| Budget Amount *help |
¥3,700,000 (Direct Cost: ¥3,700,000)
Fiscal Year 2002: ¥600,000 (Direct Cost: ¥600,000)
Fiscal Year 2001: ¥3,100,000 (Direct Cost: ¥3,100,000)
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| Research Abstract |
To study the mechanism of the oscillation, we first examined if heat shock proteins (Hsps) are involved. Neither the protein levels of major Hsps nor the expression of the b-galactosidase gene as a reporter under control of the promoter carrying heat shock element oscillated during the metabolic oscillation. The level of trehalose cycled with the same periodicity, as did energy metabolism. This oscillation was not found in a GTS1-deleted mutant. These results suggest that heat resistance oscillation is induced by fluctuations in trehalose level and not by oscillatory expression of Hsps.
We screened a yeast cDNA library for proteins that bind to Gts1p using yeast two-hybrid system and cloned multiple TDH cDNAs encoding Gliceraldehyde-3-phosphate dehydrogenase (GAPDH). The zinc-finger and dimerization sites of Gts1p were required for full ability to bind GAPDH and Gts1ps mutated at these sites lost the ability to regulate ultradian oscillations of energy metabolism. Of the three TDH genes
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, only TDH1 fluctuated at the mRNA level in continuous culture and its deletion resulted in disappearance of the oscillation. This loss of biological rhythms in THD1-deleted mutant was rescued by the expression of TDH1 but not of TDH2 or TDH3 under control of the TDH1 promoter. Thus, Gts1p plays a role in the regulation of the metabolic oscillation by interacting with the GAPDH.
Gts1p contains sequence motifs similar to N-deguron and the ubiquitin association domain. When the lysine residue at the putative ubiquitination site of N-deguron was mutated, both protein level and half-life of mutant Gts1p increased. During continuous culture, the protein level of the mutant Gts1p elevated and did not fluctuate, leading to disappearance of metabolic oscillation. When ubiquitin association domain was mutated, polyubiqutination level was decreased. Gts1p is degraded through conjugation with ubiquitin, and the UBA domain promoted the degradation of ubiqutinaed Gts1p, causing a fluctuation in protein level, which is required for the maintenance of metabolic oscillation. Less
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