Diseño de instrumentos de reconstrucción

As mentioned, idh1Δ or idh2Δ strains showed a slow growth phenotype on non-fermentable carbon sources but they grew equal to the wild type when provided with the fermentable carbon source glucose (Cupp and McAlister-Henn, 1991; Zhao and McAlister-Henn, 1996a). In the experiments in this work a growth defect for idh2Δ strains was seen when supplemented with glycerol but also when glucose was used. Of note is that the growth defect was only observed on minimal medium, indicating that this medium was deficient in nutrients that were necessary for wild type growth.

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α-ketoglutarate, the product of isocitrate dehydrogenase activity, is an important precursor for glutamate synthesis. Glutamate is synthesised from

α-ketoglutarate and vice versa by the glutamate dehydrogenase. It was found that idh2Δ strains grew slow on minimal glucose media lacking glutamate (McCammon and McAlister-Henn, 2003). Glutamate synthesis may be impaired in these strains because Idh2 is missing and α-ketoglutarate synthesis is reduced. This may render cells auxotroph for glutamate to some extent and reduce growth due to a lack of protein building blocks. As the minimal medium that was used was missing glutamate this may explain the growth defect seen for idh2Δ strains.

NADH is another product of isocitrate dehydrogenase. Without an active enzyme the level of NADH was reduced (Elzinga et al., 1993). Consequently, the level of NAD+ _{would increase. In a wild type strain where both Idh1 and Idh2 are} present an increase in NAD+ promotes isocitrate dehydrogenase function. But as Idh2 is missing the cell is unable to resume the citric acid cycle. As NADH is used for ATP production in the oxidative phosphorylation pathway this would reduce energy levels inside the cell. And as the citric acid cycle is inhibited in idh2Δ strains this suggests that they instead use fermentation to produce ATP. Wild type strains, on the other hand, may use a mix of both respiratory (via the citric acid cycle) and fermentative metabolism. As the respiratory pathway is more efficient this might confer a growth advantage to wild type strains. Therefore, this would result in a relative slow growth for idh2Δstrains.

As a first test to determine if reduced α-ketoglutarate synthesis or low NADH levels are causing the growth defect of idh2Δ strains the growth assays above were repeated by supplementing the minimal media with glutamate. The result of a growth assay is shown in Figure D16. Adding glutamate to the media abolished the growth defect of IDH2 deletion strains under non-starvation conditions and they grew like the wild type. While glutamate synthesis was not restored due to a lack of IDH2 and α-ketoglutarate, the cells used the supplemented glutamate and this allowed protein synthesis and cellular growth. Alternatively, glutamate was converted into α-ketoglutarate by the glutamate dehydrogenase, compensating for the loss of Idh2 function. This may have allowed the citric acid cycle as well as NADH and ATP production to resume. Thus, this result supported both the idea of reduced α-ketoglutarate synthesis and low energy levels.

Chapter D: Finding potential Gcn2 regulators

141 The minimal medium used herein contained histidine and methionine. Histidine can be converted into glutamate and methionine can be converted into succinyl-CoA, the next product in the citric acid cycle after α-ketoglutarate. This suggested that these amino acids might compensate for the loss of Idh2 function to some extent but may not increase NADH levels to wild type levels. In turn, this supported the idea that the growth defect of idh2Δ strains was largely due to low

energy levels inside the cell and less due to reduced α-ketoglutarate synthesis. On the other hand, it is possible that the cytosolic NADP-specific isocitrate dehydrogenase (subunits Idp1 and Idp2) may be able to compensate for a lack of Idh2 or Idh1 (Cupp and McAlister-Henn, 1992). This supported the idea that the growth defect was caused by reduced glutamate synthesis.

Under starvation conditions when glutamate was not supplemented a growth defect was observed for idh2Δ strains compared to the wild type (cf. Figure D14). It was possible that this growth defect was caused either by lack of

α-ketoglutarate or NADH or both instead of an impaired starvation response. In order to test this, the starvation media was supplemented with glutamate. The growth defect of idh2Δ strains was restored to wild type levels under these conditions (Figure D16). This indicated that idh2Δ strains were able to overcome the starvation and suggested that the GAAC was active. Again, a lack of

α-ketoglutarate or a lack of NADH are possible explanations for the growth defect of idh2Δ strains grown under glutamate-deficient starvation conditions.

Transforming the wild type and idh2Δ strains with either of the two Tiling Collection plasmids rendered them slow growing under non-starvation and starvation conditions and when glutamate was missing compared to strains transformed with the vector control (cf. Figure D14). This growth defect for strains containing a Tiling Collection plasmid was also seen for media supplemented with glutamate (Figure D16). This supports the idea that overexpression of BAG7 causes slow growth. Because idh2Δ strains with the vector control grew like the wild type and there was no impaired growth under starvation conditions no evaluation in regards to Idh2 complementation can be made from this growth assay. The comparison of minimal and starvation media indicated that transformation with the IDH2_E4 plasmid weakly increased growth for the wild type and idh2Δ strains while the presence of the IDH2_F4 plasmid weakly reduced their growth (Figure D17). This suggested that under starvation conditions with

Chapter D: Finding potential Gcn2 regulators

142 glutamate supplementation the overexpression of BAG7 in the wild type resulted in no or only a very weak growth defect. Similarly, there was only a small growth difference between idh2Δ strains with the plasmids and with the vector control when comparing minimal and starvation media (Figure D17). This indicated that reintroducing IDH2 does not strongly restrict or increase growth, presumably because isocitrate dehydrogenase activity was not changed (Cupp and McAlister- Henn, 1991).

To conclude, supplementing idh2Δ strains with glutamate restored their growth to wild type levels under both non-starvation and starvation conditions. In addition, while transformation of the wild type and idh2Δ strains with the Tiling Collection plasmids resulted in a slow growth this was not exacerbated under starvation conditions.

Figure D16: Supplementing idh2Δ strains with glutamate abolished their growth defect

under starvation conditions. The two plasmids used are IDH2_E4 and IDH2_F4.

E: glutamate. BY4741 is the wild type strain. Strains without the plasmid contain an empty vector. Each panel shows the result of a single petri dish.

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143

Figure D17: Supplementing idh2Δ strains with glutamate abolished their growth defect

under starvation conditions. Quantification of the growth assays was done as in Figure D6. The two plasmids used are IDH2_E4 and IDH2_F4.

D.5.4 Discussion

This study provided evidence that Idh2 may be involved in the starvation response, based on an impaired starvation response in growth assays and reduced eIF2α phosphorylation of idh2Δ strains. However, glutamate supplementation restored wild type growth for idh2Δ strains in the growth assays. While there were two possible explanations for the growth defect on media lacking glutamate (low α-ketoglutarate levels or low NADH levels), it was not possible to distinguish between them in these experiments. It was conceivable that both are valid and that both contributed to the growth defect, although to an unknown extent. 0.0 0.2 0.4 0.6 0.8 1.0 1.2

WT WT+E4 idh2Δ+E4 idh2Δ gcn2

R ela ti ve gr ow th 0.0 0.2 0.4 0.6 0.8 1.0 1.2 WT WT+F4 idh2Δ+F4 idh2Δ gcn2 R ela ti ve gr ow th

Chapter D: Finding potential Gcn2 regulators

144 The enzyme glutamate dehydrogenase converts glutamate to

α-ketoglutarate. To further test if the growth defect is caused by low glutamate or low energy levels, a strain deleted for IDH2 and in addition for the genes encoding glutamate dehydrogenase subunits GDH1 or GDH2 could be used. A negative genetic interaction between Idh2 and Gdh2 was already reported, i.e. the double deletion strain had a stronger growth defect than expected from the multiplicative effect of combining two single mutants, indicating a close functional relationship (Szappanos et al., 2011). On media supplemented with glutamate, an idh2Δ/gdh2Δ double deletion should inhibit the conversion of glutamate into α- ketoglutarate. Consequently, the citric acid cycle should remain inhibited and the cells should grow slow compared to IDH2 single deletion strains.

To further study the involvement of Idh2 in the starvation response a plasmid containing only IDH2 but no other protein coding genes needs to be used. This way the growth-inhibitory effects of BAG7 overexpression can be excluded from the experiment. In addition, it was possible that Bag7 or the gene product of YOR135C (Irc14) may interfere with amino acid biosynthesis or the GAAC. For example, their overexpression could have interfered with Gcn2 activation. Bag7 is involved in actin cytoskeleton organisation and cell wall biosynthesis by stimulating the GTPase Rho1 (Roumanie et al., 2001; Schmidt et al., 2002). Interestingly, Rho1 has been implicated in the control of bud growth (Cabib et al., 1998; Schmidt and Hall, 1998). In addition, overexpression of Bag7 rendered cells sensitive to rapamycin, a drug that inhibits the TOR stress-response pathway (Butcher et al., 2006). One study found that RHO1 expression was decreased under SM-induced starvation conditions, suggesting that bud growth was reduced to preferably synthesise proteins involved in the starvation response (Jia et al., 2000). Taking into account that the cytoskeleton may regulate Gcn2 activity and that Gcn2 was proposed to be inhibited by Yih1 in the bud (Sattlegger et al., 2004) this may suggest a connection between Bag7/Rho1 and the GAAC. However, no further evidence for such a connection exists in the literature and neither Bag7 nor Rho1 were identified in the Yih1 interactome. Strains deleted for BAG7 or RHO1 were not tested for SM sensitivity in the large-scale study by Parsons et al. (2004). Based on these findings it is conceivable that Bag7 overexpression may interfere with Gcn2 activation and this supports the idea that a plasmid only containing the IDH2 gene would be needed. Not much is known about the function of YOR135C

Chapter D: Finding potential Gcn2 regulators

145 but it may be involved in DNA metabolism (Alvaro et al., 2007). A YOR135C deletion mutant was tested negative for SM sensitivity, suggesting that it is not involved in Gcn2 activation (Parsons et al., 2004).

A separate approach to study the connection between Idh2 and the GAAC is to use other starvation-inducing drugs instead of SM such as 3AT (causes histidine starvation) or L-methionine-S-sulfoximine (MSX, causes glutamine starvation). Yeast strains that cannot activate Gcn2 were unable to grow under such conditions (Cambiaghi et al., 2014; Rolfes and Hinnebusch, 1993). If Idh2 is involved in the starvation response then cells without Idh2 should grow slow on medium supplemented with either 3AT or MSX. This growth defect should then be restored to wild type levels by reintroducing IDH2 on a plasmid.

In order to further elucidate the role of Idh2, strains deleted for IDH1, the other isocitrate dehydrogenase subunit, could be used as this resulted in a similar growth defect as for idh2Δ strains (Cupp and McAlister-Henn, 1992). Thus, growing idh1Δ under starvation conditions should result in impaired growth and this should be complemented with glutamate supplementation in a similar way as for idh2Δ strains. Furthermore, an idh1Δ/idh2Δ double deletion strain may be used which should also result in a growth defect. There is some evidence that a double deletion may exacerbate the growth defect under certain conditions such as when they were provided with non-fermentable carbon sources (Cupp and McAlister- Henn, 1992; Zhao and McAlister-Henn, 1996b). Thus, it may be possible that this increased growth defect is also seen under starvation conditions.