Regulation of the Gts1p Level by the Ubiquitination System to Maintain Metabolic Oscillations in the Continuous Culture of Yeast

Abstract

Yeast cells exhibit sustained ultradian oscillations of energy metabolism in coupling with cell cycle and stress resistance oscillations in continuous culture. We have reported that the rhythmic expression of Gts1p is important for the maintenance of ultradian rhythms. Structurally, Gts1p contains sequence motifs similar to N-degron and the ubiquitin association domain, raising the possibility that the Gts1p level is regulated by degradation via ubiquitination. When the lysine residue at the putative ubiquitination site of the N-degron was substituted with arginine, both the protein level and half-life of mutant Gts1p increased. During continuous culture, the protein level of the mutant Gts1p was elevated and did not fluctuate, leading to the disappearance of metabolic oscillation within a day. Furthermore, using three Gts1ps containing mutations in the ubiquitin association domain, we showed that the lower the binding activity of the mutant Gts1ps for polyubiquitin in vitro, the higher the protein level in vivo. Expression of the mutant Gts1ps in the continuous culture resulted in an increase in Gts1p and early loss of the oscillation. Therefore, Gts1p is degraded through conjugation with ubiquitin, and the UBA domain promoted the degradation of ubiquitinated Gts1p, causing a fluctuation in protein level, which is required for the maintenance of metabolic oscillations.In an open system using a bioreactor, yeast cells exhibit sustained ultradian oscillations of energy metabolism in a continuous (chemostat) culture under aerobic and glucose-limited conditions (1-4). The energy metabolism pathway has been proven to be an autogenous oscillator under extreme nonequilibrium energy conditions according to the theory of "dissipative structures" established by Prigogine and others (5-7). In brief, the metabolic pathway oscillates autonomously under the primary control of phosphofructokinase, transferring energy from glucose to NADH, which acts as the feed-forward activator, and then from NADH to ATP, which acts as the feedback inhibitor. After ATP as an inhibitor is consumed as fuel for various ATPases, glucose again begins to enter the glycolytic pathway. The oscillations are detectable as a periodic change in the factors involved in energy metabolism such as dissolved oxygen (DO) 1 levels, CO 2 production, glucose and ethanol concentrations, and amounts of storage carbohydrates. DO oscillation is due to the periodic change between respiratory and respiro-fermentative phases in which oxygen demands are relatively high and low, respectively. DO oscillations arise spontaneously in concert with cell division and are dependent on a high cell density (ϳ5 ϫ 10 8 cells/ml), regulating the cell density throughout the oscillation (8). We have reported that cellular responses to various stress conditions, such as heat, oxidative agents, and cytotoxic compounds, are regulated in concert with metabolic oscillation (8) and that the coupling 2 was disrupted by inactivation of the GTS1 gene (9). Furthermore, we suggested that the rhy...

Cbl-b interacts with ubiquitinated proteins; differential functions of the UBA domains of c-Cbl and Cbl-b

“…The relative contribution of the three helixes of the UBA b to ubiquitin binding is confirmed by mutations in the predicted hydrophobic patch of the UBA b . Mutation of two residues in the loop linking helixes 1 and 2 (G943A and Y944Q; Figure 3a) abolished ubiquitin binding, as has been demonstrated for the equivalent resides in other UBA domains (Wilkinson et al, 2001;Saito et al, 2002). In contrast, mutation of L967A, which is also predicted to contribute to the hydrophobic patch, failed to affect ubiquitin binding (data not shown), suggesting that the first two helixes of the UBA b make a greater contribution to ubiquitin binding than the third helix.…”

“…For example, the yeast E3 ligase, Rsp5, binds the CUE domain of Vsp9 and ubiquitinates it Shih et al, 2003). Interestingly, the UBA domain of the yeast protein, Gts1p, is involved in its ubiquitination and degradation (Saito et al, 2002), suggesting that, like other ubiquitin binding domains, the UBA domain may mediate intermolecular ubiquitination. Further studies will be necessary to determine if this is the case for the UBA b .…”

“…Subsequently, it was shown that the UBA domains of a number of proteins interact with ubiquitin (Bertolaet et al, 2001b;Chen et al, 2001;Wilkinson et al, 2001;Funakoshi et al, 2002;Meyer et al, 2002;Rao and Sastry, 2002;Saito et al, 2002;Ciani et al, 2003;Raasi and Pickart, 2003;Ryu et al, 2003;Chim et al, 2004). In addition to binding ubiquitin, UBA domains are capable of interacting with ubiquitin-like domains (Ryu et al, 2003) and the ubiquitin-like protein, NEDD8 (Tanaka et al, 2003).…”

Ultradian metabolic rhythm in the diazotrophic cyanobacterium Cyanothece sp. ATCC 51142

“…Aerobically growing, dense continuous cultures of S. cerevisiae exhibit autonomous energy-metabolite ultradian oscillations of ca. 2-5 h periods that are observed under low nitrogen and low carbon conditions (18,(24)(25)(26). Although yeast are eukaryotic organisms that separate incompatible processes in different compartments, they also use temporal separation to carry out incompatible metabolic reaction and further alleviate futile cycles (27).…”

The Gts1 protein stabilizes the autonomous oscillator in yeast

“…Overexpression of the Gts1p produced a pleiotropic phenotype with essentially the opposite effects on the phenotype (Yaguchi et al , 1996). The properties of the Gts1p may be influenced by phosphorylation (Yaguchi et al , 2000) and ubiquitination (Saito et al , 2002).…”