There was a large increase in the L-leucine uptake and also in the oxygen consumption in yeast grown under catabolite derepression, as reported previously [5
]. There was a 16.6 ± 0.9-fold increase in L-[U-14
C]leucine oxidation in yeast undergoing catabolite derepression than those grown in glucose under CCR (). The accompanying 15-fold increase in the oxygen consumption in the WT yeast grown in galactose compared to glucose medium, we have reported previously [5
], was also observed for other WT strains used in this study (data not shown).
Figure 1 (a) L-[14C]leucine (750μM) oxidation in WT and amino acid permease deleted yeast strains. Yeast was grown in either 2% glucose (filled bars) or 2% galactose (hollow bars). L-[14C]leucine uptake and oxidation were performed as described (more ...)
Is the pool of oxidized leucine derived largely from the increased L-leucine uptake during catabolite derepression? If the source of oxidized leucine was largely derived from the increased uptake, there should be a decrease in the leucine oxidation in yeast from which major amino acid permeases were deleted. The rate of leucine oxidation was proportionally decreased in strains from which the major TOR regulated amino acid permeases BAP1, TAT1, and GNP1 were deleted (). There was a good corelation between the decrease in leucine oxidation and the decrease in uptake () when normalized to L-[U-14C]leucine oxidation and uptake, respectively, in the appropriate strain. These results indicate that the rate of leucine oxidation is correlated to the uptake during catabolite derepression.
The α-ketoglutarate/glutamate oxido-reductive deamination reaction catalysed by glutamate dehydrogenase plays a key role in nitrogen flow and hence amino acid carbon entry into oxidative metabolism. Concomitant with the increase in leucine oxidation we also observed a significant increase in intracellular glutamate concentration in galactose grown yeast (glucose 4.5 ± 0.25 and galactose 38.2 ± 4.9, n = 3, P < 0.05).
An interesting corollary is that in WT yeast 14CO2 production from L-[U-14C]leucine is dependent upon the presence of glucose or galactose (). Yeast incubated with L-[U-14C]leucine but without a carbohydrate source (–CHO) released only nominal amounts of 14CO2. Addition of glucose or galactose to yeast from either ± CCR cultures (+CHO) resulted in measurable leucine oxidation (). Deoxyglucose (+DOG) did not mimic this response indicating the requirement for a metabolizable carbohydrate source to achieve measurable leucine oxidation.
Figure 2 Presence of glucose or galactose in the medium is essential to measure L-[14C]leucine (750μM) oxidation in WT (M3750) yeast. Yeast was grown in 2% glucose (filled bars) or 2% galactose (hollow bars) and incubated in the assay medium with (more ...)
Transamination of leucine results in the formation of the branched chain keto acid, α
-KIC). The key regulatory enzyme controlling entry of keto acids into oxidative metabolism is BckDH. The basal activity of BckDH was measured in whole cell extracts (after 0.05% treatment with Triton X-100 to release the enzyme from mitochondria) of WT yeast grown in 2% glucose or galactose medium; the BckDH activity determined was basal rather than total (dephosphorylated). The activity of BckDH was linear for at least 6
min (). There was approximately a 30-fold increase in the basal BckDH activity in yeast grown in 2% galactose compared to those grown in glucose.
Figure 3 Time course of BckDH basal activity in WT yeast, grown in 2% glucose (dashed line) or 2% galactose (solid line). Representative trace of NADH formation in cell homogenates using α-ketoisovalerate as a BCKD substrate (added at arrow). Dashed line (more ...)
BckDH is a member of the 2-oxo acid dehydrogenase multienzyme complexes that contains dihydrolipoamide dehydrogenase (Lpd1). An increase in protein expression, measured by fluorescence microscopy, for Lpd1 was also observed. Analysis of images (not shown) from Lpd1-GFP strains indicates a 3.0 ± 0.5-fold increase Lpd1 expression in yeast grown in galactose compared to those grown in glucose. These results indicate that leucine catabolic pathways are upregulated during catabolite derepression.
The yeast grown with galactose as sole carbon source is in an oxidative state of metabolism with increased oxygen consumption [5
]. To confirm if the yeast relied upon the activity of TCA cycle during catabolite derepression for growth and that any residual fermentative component of metabolism did not account for growth we used a strain with the cytochrome c1 gene deletion. Cyt1 is a subunit of complex III in the mitochondrial electron transport chain. There was no regression in growth of Δcyt1
strain compared to WT when grown in medium with 2% glucose (). However, there was no growth visible for the Δcyt1
yeast when grown in galactose medium (), even after 72 hours of incubation. Similar results were observed for growth in liquid medium (data not shown). These results indicate that reductive substrate-electron transfer is critical for growth with galactose as the sole carbon source. We further investigated the role of the TCA cycle and electron transport by examining the expression of citrate synthase and Cyt1 from the Yeast-GFP clone collection [10
] in yeast grown in 2% glucose or galactose medium. The Yeast-GFP clone collection is designed to express full-length proteins, tagged at the carboxy-terminal end with GFP, from their endogenous promoters. A very faint GFP signal was observed for Cyt1 protein in yeast grown in 2% glucose (). This expression was increased by 2.0 ± 0.24-fold when WT yeast was grown in 2% galactose YNB (). Cs1 expression was visible in WT yeast grown in 2% glucose medium (); however this was significantly (P
< 0.05, n
= 20) increased by 30 ± 5% in yeast grown in galactose medium ().
Activity of the TCA cycle is essential when WT yeast is grown with galactose as sole carbon source. Image of WT (BY4741) and Dcyt1 grown on agar plates with 2% glucose or galactose added to the YNB medium.
Figure 5 Direct fluorescence was measured in yeast strains with GFP-tagged CYT1 and CIT1 using Zeiss LSM510 and Bio-Rad Radiance 2100 confocal systems, respectively. (a) Cyt1-GFP fluorescence in yeast grown in 2% glucose YNB compared to (c) in 2% galactose YNB (more ...)