Glucose Withdrawal Results in a Loss of Polyribosomes
When extracts were prepared from yeast grown and washed for 10 min in standard glucose-containing medium (YPD), they exhibited a normal polyribosome profile (Figure A). Such a profile includes an accumulation of multiple ribosomes bound to single mRNAs (polysomes) toward the bottom of the gradient. When cells grown in YPD were washed for 10 min in YP lacking glucose, however, polysomes were lost and the level of single 80S ribosomes increased (Figure A). This change in polysome profile has been noted previously for many temperature-sensitive strains bearing mutations in key translation initiation factors upon transfer to the restrictive temperature and is indicative of an inhibition of translational initiation (Hartwell and McLaughlin, 1969
; Mathews et al., 1996
). We also found that polysomes could be maintained in cells washed in buffer provided that glucose and amino acids were present (our unpublished results).
Figure 2 Glucose or fructose removal from yeast medium specifically inhibits translation initiation. (A) Polyribosome traces from the wild-type strain (yAS2568). Yeast was grown in YPD and resuspended in YP medium lacking (YP) or containing (YPD) glucose for the (more ...)
Glucose Withdrawal Inhibits Translation Rapidly and Reversibly
The kinetics of polysome redistribution upon glucose withdrawal was investigated. Extracts from cells that had been washed for increasing amounts of time in the absence of glucose were examined on polysome gradients (Figure A). A redistribution of polysomes into the 80S peak was evident after 1 min of glucose depletion, and after 2.5 min this redistribution was almost complete.
The rate of protein synthesis was measured by [35S]methionine incorporation to confirm that the redistribution in polysomes was associated with an inhibition of protein synthesis. Cells were transferred to medium containing or lacking glucose and labeled with [35S]methionine for the next 10 min (Figure D). Yeast cells incorporated [35S]methionine linearly in the presence of glucose. However, upon transfer to medium lacking glucose, there was an almost complete inhibition of further [35S]methionine incorporation. These data, together with the nearly complete loss of polysomes in the absence of glucose, indicate that translation initiation is inhibited by glucose removal.
We also asked how quickly the polysome profile could be restored by the readdition of glucose to inhibited cells (Figure B). As expected, a 10-min wash without glucose (YP 10′) drastically reduced polysome levels. Subsequent addition of glucose-containing medium led to a marked increase in polysomes after 1–2.5 min and a complete restoration of polysomes after 5 min. Therefore, glucose removal causes a sudden inhibition of translation initiation that can be rapidly reversed.
The Polysome Redistribution Is Carbon Source Specific
We next investigated whether the loss of polyribosomes was specific to glucose withdrawal or a general consequence of carbon source depletion. Accordingly, yeast were grown on various carbon sources (glucose [D], fructose [F], galactose [G], maltose [M], sucrose [S], and raffinose [R]) and then washed for 10 min in the presence or absence of the carbon source. Only yeast growing on glucose or fructose before polysome analysis exhibited polysome loss upon carbon source depletion (Figure , B, C, and E). Yeast cells grown on other carbon sources had nearly normal levels of polysomes, even after carbon source removal. These polysomes are indicative of elongating ribosomes, because after galactose withdrawal the yeast continues to incorporate [35S]methionine at approximately half the rate observed when galactose is maintained (Figure D).
We also determined how long it took after galactose removal for a decrease in translation to become evident (Figure E). After 15 min, there was no major effect of galactose removal upon translation, but at times thereafter, a gradual decrease in the level of polysomes was observed. It is interesting to compare the polysome profile 1 min after glucose removal (Figure A) with that produced 40 or 60 min after galactose removal (Figure E); they have a very similar pattern, even though the length of time in the absence of a carbon source was vastly different.
We conclude from these data that the rapid inhibition of translation upon carbon source withdrawal is specific to the removal of either glucose or fructose. This carbon source specificity is identical to that of many previously described glucose-dependent signal transduction pathways (Thevelein, 1994
). We also found that 2-deoxyglucose had a general inhibitory effect on translation and therefore could not be used to determine whether the absence of glucose or a glucose metabolite leads to translational inhibition (our unpublished results).
Translational Inhibition Is Not Caused by a Gross Decrease in mRNA Abundance
One possible explanation for the inhibition of translation initiation upon glucose withdrawal is that it occurs indirectly via a massive decay of mRNA. Therefore, we assessed the abundance of specific mRNAs with a range of half-lives (11 min for PAB1
, 45 min for PGK1
, 30 min for ACT1
, 43 min for CYH2
, and 2.5 min for MFA2
[Herrick et al., 1990
]) by Northern blot analysis after glucose removal (Figure A). In general, the level of mRNAs did not decrease when glucose was withdrawn. In fact, the level of certain mRNAs (e.g., CYH2
) increased when glucose was removed. This effect could be due to the inhibition of translation, which presumably has already taken place and leads to the stabilization of certain mRNAs (Peltz et al., 1992
; Beelman and Parker, 1994
). These results rule out the formal possibility that glucose withdrawal leads to translational inhibition as a result of increased mRNA instability.
Figure 3 Neither mRNA degradation nor transcription contributes to the inhibition of translation upon glucose withdrawal or the recovery after glucose readdition. (A) Northern blot stained with methylene blue to visualize the rRNA levels (upper panel). The same (more ...)
Neither Translational Inhibition nor Recovery Requires New Transcription
temperature-sensitive mutant was used to assess whether an additional round of gene expression is a requirement for the process of translational inhibition. This strain harbors a mutation in an RNA polymerase II gene, and after a shift to the restrictive temperature, it is rapidly inhibited for the transcription of most mRNAs (Nonet et al., 1987
; Herrick et al., 1990
or its isogenic wild-type parent was shifted to 37°C 5 min before a 10-min incubation in medium with or without glucose (Figure B). In wild-type yeast, this temperature shift did not affect the polysome profile in the presence of glucose (Figure Ci), and the removal of glucose still inhibited translation (Figure Cii). For the rpb1-1
mutant, the 15-min 37°C incubation would be expected to lead to a substantial decrease in the quantity of mRNA and therefore polysomes, because the average half-life of a yeast mRNA is ~15 min. Indeed, as shown in Figure Civ, after 15 min at 37°C the rpb1-1
strain contains approximately half the polysomes present in the wild-type parent. However, even allowing for this decrease in polysomes after transcriptional shutdown, there was still a rapid additional decrease in polysome levels when glucose was removed from the medium (Figure Cv).
A similar result was observed for the recovery of translational activity after the readdition of glucose. In these experiments, glucose was removed from yeast for 10 min to allow for the inhibition of translation. Halfway through this period, the yeast cells were shifted to 37°C to completely inhibit mRNA transcription in the rpb1-1 strain. The levels of polysomes were then assayed after readdition of glucose for another 10 min (Figure B). In the wild-type strain (Figure Ciii), the shift in temperature to 37°C did not affect its ability to recover polysomes after readdition of glucose. For the rpb1-1 strain (Figure Cvi), polysomes recovered to almost the same level as that seen when transcription was inhibited in the presence of glucose (Figure Civ). These results indicate that new transcription is not required for glucose withdrawal to inhibit translation or for glucose readdition to stimulate translation.
The Inhibition of Translation by Glucose Withdrawal Does Not Occur through Previously Described Translational Inhibitory Mechanisms
As described in the INTRODUCTION, some prominent examples of signal-mediated translational inhibition have been discovered. First, starvation for amino acids leads to a global inhibition of translation (Tzamarias et al., 1989
). We confirmed this result (Figure A) with the use of a wild-type strain in which the withdrawal of amino acids reduced the level of polysomes. Surprisingly, glucose removal had a more severe effect than even amino acid depletion, because it generated a greater reduction in polysomes. The amino acid starvation pathway of translational inhibition requires the Gcn2p protein kinase, which phosphorylates the α-subunit of eIF2 (a translation initiation factor encoded by the SUI2
gene). The mutation of Ser51 to Ala in Sui2p disrupts this regulatory circuit (Dever et al., 1992
). This mutation was used to investigate whether the translational inhibition by glucose withdrawal occurs by a similar mechanism. Consistent with previous results, the SUI2-S51A
strain was resistant to the inhibition of translation caused by amino acid withdrawal (Figure A). However, the mutant was sensitive to the removal of glucose at the translational level (Figure A). Similar effects were found with a gcn2
null strain (our unpublished results). These results support the conclusion that glucose removal and amino acid starvation elicit translational inhibition via different mechanisms.
Figure 4 Mutants in other translational control pathways are still inhibited for translation upon glucose withdrawal. (A) Polyribosome traces from the wild-type (yAS2570) and SUI2-S51A (yAS2571) strains. Yeast was grown in SCD-Leu medium and washed for 10 min (more ...)
Another signal-mediated translational inhibitory response is induced by inactivation of the TOR signaling pathway (Barbet et al., 1996
). For example, rapamycin or nutrient starvation inhibits the TOR proteins, which are normally capable of phosphorylating Tap42p. A specific mutation, tap42-11
, confers rapamycin resistance (Di Como et al., 1996
). If the inhibition of translation by glucose removal is brought about via inactivation of Tor1p and Tor2p, we reasoned that the tap42-11
mutant would be at least partially resistant to glucose withdrawal. However, the tap42-11
mutant exhibited the same level of translational inhibition on glucose withdrawal as its isogenic parent (Figure B). The kinetics and magnitude of the rapamycin-dependent inhibition of translation also bear little relation to those of the inhibition of translation by glucose depletion (Barbet et al., 1996
; Powers and Walter, 1999
). Previously, even 30 min after rapamycin treatment only a limited decrease in polysomes was observed (Powers and Walter, 1999
), whereas there was almost complete loss of polysomes 2.5 min after glucose depletion in the present study. It has also been suggested that rapamycin leads to the selective degradation of the translation initiation factor eIF4G (Berset et al., 1998
). When glucose was withdrawn from yeast cells, we found no evidence that eIF4G was degraded (our unpublished results). These results suggest that the inhibition of translation by glucose removal is unlikely to result from the inactivation of the TOR pathway.
Specific Glucose Repression Mutants Exhibit Resistance to Translational Inhibition by Glucose Withdrawal
Growth in the presence of glucose represses the expression of a wide variety of genes whose products are involved in the use of alternative carbon sources and oxidative metabolism (Gancedo, 1998
). This process involves the signal transduction pathway summarized in Figure C. One of the most significant changes that occurs in yeast cells depleted of glucose is the activation of the glucose derepression pathway. The absence of glucose generates an active Snf1p protein kinase complex that inhibits the activity of several transcriptional repressors. This leads to the derepression of many glucose-repressed genes. We analyzed a series of mutants in the glucose repression/derepression pathway to test whether this pathway is involved in the inhibition of translation upon glucose removal. These mutants have been isolated and characterized during many years of study of this pathway (Entian, 1980
; Celenza and Carlson, 1986
; Niederacher and Entian, 1987
; Schüller and Entian, 1987
; Vallier and Carlson, 1994
; Tu and Carlson, 1994
; Devit et al., 1997
; Sherwood and Carlson, 1997
A number of mutants in the glucose repression pathway that give constitutive derepression were identified as translationally resistant to the removal of glucose. For example, as shown in Figure and Table , reg1Δ, hxk2Δ, and ssn6-Δ6 were resistant to translational inhibition on glucose removal. However, other glucose repression mutations, such as glc7-T152K, mig1Δ, gsf1-1, and gsf2-Δ1, did not give resistance to the translational inhibition by glucose withdrawal, even though these mutants are derepressed.
Figure 5 Polyribosome analyses for a selection of strains used in this study. Examples of polyribosome traces generated with the use of the wild-type strain (yAS2572 [FY250]) and various mutant strains (yAS2576 [reg1Δ], (more ...)
A summary of the sensitivity/resistance of various mutant yeast strains to glucose removal at the translational level
Reg1p is a regulatory subunit of the Glc7p protein phosphatase 1. It is thought to directly maintain Snf1p in an inactive state via its dephosphorylation (Tu and Carlson, 1995
; Ludin et al., 1998
). Therefore, it was unexpected that a reg1
mutant was resistant to glucose removal but the glc7-T152K
mutant (which has been shown to be defective in Reg1p binding) was not. To further examine this discrepancy, we analyzed a series of strains containing mutations in the GLC7
gene (see Ramaswamy et al.
 for a full listing of the mutants used). One of these mutations, glc7-Q48K
, conferred mild resistance to the inhibition of translation by glucose withdrawal by both the polysome and [35
S]methionine-labeling assays (Table ). This mutant also exhibited constitutive derepression, as judged by its growth on sucrose in the presence of 2-deoxyglucose. Interestingly, the Gln48 residue lies in a similar region of the modeled structure of PP1A (based on the rabbit protein phosphatase type 1) as Thr152 and many other residues involved in glucose repression (see Baker et al., 1997
). The identification of a glc7
mutation that confers resistance to glucose withdrawal is consistent with current models of Reg1p action (Ludin et al., 1998
; Alms et al., 1999
). As such, the resistance of the reg1Δ
mutant would be via the inactivation of Glc7p toward specific substrates.
It has been shown that deletion of Hxk2p hexokinase, but not Glk1p and Hxk1p (which are also responsible for phosphorylating glucose to give glucose-6-phosphate), leads to constitutive derepression (Entian, 1980
). This result has been interpreted to indicate that further metabolism of glucose to glucose-6-phosphate is not the primary signaling event for glucose repression. We tested a mutant strain deleted for both hxk1
and found that protein synthesis was still inhibited upon glucose removal (Table ). This finding shows that resistance to the inhibition of translation follows the same hexokinase specificity as the glucose repression pathway (i.e., only hxk2Δ
has a phenotype). It was also suggested recently that Reg1p/Glc7p can dephosphorylate Hxk2p and that this event is involved in glucose repression (Randez-Gil et al., 1998
; Alms et al., 1999
). Therefore, we tested a strain containing a mutation at the site of Hxk2p phosphorylation (Ser15 to Ala15) that has been reported to be derepressed (Randez-Gil et al., 1998
). This mutation did not lead to resistance to glucose withdrawal at the translational level (Table ). We conclude from this result that maintenance of Hxk2 Ser15 phosphorylation in a reg1Δ
strain is not the reason why cells are resistant to the translational inhibition induced by glucose removal.
As only a subset of glucose repression mutants exhibited resistance to translational inhibition by glucose withdrawal, it seemed possible that these mutations either had common effects outside glucose repression or generated a higher level of derepression than the mutants that had no effect. If the glucose repression pathway is involved in the resistance phenotype, then a SNF1
null mutation in the reg1Δ
mutant (in which constitutively active Snf1p is normally maintained [Jiang and Carlson, 1996
; Ludin et al., 1998
]) would be expected to reestablish sensitivity to translational inhibition on glucose removal. As shown in Figure and Table , translation was inhibited in both the hxk2Δ snf1Δ
and reg1Δ snf1Δ
mutants upon glucose removal to the same extent as in the wild type (FY250). This result demonstrates that the constitutive Snf1p activity associated with the reg1Δ
mutants is required for their resistance to glucose removal at the translational level. In combination with the involvement of proteins throughout the repression pathway (from Hxk2p at the cell membrane to the transcriptional repressor Ssn6p), this suggests that derepression of the glucose repression pathway leads to the observed resistance phenotype. Thus, it seems likely that the level of constitutive derepression in a mutant is critical in determining whether it is resistant or sensitive to glucose removal at the translational level.
Our observation that a transcriptional repression mutant (ssn6Δ) is resistant to the translational inhibition upon glucose removal is difficult to integrate with the finding that new transcription is not required for the inhibition of translation or its recovery after glucose readdition (Figure C). This contradiction suggests that mutants in the glucose repression pathway could be acting in an indirect way to confer resistance to the effects of glucose withdrawal upon translation (see DISCUSSION).
The snf3Δ rgt2Δ Mutant Is Resistant to the Translational Inhibition upon Glucose Withdrawal
A different pathway has been described in which the presence of glucose is sensed by two integral membrane proteins, Rgt2p and Snf3p. These are high- and low-affinity glucose sensors, respectively, and they signal the presence of glucose to allow for the transcription of the HXT
genes (Figure D) (Özcan et al., 1996a
). This pathway involves Grr1p, which forms part of the SCF ubiquitin-conjugating complex that is involved in protein degradation. It has been proposed that this complex targets the transcriptional repressor Rgt1p for degradation and thus allows HXT
gene expression (Li and Johnson, 1997
). We chose to investigate whether these glucose sensors were responsible for signaling changes in glucose levels in the medium to the translational apparatus.
The constitutive activation of this pathway via the deletion of rgt1
or the presence of dominant active SNF3-1
mutations did not prevent the rapid inhibition of translation induced by glucose depletion (Table ). Inactivation of the pathway via deletion of grr1
, or rgt2
also did not prevent the response to glucose. However, the snf3Δ rgt2Δ
double mutant did exhibit resistance (Figure and Table ). This strain, however, exhibits complex phenotypes. For instance, the strain grows poorly on glucose, perhaps explaining the lower level of polysomes even in the presence of glucose. In addition, like the reg1Δ
mutants, this strain is constitutively derepressed. One explanation for this is that this mutant reacts as if glucose were limiting as a result of the decreased glucose transport caused by the constitutive transcriptional repression of the HXT
genes (Schmidt et al., 1999
). Interestingly, the grr1Δ
mutant (which is also constitutively derepressed for a similar reason [Flick and Johnston, 1991
; Vallier et al., 1994
]) is sensitive to glucose removal at the translational level. Because Grr1p lies downstream of the Rgt2p and Snf3p glucose sensors, it seems unlikely that the resistance observed in the snf3Δ rgt2Δ
mutant is a consequence of its derepression phenotype. However, to test this directly, we deleted SNF1
in the snf3Δ rgt2Δ
background to abolish derepression (Schmidt et al., 1999
). In the snf3Δ rgt2Δ
mutant, deletion of SNF1
does not reestablish the inhibition of translation upon glucose removal (Figure and Table ). This result demonstrates that, in contrast to the reg1Δ
mutants, the snf3Δ rgt2Δ
mutant is not resistant as a consequence of constitutively active Snf1p. This opens the possibility that the Snf3p and Rgt2p glucose sensors may be directly involved in signaling the levels of glucose in the medium to the translational machinery.
Low Levels of cAPK Activity Prevent the Inhibition of Translation on Glucose Removal
Other signal transduction pathways in which glucose has been implicated as a signaling nutrient converge on the cAPK (Figure E). Two tpkw
mutants (tpk1w1 tpk2Δ tpk3Δ
and tpk1Δ tpk2w1 tpk3Δ
), in which two of the genes encoding cAPK were deleted and the third was severely impaired, were resistant to the effects of glucose removal at the translational level (Figure and Table ). These mutants have an undetectable level of cAPK activity and therefore represent an extreme inhibition (Cameron et al., 1988
). Interestingly, another of these tpkw
mutants (tpk1Δ tpk2Δ tpk3w1
) was not resistant to the inhibition of translation caused by glucose depletion (Table ). This mutant was also previously shown to have the highest cAPK activity of these three mutants (Nikawa et al., 1987
In low-cAPK mutants such as the tpkw
mutants, the transcription factors Msn2p and Msn4p are constitutively nuclear, and as a result, the stress-controlled genes are constitutively expressed. Interestingly, the removal of glucose has been shown to induce the stress response, and this is mediated via the relocalization of the Msn2p and Msn4p transcription factors from the cytoplasm to the nucleus. This relocalization is thought to require inactivation of cAPK upon glucose removal (Görner et al., 1998
). We examined whether the resistance to glucose removal at the translational level in low-cAPK mutants is a result of the constitutive activity of the stress response pathway in these mutants. We used a strain that is deleted for MSN2
as well as all of the cAPK genes (TPK1
, and TPK3
). In this strain, the deletion of the Msn2p and Msn4p transcription factors abolishes the constitutive stress response that is characteristic of low-cAPK mutants (Smith et al., 1998
). However, this strain is still largely resistant to the translational inhibition caused by glucose removal (Figure and Table ). In addition, when glucose is removed from a wild-type strain in which the stress response genes are preinduced (via heat, salt, or ethanol stress), translation is still largely inhibited (our unpublished results). Therefore, the activity of the stress response pathway does not explain the translational resistance to glucose withdrawal that is evident in low-cAPK mutants.
In [35S]methionine incorporation assays, the rate of incorporation in the presence of glucose for the tpkw mutants was significantly lower than that for the wild-type parent (our unpublished results). This correlates with their slow growth (Table ). However, it was clear that in the absence of glucose these mutants incorporated [35S]methionine, whereas the wild-type strain showed impaired incorporation (Table ). In addition, the tpk1Δ tpk2Δ tpk3Δ msn2Δ msn4Δ strain has only a slightly impaired growth rate compared with wild-type strains and yet is still resistant to glucose withdrawal at the translational level (Table ).
Two pathways have been described that regulate cAPK activity in response to glucose. These are the RAS/cAMP pathway and the fermentable growth medium–induced pathway. The RAS/cAMP pathway is involved in the metabolic switch that occurs when cells are transferred onto glucose (Jiang et al., 1998
), and the fermentable growth medium–induced pathway is involved in the maintenance of high cAPK activity during growth on glucose (Thevelein, 1991
; Crauwels et al., 1997
). Neither activating nor inhibitory mutations in various components of these pathways (Bcy1Δ
, and sch9Δ
) were found to have a significant effect on the level of translational inhibition caused by glucose removal (Table ). It seems likely that the inhibitory mutations in these pathways do not reduce the cAPK activity sufficiently to allow resistance to glucose removal at the translational level.
An Analysis of ATP Levels after Removal of the Carbon Source
Our identification of yeast mutants resistant to the inhibitory effects of glucose withdrawal on translation supports the hypothesis that this translational inhibition is a signal-mediated event. However, our interpretations are complicated by the fact that glucose is also the main energy source of the cells (Thevelein, 1994
). Therefore, an alternative possibility is that upon glucose removal the energy levels in the cell decrease to such an extent that translation can no longer proceed, because translation is one of the major energy-consuming processes in the cell.
To investigate whether energy depletion explains the translational inhibition, we measured ATP levels with the use of a luciferin/luciferase assay. This assay has the advantage that it can be used to assess the ATP levels quickly with a minimum of cellular manipulations (Simpson and Hammond, 1989
). Figure shows the results of time-course experiments whereby yeast was incubated in the presence or absence of glucose, with the amount of ATP expressed as a percentage of the starting ATP level of the culture. The precise starting ATP levels for all the strains tested were remarkably similar, falling in a range between 250 and 350 amol/cell. This correlates well with previous measures of the intracellular ATP level in yeast (Simpson and Hammond, 1989
Figure 6 Measurement of intracellular ATP levels after glucose withdrawal from the growth medium. The percentage of the starting intracellular ATP level for 30 min after transfer of yeast to medium with glucose (□) or without glucose (♦) is plotted. (more ...)
We found that after glucose removal, wild-type strains (FY250 [yAS2572], SP1 [yAS962], and W3031A [yAS2568]) exhibited a rapid (after 1 min or less) decrease in ATP level to ~20% of the starting value (Figure ). This is consistent with the results of Wilson et al. (1996)
, who showed that the AMP/ATP ratio increases immediately after glucose removal. A decrease to ~70% of the starting level of ATP also was observed in the presence of glucose. It is not clear why this decrease occurred, but it may relate to the cellular manipulations during the experiment. The decrease in ATP level that was observed upon glucose removal coincides with the translational inhibition described above. However, there was a variation in the rate of recovery of the ATP level (Figure , compare FY250, SP1, and W3031A). In some wild-type strains, the ATP level quickly recovered to >70% of that found before glucose withdrawal after 10 min (Figure , W3031A) (our unpublished results). Only in one wild-type background, FY250, did the low ATP level persist in the absence of glucose (Figure ). The recovery of ATP levels is consistent with the results of Ditzelmüller et al. (1983)
, who found that ATP levels remained remarkably constant under a range of different growth conditions. Even though there was a wide variation between strain backgrounds in the level of ATP reduction and recovery after glucose removal, all of these strains were equally inhibited at the translational level. Therefore, it is possible that the decrease in ATP is either required for or generated by the inhibition of translation. However, it does not seem likely that reduced energy levels account directly for the translational inhibition upon glucose removal, because ATP levels recover rapidly to near normal levels in some inhibited strains.
The removal of galactose from a wild-type culture grown on galactose (W3031A gal) or the removal of glucose from the reg1Δ, hxk2Δ, and tpk1w1 tpk2Δ tpk3Δ mutants growing on glucose did not result in an initial decrease in ATP. As under these conditions strains are resistant to the translational inhibition upon carbon source removal, this might suggest that the initial decrease in ATP is involved in the translational inhibition. However, some of the glucose repression mutants that were not resistant to the translation inhibition exhibited no decrease in ATP after glucose removal. The most convincing example of this involves the grr1Δ strain, which exhibited a complete inhibition of translation on glucose removal (Figure ) but did not show a significant decrease in ATP when glucose was withdrawn (Figure ). This result supports the conclusion that the decrease in ATP resulting from glucose removal is not required to generate the inhibition of translation.
Since GTP hydrolysis is also required for both the initiation and the elongation steps of translation, the levels of GTP could affect the translational capacity of the cell after glucose removal. We do not favor this hypothesis for several reasons. First, our identification of yeast mutants that have no connection with the regulation of GTP levels and yet are resistant to the inhibitory effects of glucose withdrawal on translation does not support this hypothesis. In addition, the “runoff” of polysomes observed after glucose removal requires that translational elongation continues while initiation is inhibited (Mathews et al., 1996
). Translational elongation of a polypeptide requires at least two GTP molecules per amino acid added, whereas initiation requires only one or two GTP molecules per polypeptide chain (Merrick and Hershey, 1996
). The fact that translational elongation continues after glucose removal and allows for the runoff of polysomes is highly suggestive that GTP levels are not limiting. Finally, the level of GTP is intrinsically linked to the level of ATP, because GDP is converted to GTP by the enzyme nucleoside diphosphate kinase, which uses ATP as the phosphate donor (Parks and Argawal, 1973
). As with ATP levels, the levels of GTP have been shown to remain relatively constant as long as cell viability is maintained (Ditzelmüller et al., 1983
). As the ATP levels do not correlate with the inhibition of translation upon glucose removal, it seems likely that GTP levels will also fail to correlate.