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The body can't just rely on glucose—other carbohydrates, proteins, and lipids are also important sources of energy The next 4 chapters will cover these additional pathways involved in energy production and storage Diseases resulting from dysfunction of these side pathways are a big part of how the Step 1 likes to test metabolism.
Chapter 5 focused on the main path from glucose to ATP production glycolysis, TCA cycle, and oxidative phosphorylation as well as glucose synthesis by gluconeogenesis Chapter 6 focuses on other carbohydrates Fructose and galactose are common dietary carbohydrates that enter into the glycolysis pathway for energy production Glycogen is a polysaccharide, consisting of multiple glucose subunits, for energy storage.
Table Graphic Jump Location Table Summary of pathways. View Table Favorite Table Download.
Figure Gal4p bound 98 targets and Tye7p bound targets with the single probe full-genome array Tables S2 and S3 , so Tye7p appears to function as a more global regulator. The results are similar between the two factors except, as is discussed below, Gal4p bound more targets involved in pyruvate metabolism.
Although Tye7p bound a large number of targets, no GO process is enriched other than carbon metabolism. The P -value of enrichment for each GO category is indicated. The common targets are highly enriched for glycolysis genes. The Gal4p independent targets include the five components of the PDH while the Tye7p independent targets contain the three components of the trehalose synthase complex.
Smoothed peak intensity curves of the tiling array binding events were created to estimate the largest fold enrichments and thus the most significant targets. For Gal4p, the 13 bona fide glycolytic pathway promoters are in the top 68 smoothed peak intensities while for Tye7p they are in the top 52 peaks Tables S4 and S5. Therefore, the glycolytic promoters are among the most significant targets for both factors. Although Gal4p and Tye7p bound many common targets, there were a significant number of individual binding events, some of which are related to carbohydrate metabolism Figure 3B.
The clearest example is that Gal4p bound the promoter sequences of the five genes encoding the pyruvate dehydrogenase complex PDH while Tye7p bound the promoter sequences of the three genes for the trehalose synthase complex.
Tye7p also bound several genes involved in glycogen and glycerol metabolism. A summary of selected metabolic binding targets under glucose growth conditions is shown in Figure 4A and Table S6. Many of the Gal4p and Tye7p targets are linked to the glycolytic pathway suggesting that these factors also regulate the input and output fluxes of the pathway. Black represents no binding while red represents binding with the color brightness indicating the degree of enrichment. Binding to the glycolytic genes was so strong that binding to two neighboring probes was sometimes observed.
In such cases, both values were included as these occurred in situations where a shared promoter region contained two probes but the tiling array confirmed the presence of a single binding site. The fold enrichments of these and other metabolic targets are given in Table S6.
B Transcription profile of selected metabolic genes for the tye7 and gal4tye7 strains. Black represents no change in expression, green is down-regulated, and red is up-regulated with the color brightness indicating the degree of expression change.
The expression levels of these and other metabolic genes are given in Table S7. ChIP-CHIP is a whole-genome approach for determining binding locations of a transcription factor; however, it is insufficient to give a complete picture of a factor's biological function. As was observed with GAL4 , the binding and transcription profiles can provide different insights. Therefore, to complement the ChIP-CHIP analysis, transcription profiling comparing wild type and mutant strains was performed under glucose growth conditions.
The expression levels of selected carbohydrate metabolic genes is illustrated in Figure 4B and Table S7. As expected, not all targets bound by ChIP-CHIP showed differential expression and not all differentially regulated genes showed direct binding of the transcription factors; however, in general the most significantly bound targets were down-regulated in the absence of the factor.
The glycolytic and fermentation genes were down-regulated in the tye7 strain confirming Tye7p's role as an activator of fermentative metabolism. The involvement of Gal4p in the activation of glycolytic gene expression was masked in the gal4 expression profiles, most likely because the absence of GAL4 caused an up-regulation of TYE7 . Therefore, Tye7p appears able to significantly compensate for the loss of Gal4p, further supporting the idea that it plays a more central role in glycolytic gene regulation than does Gal4p.
Expression of the trehalose metabolic genes was down-regulated in the tye7 strain but not further reduced in the gal4tye7 strain indicating that Gal4p is not involved in their activation. Trehalose is a glucose disaccharide that has a role as a storage carbohydrate in yeast. Another important storage molecule in yeast is glycogen. These genes were not down-regulated in the tye7 strain some were slightly up-regulated along with GAL4 but were reduced in the gal4tye7 strain.
The down-regulated genes were analyzed for GO enrichment. As expected, all the categories were related to carbon metabolism. Generally, the two deletion strains had similar results with glycolysis tye7 P -value: 1. Tye7p directly activated many genes involved in trehalose and glycogen metabolism independently of Gal4p. As well, the genes encoding the glycolytic-committing enzyme phosphofructokinase PFK1 and PFK2 were among the top six most down-regulated genes in the tye7 strain while the gluconeogenesis-specific gene FBP1 was moderately down-regulated.
Therefore, it appears that Tye7p regulates the flux between energy storage and energy production at the glucosephosphate branch point Figure 1. To support this claim, we investigated whether the levels of trehalose and glycogen were different in the tye7 strain compared to the wild type. Since exponentially growing cells have low trehalose levels that rapidly accumulate during stationary phase  , trehalose amounts were determined from cells at both phases. We observed significantly increased levels of trehalose in the tye7 strain for both stationary and logarithmic phase cells Figure 5A.
Additionally, iodine staining showed that the glycogen content in the tye7 cells was higher than that of the wild type Figure 5B. The higher storage carbohydrate levels correlate with the expression profile as FBP1 and the majority of genes involved in trehalose and glycogen metabolism were down-regulated 2—3 fold while PFK1 and PFK2 were down-regulated approximately 6 and 9 fold, respectively. Therefore, the glucosephosphate flux in the tye7 strain would favor trehalose and glycogen synthesis. In contrast, the gal4 strain showed wild type levels of both storage carbohydrates, suggesting that Tye7p alone regulates the cell's decision to commit to glycolysis or energy storage.
A The trehalose content of wild type BWP17 , tye7 , and gal4 cells at both stationary and logarithmic phases were measured. Trehalose levels nmol trehalose per mg cell protein were reported relative to the wild type in stationary phase. Both wild type and gal4 cells had no detectable trehalose levels in the logarithmic phase and were not included in the graph. B Wild type BWP17 , tye7 , and gal4 cells were exposed to iodine vapor to indicate the glycogen content as iodine vapor stains cells brown upon reacting with glycogen.
The tye7 cells stained a darker brown than the wild type indicating that there is more glycogen present.
It is clear that Gal4p and Tye7p are important for fermentative growth when glucose, fructose or mannose is the carbon source. Although there was no phenotype during growth on galactose or glycerol, we investigated the effect of these carbon sources on binding to identify any differences Dataset S1. Figure 6A illustrates the ChIP-CHIP results of selected carbohydrate metabolic targets during growth on galactose and glycerol media with the behavior during growth on glucose included as a comparison. Heat map displays were created as described in Figure 4. B Transcription profiles of selected metabolic genes for tye7 1 and gal4tye7 2 strains under glucose Glu , galactose Gal , and glycerol Gly carbon sources.
A striking trend was the difference in binding between Gal4p and Tye7p under the various carbon sources.
Whereas the peak intensity of Gal4p binding changed based on the carbon source, the peak sizes of Tye7p binding were largely unaffected Figure 7A. This pattern was consistent for the majority of targets resulting in a decrease in the overall number of Gal4p binding targets from glucose to galactose to glycerol and a similar overall number of binding sites for Tye7p with the different carbon sources Figure S2.
Therefore, Gal4p binds its few targets in a carbon source-dependent manner while Tye7p appears to be a more global regulator that binds its targets independently of the carbon source the cells are growing on. This difference in target binding dependent on the carbon source could be a direct result of the protein levels of the transcription factors. These results further support the inference that Tye7p is the more central regulator of carbohydrate metabolism.
The ORF and bp upstream region is shown. Values for each data point were determined by taking the mean fold enrichment of each probe and the surrounding four probes. The dashed blue and green lines represent the Tye7p and Gal4p binding sites, respectively approximated over the three carbon sources. The consensus motifs and their positions are indicated for each factor.
B Consensus motifs based on the top 30 peak intensities from the tiling array for Gal4p and Tye7p on glucose media. Another trend was that Gal4p showed stronger binding to the promoters of genes encoding the glycolytic pathway enzymes that acted in the later part of the pathway from TPI1 on compared to the early part of the pathway, regardless of the carbon source of the growth medium Figure 6A.
This setting proved to be particularly useful for research on vertebrate development [ 1 ] and modeling of human disease [ 2 ], namely the hematopoietic [ 3 , 4 ] and the cardiac systems [ 5 , 6 ]. Under aerobic conditions, Crabtree-negative cells predominately oxidize pyruvate to carbon dioxide through the TCA cycle. Coordinately regulating this flux ensures that the cell is committed either to storing energy or producing energy as to avoid futile cycling; therefore, it is logical that the key glycolytic activator also regulates trehalose and glycogen metabolism. Associated Data Supplementary Materials ijmss Plant Cell Rep.
Interestingly, this difference in binding within the pathway corresponds at the point where the six carbon glucose molecule has been converted to two three carbon products and also represents the separation between the initial ATP consuming steps and the later energy producing steps Figure 1. Therefore, while Tye7p appears to be involved in committing the cell to glycolysis, Gal4p appears to focus on the later part of the pathway to promote energy production once the commitment is made.
The binding distribution curves created with the tiling array were also used to predict the motif that CaGal4p and CaTye7p recognize by looking for sequences enriched around the binding sites of the top peak intensity targets. Analysis of the top CaGal4p binding targets revealed enrichment for this motif Figure 7B.
To gain further insight into how Gal4p and Tye7p regulate their targets in response to different carbon sources, transcription profiles comparing wild type and deletion strains with galactose and glycerol as the sole carbon source were performed. Figure 6B illustrates the expression profiles of selected carbohydrate metabolic targets during growth on galactose and glycerol media with the behavior during growth on glucose included as a comparison complete lists of down-regulated genes in Tables S8 , S9 , S10 , S11 , S12 and S The glycolytic genes were down-regulated in the tye7 strain under galactose and glycerol growth conditions but not as significantly as with glucose-containing media.
Gal4p strongly activated the glycolytic genes on glucose and galactose media but had only minimal effect when glycerol was the carbon source. This result correlates with the location profiling data as Gal4p displayed reduced binding under glycerol growth conditions.