**3. Glucose-accelerated protein degradation in** *Candida albicans*

The previous section examined the impact of glucose on Pck1 and Icl1 expression and the assimilation of alternative carbon sources such as lactic and oleic acid in *C. albicans.* So far, the author's data confirmed that transcript profiling results were described by Yin *et al*. [54] and Rodaki *et al*. [55]. Numerous *S. cerevisiae* transcripts, including those encoding gluconeogenic enzymes (*FBP1* and *PCK1*), are repressed by glucose [54]. Similarly, 180 genes in *C. albicans*, including gluconeogenic and glyoxylate cycle enzymes were, down-regulated, even in response to very low concentrations of glucose (0.01%) [55]. The results presented in the previous section confirmed that the *ScICL1* and *ScPCK1* genes in *S. cerevisiae* and the *CaICL1* and *CaPCK1* genes in *C. albicans* are exquisitely sensitive to glucose.

While *S. cerevisiae* and *C. albicans* displayed similar responses at the transcriptional level, they diverged significantly at the post-transcriptional and metabolic levels. The *C. albicans* Icl1 and Pck1 enzymes were expressed during growth on lactate and oleic acid and were not destabi‐ lised by the addition of 2% glucose. In contrast, their orthologues in *S. cerevisiae* were rapidly destabilised by glucose. Consequently, this appeared to affect carbon assimilation, allowing *C. albicans* to continue to assimilate alternative carbon sources even after exposure to glucose. In contrast, following glucose addition to *S. cerevisiae* cells, Icl1 and Pck1 were degraded and the cells stopped assimilating lactic acid or oleic acid. These new findings showed that there are fundamental differences in the regulation of carbon assimilation in *C. albicans* compared to *S. cerevisiae* [63]. The next step is to examine the basis for these differences and in particular, possible differences in glucose-accelerated protein degradation between *C. albicans* and *S. cerevisiae.* The focus of these studies was on Icl1.

Entian and Schüller [46] reported the genetic characterisation of *C. albicans* gluconeogenic and glyoxylate cycle genes. The *C. albicans FBP1, PCK1, MLS1* and *ICL1* genes were all isolated by functional complementation of the corresponding *S. cerevisiae* deletion mutants. Remarkably, the regulation of the heterologously expressed *C. albicans* gluconeogenic and glyoxylate cycle genes in *S. cerevisiae* was similar to that of their *S. cerevisiae* orthologues. Therefore, in this project we expressed *C. albicans ICL1* in *S. cerevisiae* and tested whether CaIcl1 is destabilised by glucose in *S. cerevisiae.* The other aims of this section are to test whether *C. albicans* has retained the ability to destabilise target proteins in response to glucose and to examine the signals and mechanisms that trigger glucose-mediated destabilisation of target proteins in *C. albicans.*

To test whether *C. albicans* is able to degrade proteins in response to glucose, the *S. cerevisiae ICL1* gene was expressed in *C. albicans.* To achieve this, one *C. albicans ICL1* allele was replaced with a tagged *S. cerevisiae ICL1* ORF. The *ScICL1* locus in *S. cerevisiae* was first tagged using primers with Myc3-URA3, and the genomic DNA from this tagged was PCR amplified using primers to create the *CaICL1p-ScICL1-MYC3-URA3* cassette [63]. This cassette was transformed into the *CaICL1* locus in *C. albicans ICL1/ICL1*. Before going further, it was necessary to test whether the *ScICL1-MYC3-URA3* sequence was integrated accurately into the *CaICL1* genomic locus. Three primer pairs were designed to amplify overlapping fragments of the *CaICL1p-ScICL1-MYC3-URA3* locus based on the *in silico* sequence [63]. PCR amplification using these primers yielded the desired bands, establishing that the newly created strain *ScICL1-MYC3- URA3* sequence had integrated correctly into the *CaICL1* locus in the *C. albicans* genome [63].

Then Western blots were performed to test whether the ScIcl1-Myc3 protein was detectable in these *C. albicans* transformants. The two positive clones were grown to stationary phase overnight on an alternative carbon source in the absence of glucose. Controls were included to confirm expression of the tagged ScIcl1 in *C. albicans.* In both new strains, an Icl1 band of the predicted size (62 kDa) was observed, which was the right size compared to the controls.

To further ensure the correct replacement of the *C. albicans ICL1* ORF with the tagged *S. cerevisiae ICL1* ORF, the functionality of this *ScICL1* ORF was tested in *C. albicans.* The *ScICL1- MYC3-URA3* cassette was amplified from genomic DNA and transformed into *C. albicans ICL1/ icl1* cells selecting heterozygote for uridine prototrophs. Once again, correct insertion of the *ScICL1-MYC3-URA3* sequence was confirmed by diagnostic PCR using the three primer pairs as before and also tested by another three primer pairs to confirm the construction of the *C. albicans ScICL1-MYC3-URA3/icl1* strain [63].

Western blots were performed to confirm the expression of the Myc-tagged *ScICL1* ORF *S. cerevisiae* in *C. albicans ScICL1-MYC3-URA3/icl1* background. Two positive clones were grown to stationary phase on lactate-containing medium and protein subjected to Western blotting. This showed the expression of the tagged ScIcl1 of about 62 kDa in *C. albicans.* As expected the ScIcl1 protein was not expressed during growth on glucose because it was expressed from the endogenous *CaICL1* promoter that is glucose repressed [63].

lised by the addition of 2% glucose. In contrast, their orthologues in *S. cerevisiae* were rapidly destabilised by glucose. Consequently, this appeared to affect carbon assimilation, allowing *C. albicans* to continue to assimilate alternative carbon sources even after exposure to glucose. In contrast, following glucose addition to *S. cerevisiae* cells, Icl1 and Pck1 were degraded and the cells stopped assimilating lactic acid or oleic acid. These new findings showed that there are fundamental differences in the regulation of carbon assimilation in *C. albicans* compared to *S. cerevisiae* [63]. The next step is to examine the basis for these differences and in particular, possible differences in glucose-accelerated protein degradation between *C. albicans* and *S.*

Entian and Schüller [46] reported the genetic characterisation of *C. albicans* gluconeogenic and glyoxylate cycle genes. The *C. albicans FBP1, PCK1, MLS1* and *ICL1* genes were all isolated by functional complementation of the corresponding *S. cerevisiae* deletion mutants. Remarkably, the regulation of the heterologously expressed *C. albicans* gluconeogenic and glyoxylate cycle genes in *S. cerevisiae* was similar to that of their *S. cerevisiae* orthologues. Therefore, in this project we expressed *C. albicans ICL1* in *S. cerevisiae* and tested whether CaIcl1 is destabilised by glucose in *S. cerevisiae.* The other aims of this section are to test whether *C. albicans* has retained the ability to destabilise target proteins in response to glucose and to examine the signals and mechanisms that trigger glucose-mediated destabilisation of target proteins in *C.*

To test whether *C. albicans* is able to degrade proteins in response to glucose, the *S. cerevisiae ICL1* gene was expressed in *C. albicans.* To achieve this, one *C. albicans ICL1* allele was replaced with a tagged *S. cerevisiae ICL1* ORF. The *ScICL1* locus in *S. cerevisiae* was first tagged using primers with Myc3-URA3, and the genomic DNA from this tagged was PCR amplified using primers to create the *CaICL1p-ScICL1-MYC3-URA3* cassette [63]. This cassette was transformed into the *CaICL1* locus in *C. albicans ICL1/ICL1*. Before going further, it was necessary to test whether the *ScICL1-MYC3-URA3* sequence was integrated accurately into the *CaICL1* genomic locus. Three primer pairs were designed to amplify overlapping fragments of the *CaICL1p-ScICL1-MYC3-URA3* locus based on the *in silico* sequence [63]. PCR amplification using these primers yielded the desired bands, establishing that the newly created strain *ScICL1-MYC3- URA3* sequence had integrated correctly into the *CaICL1* locus in the *C. albicans* genome [63]. Then Western blots were performed to test whether the ScIcl1-Myc3 protein was detectable in these *C. albicans* transformants. The two positive clones were grown to stationary phase overnight on an alternative carbon source in the absence of glucose. Controls were included to confirm expression of the tagged ScIcl1 in *C. albicans.* In both new strains, an Icl1 band of the predicted size (62 kDa) was observed, which was the right size compared to the controls. To further ensure the correct replacement of the *C. albicans ICL1* ORF with the tagged *S. cerevisiae ICL1* ORF, the functionality of this *ScICL1* ORF was tested in *C. albicans.* The *ScICL1- MYC3-URA3* cassette was amplified from genomic DNA and transformed into *C. albicans ICL1/ icl1* cells selecting heterozygote for uridine prototrophs. Once again, correct insertion of the *ScICL1-MYC3-URA3* sequence was confirmed by diagnostic PCR using the three primer pairs as before and also tested by another three primer pairs to confirm the construction of the *C.*

*cerevisiae.* The focus of these studies was on Icl1.

*albicans ScICL1-MYC3-URA3/icl1* strain [63].

*albicans.*

210 Genital Infections and Infertility

The phenotype of this *C. albicans ScICL1-MYC3-URA3/icl1* mutant was then tested by growing in different carbon sources: glucose, fructose, lactate, oleic acid, pyruvate and acetate. The mutants were compared with control *C. albicans ICL1/ICL1, ICl1/icl1* and *icl1/icl1* strains. The presence of the *ScICL1* gene in a *C. albicans icl1* background was sufficient to restore growth on lactate, oleic acid, pyruvate and acetate. This growth was comparable to the positive *ICL1/ ICL1* control strain and contrasted with the negative *icl1/icl1* control, which was only able to grow on glucose and fructose. This indicated that the tagged *ScICL1* was functional in *C. albicans* [63].

Having confirmed the genotype, expression and functionality of the *ScICL1* ORF in *C. albicans*, the next step was to test the effects of glucose on the levels of the ScIcl1 protein when expressed in *C. albicans*. The *C. albicans ScICL1-MYC3-URA3/*/icl1 strain was first grown on alternative carbon sources (lactate or oleic acid), and then glucose was added to a final concentration of 2%. Samples were then taken at regular intervals, proteins extracted, and ScIcl1-Myc3 levels examined by Western blotting. Control cultures to which no glucose was added, were also examined. This showed that ScIcl1 levels remained high in *C. albicans* cells grown on the alternative carbon sources. However, following the addition of 2% glucose to *C. albicans* cells, ScIcl1 was degraded. This indicates that *C. albicans* has retained the capacity to destabilise target proteins in response to glucose [63].

*C. albicans* is clearly capable of degrading target proteins following exposure to glucose. Therefore, why is CaIcl1 not degraded following glucose addition? Has CaIcl1 lost the specific signal that would target it for glucose-accelerated degradation? To test this, the CaIcl1 protein was expressed in *S. cerevisiae.*

To achieve this, the *S. cerevisiae ICL1* ORF was replaced with a Myc3-tagged *C. albicans ICL1* ORF. The *C. albicans ICL1-MYC3-URA3* was PCR amplified using the primers and transformed into *S. cerevisiae* strain (Ura- , Leu- ) selecting for uracil prototrophs. Correct integration of the *CaICL1-MYC3-URA3* cassette into the *ScICL1* locus was confirmed by diagnostic PCR using three primer pairs. In this way, a new *S. cerevisiae* strain was constructed containing a *ScICL1p-CaICL1-MYC3-URA3* mutation [63].

Western blots were performed to test whether the CaIcl1-Myc3 protein of the predicted 61 kDa size was expressed in *S. cerevisiae*. Cells were grown on alternative carbon sources in the absence of glucose and compared to positive and negative controls. These Western blots confirmed that CaIcl1 61 kDa was expressed in *S. cerevisiae* during growth on lactate or oleic acid and that CaIcl1-Myc3 was in the correct size compared to the positive control in which CaIcl1-Myc3 was expressed in *C. albicans.* Interestingly, CaIcl1-Myc3 was expressed in *S.* *cerevisiae* cells grown on lactate or oleic acid, but not in cells grown on glucose. This was to be expected when the *CaICL1-MYC3* ORF was expressed from the *ScICL1* promoter [63].

Having confirmed the genotype of the *S. cerevisiae* mutant expressing the *C. albicans ICL1* ORF, the next step was to test the effects of glucose on the CaIcl1-Myc3 protein levels in *S. cerevi‐ siae*. The *S. cerevisiae* strain was grown on lactate or oleic acid, and then glucose was added to a final concentration of 2%. Cells were harvested at various time periods thereafter, protein extracts prepared, and CaIcl1-Myc3 protein levels measured by Western blotting. The results showed that CaIcl1-Myc3 remained at high levels in *S. cerevisiae* during growth on the alter‐ native carbon sources. Even upon the addition of 2% glucose, the levels of CaIcl1-Myc3 remained high in *S. cerevisiae.* Minimal decay of the CaIcl1-Myc3 protein was observed even 4 hours after glucose addition. These data suggest that the *C. albicans* Icl1 protein has lost the signals that trigger destabilisation in response to glucose [63].

The above work suggested that *C. albicans* has retained the ability to degrade target proteins in response to glucose, but that CaIcl1 has lost the specific signal(s) that trigger this glucoseaccelerated protein degradation. What is the nature of this degradation signal that has been lost by CaIcl1?

Ubiquitination is known to play a role in the glucose-accelerated degradation of gluconeogenic enzymes in *S. cerevisiae* [57]. Previously, Entian and Barnett [45] demonstrated that Ubc8 functions in the catabolite degradation of fructose-1,6-bisphosphatase in *S. cerevisiae.* Earlier, Johnson *et al.* [69] showed that ubiquitin acts as a degradation signal in *S. cerevisiae.* Therefore, consensus ubiquitination target sites were examined in CaIcl1 and ScIcl1 using Ubpred (Predictor of protein ubiquitination site, from htt/www.ubpred.org/index.html) [70 - 73].

Based on this bioinformatic comparison, the ScIcl1 sequence contains strong consensus ubiquitination sites at amino acids 158 and 551, but there is a lack of high confidence ubiqui‐ tination targets in CaIcl1. This prediction was based on high level of confidence which is described in Ubpred system containing score range 0.84 ≤ s ≤ 1.00, 0.197 for sensitivity and 0.989 for specificity. These included the hydrophobic nature of the ubiquitination target site for the high confidence prediction (TEDQFKENGVKK), which is contrast to the low- and medium- confidence sites that contain acidic and basic residues in the putative ubiquitination site (NGVKK; FNWPKAMSVD) [70 - 73]. Therefore, the presence of consensus ubiquitination sites in these proteins correlated with glucose-accelerated degradation.

Hence, the next step is to test whether ScIcl1 decay rates in *C. albicans* are affected by inacti‐ vation of polyubiquitin (*UBI4*).

To achieve this, the experimental goal was to introduce the Myc3-tagged *S. cerevisiae ICL1* ORF into a *C. albicans ubi4/ubi4* mutant [74]. The *ScICL1-MYC3-URA3* cassette was PCR amplified using primers and transformed into *C. albicans ubi4/ubi4* cells selecting for uridine prototrophs. The correct insertion of the *ScICL1-MYC3-URA3* into the *CaICL1* locus was confirmed by diagnostic PCR with the same primer pairs as before. These amplified the conjoined sequence of the *CaICL1* promoter and the *ScICL1* ORF. The successful and accurate insertion of the *ScICL1-MYC3-URA3* cassette into *C. albicans ubi4/ubi4* mutant was confirmed in this way.

Western blotting was then performed to test whether a ScIcl1-Myc3 protein of the predicted size was expected in the *ubi4/ubi4* cells. Cells were grown overnight on the alternative carbon sources (lactate and oleic acid) in the absence of glucose. A new Myc3-containing protein of 62 kD was observed indicating that the ScIcl1-Myc3 protein was expressed on YPL and was the right size [63].

*cerevisiae* cells grown on lactate or oleic acid, but not in cells grown on glucose. This was to be

Having confirmed the genotype of the *S. cerevisiae* mutant expressing the *C. albicans ICL1* ORF, the next step was to test the effects of glucose on the CaIcl1-Myc3 protein levels in *S. cerevi‐ siae*. The *S. cerevisiae* strain was grown on lactate or oleic acid, and then glucose was added to a final concentration of 2%. Cells were harvested at various time periods thereafter, protein extracts prepared, and CaIcl1-Myc3 protein levels measured by Western blotting. The results showed that CaIcl1-Myc3 remained at high levels in *S. cerevisiae* during growth on the alter‐ native carbon sources. Even upon the addition of 2% glucose, the levels of CaIcl1-Myc3 remained high in *S. cerevisiae.* Minimal decay of the CaIcl1-Myc3 protein was observed even 4 hours after glucose addition. These data suggest that the *C. albicans* Icl1 protein has lost the

The above work suggested that *C. albicans* has retained the ability to degrade target proteins in response to glucose, but that CaIcl1 has lost the specific signal(s) that trigger this glucoseaccelerated protein degradation. What is the nature of this degradation signal that has been

Ubiquitination is known to play a role in the glucose-accelerated degradation of gluconeogenic enzymes in *S. cerevisiae* [57]. Previously, Entian and Barnett [45] demonstrated that Ubc8 functions in the catabolite degradation of fructose-1,6-bisphosphatase in *S. cerevisiae.* Earlier, Johnson *et al.* [69] showed that ubiquitin acts as a degradation signal in *S. cerevisiae.* Therefore, consensus ubiquitination target sites were examined in CaIcl1 and ScIcl1 using Ubpred (Predictor of protein ubiquitination site, from htt/www.ubpred.org/index.html) [70 - 73].

Based on this bioinformatic comparison, the ScIcl1 sequence contains strong consensus ubiquitination sites at amino acids 158 and 551, but there is a lack of high confidence ubiqui‐ tination targets in CaIcl1. This prediction was based on high level of confidence which is described in Ubpred system containing score range 0.84 ≤ s ≤ 1.00, 0.197 for sensitivity and 0.989 for specificity. These included the hydrophobic nature of the ubiquitination target site for the high confidence prediction (TEDQFKENGVKK), which is contrast to the low- and medium- confidence sites that contain acidic and basic residues in the putative ubiquitination site (NGVKK; FNWPKAMSVD) [70 - 73]. Therefore, the presence of consensus ubiquitination

Hence, the next step is to test whether ScIcl1 decay rates in *C. albicans* are affected by inacti‐

To achieve this, the experimental goal was to introduce the Myc3-tagged *S. cerevisiae ICL1* ORF into a *C. albicans ubi4/ubi4* mutant [74]. The *ScICL1-MYC3-URA3* cassette was PCR amplified using primers and transformed into *C. albicans ubi4/ubi4* cells selecting for uridine prototrophs. The correct insertion of the *ScICL1-MYC3-URA3* into the *CaICL1* locus was confirmed by diagnostic PCR with the same primer pairs as before. These amplified the conjoined sequence of the *CaICL1* promoter and the *ScICL1* ORF. The successful and accurate insertion of the *ScICL1-MYC3-URA3* cassette into *C. albicans ubi4/ubi4* mutant was confirmed in this way.

sites in these proteins correlated with glucose-accelerated degradation.

expected when the *CaICL1-MYC3* ORF was expressed from the *ScICL1* promoter [63].

signals that trigger destabilisation in response to glucose [63].

lost by CaIcl1?

212 Genital Infections and Infertility

vation of polyubiquitin (*UBI4*).

Having confirmed the genotype and the expression of the *ScICL1* ORF in *C. albicans ubi4/ ubi4* cells, the next step was to test the effects of glucose on ScIcl1-Myc3 protein levels. The *ubi4/ ubi4* cells were grown on lactate or oleic acid and then glucose was added to a final concen‐ tration of 2%. Interestingly, ScIcl1-Myc3 cells were more stable in *C. albicans ubi4/ubi4* cells than in wild type *C. albicans* cells after addition of 2% glucose. However, the inactivation of the *UBI4* (polyubiquitination) locus did not completely inhibit the degradation of the ScIcl1-Myc3 protein such that it became as stable as ScIcl1-Myc3 in wild type *C. albicans* cells in the absence of glucose. This might be because residual ubiquitination remains in *ubi4/ubi4* cells thanks to the presence of a second ubiquitin-encoding locus in *C. albicans* (*UBI3*) [74]. Nevertheless, it was concluded that the inactivation of *UBI4* (polyubiquitin) inhibits the glucose-accelerated degradation of ScIcl1 in *C. albicans.* Ubiquitination plays a role in glucose-accelerated protein decay in this fungus.

As stated above, ScIcl1 contains two high confidence putative ubiquitination sites located at residues 551 and 158, whereas CaIcl1 contains no such sites. Therefore, we reasoned that if ubiquitination plays a role in glucose-accelerated protein decay in *C. albicans*, then the addition of a ubiquitination site to CaIcl1 would confer glucose-accelerated degradation upon this protein. Therefore, the next experimental objective is to introduce the carboxyl-terminal ubiquitin site from ScIcl1 (TEDQFKENGVKK) into CaIcl1, together with the Myc3 tag into wild type polyubiquitin containing *C. albicans* cells [63].

To achieve this, a *ScUBI-*site-*MYC3-URA3* cassette was PCR amplified from *S. cerevisiae* genomic DNA and transformed into *C. albicans ICL1/ICL1* cells. To confirm the correct integration of this cassette at the 3´-end of the *CaICL1* ORF in these cells, uridine prototrophic transformants were subjected to diagnostic PCR using the same primer pairs as before and new primer pairs. This PCR amplification yielded the desired bands, establishing that the *ScUBI-MYC3-URA3* was correctly integrated at the *C. albicans ICL1* locus [63].

Having established the genotype of the new strain (*C. albicans ICL1-ScUBI-*site-*MYC3-URA3*), the next step is to confirm the expression of the CaIcl1 protein carrying the carboxyl-terminal ubiquitination site and the Myc*3* tag. Therefore, Western blots were performed to test the presence and size of the tagged protein. Five positive *C. albicans* clones were grown on an alternative carbon source (lactate) in the absence of glucose, protein extracts were made, and Western blots were performed, probing for the Myc epitope. A new Icl1-Myc*<sup>3</sup>* band of about 61 kD was observed, confirming that the Icl1 protein was expressed on YPL and that it had a similar size to the positive control ScIcl1-Myc3 [63].

Having confirmed the genotype of the new strain and the expression of the CaIcl1 protein with the carboxyl-terminal ubiquitination site in *C. albicans* cells, the next step is to test the effects of glucose on the stability of this protein. The new strain was grown on lactic or oleic acid and glucose was added to a final concentration of 2%. Cells were harvested at various time periods thereafter; protein was extracted and these were subjected to Western blotting. Interestingly the CaIcl1-Ubi-Myc3 protein was rapidly degraded following glucose addition to cells grown on lactate or oleic acid. In conclusion, the addition of a ubiquitination site to CaIcl1 accelerates its degradation in response to glucose in *C. albicans* [63].

The above observations strongly suggest that specific proteins can be targeted for degradation in *C. albicans* following exposure to glucose, and these proteins are degraded via ubiquitina‐ tion. If this is the case, it should be theoretically possible to detect ubiquitinated forms of these proteins. Hence immuno-precipitation experiments were then performed in an attempt to demonstrate ubiquitinated forms of the CaIcl1-Myc3 protein in *C. albicans.* Proteins were extracted from *C. albicans ICL1-UBI-*site-*MYC3-URA3* cells 20, 40 and 120 minutes after glucose addition. Analogous control extracts were also prepared from *S. cerevisiae* and *C. albicans* cells expressing ScIcl1+Myc3, CaIcl1+Myc3 and untagged parental strains grown on lactate plus glucose and lactate alone. These extracts were immunoprecipitated with an anti-Myc antibody that was predicted to precipitate Icl1 proteins having carboxyl-terminal Myc3 tags. These immunoprecipitates were then subjected to Western blotting with an anti-ubiquitin antibody to test whether any of these Myc3-tagged Icl1 proteins carry ubiquitin sequences. The Western blots were also probed with the anti-Myc antibody to confirm that Myc-tagged Icl1 proteins had been immunoprecipitated. This was the case. Interestingly, a weak ubiquitin-containing band of a length consistent with Icl1-Myc3 proteins was detected. Such bands were observed in three replicate experiments. These weak bands were observed for the ScIcl1-Myc3 CaIcl1- Ubi-Myc3 proteins following glucose addition (both proteins carry ubiquitination sites). However, no ubiquitination of these proteins was observed in the absence of glucose, or for the CaIcl1-Myc3 protein (which lacks a strong ubiquitination site) or for the untagged control cells [63].

These data suggest that when *S.cerevisiae* Icl1 or an artificial *C. albicans* Icl1 carrying a ubiquiti‐ nation signal is expressed in *C. albicans*, it becomes ubiquitinated and destabilised in response to glucose. However, the native *C. albicans* Icl1 protein is not destabilised by glucose. There‐ fore, in response to glucose, target proteins became ubiquitinated and then degraded in *C. albicans* [63].

What natural *C. albicans* proteins might be subjected to ubiquitin-mediated, glucose-acceler‐ ated protein degradation? To address this, bioinformatic tools were used to predict possible ubiquitination sites in glycolytic, gluconeogenic and glyoxylate cycle enzymes in *S. cerevisiae* and *C. albicans.* Five enzymes were selected for analysis: Fbp1, Pck1, Mdh1, Eno1 and Mls1. Based on this analysis, *S. cerevisiae* Fbp1, Pck1 and Eno1 appear to carry strong ubiquitination sites, while only Eno1 *C. albicans* appears to have a high confidence ubiquitination site. In conclusion, while these central metabolic pathways are highly conserved between *S. cerevi‐ siae* and *C. albicans,* these organisms appear to display significant differences in the presence of ubiquitination sites in the orthologous enzymes [63].
