplasm. this event occurs through a signaling pathway that involves the rgt and snf glucose sensors and yeast casein kinase and. when engineered to be localized to the cytoplasm, gfp-mth is degraded regardless of the presence of glucose or the glucose sensors.
 in yeast, glucose-dependent degradation of the mth protein, a corepressor of the glucose transporter gene repressor rgt is a crucial event enabling expression of several hxt. this event occurs through a signaling pathway that involves the rgt and snf glucose sensors and yeast casein kinase and. in this study, we examined whether the glucose sensors directly couple with yck1/ to convert glucose binding into an intracellular signal that leads to the degradation of mth  high levels of glucose induce degradation of mth through the rgt2/snf glucose signaling pathway. fluorescence microscopy analysis indicates that, under glucose-limited conditions, gfp-mth is localized in the nucleus and does not shuttle between the nucleus and cytoplasm. if glucose-induced degradation is prevented due to disruption of the rgt2/snf pathway, gfp-mth accumulates in the nucleus. when engineered to be localized to the cytoplasm, gfp-mth is degraded regardless of the presence of glucose or the glucose sensors. in addition, removal of grr from the nucleus prevents degradation of gfp-mth these results suggest that glucose-induced, glucose sensor-dependent mth degradation occurs in the nucleus. we also show that, like yck yck is localized to the plasma membrane via c-terminal palmitoylation mediated by the palmitoyl transferase akr however, glucose-dependent degradation of mth is not impaired in the absence of akr suggesting that a direct interaction between the glucose sensors and yck1/ is not required for mth degradation.  glucose-induced, glucose sensor-regulated degradation of mth occurs in the nucleus and does not require direct interaction of the glucose sensors with yck1/ 