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Dissection of the Regulatory Mechanism of the Ubiquitin-activating Enzyme Uba1in Autophagy Regulation

Author: ZhangShaoJin
Tutor: ZuoXiangHua; YuLi
School: Huazhong Agricultural University
Course: Animal Nutrition and Feed Science
Keywords: Autophagy The ubiquitin-activating enzyme Uba1 The target of rapamycincomplex1 Atg1 Atg13
CLC: S852.2
Type: Master's thesis
Year: 2012
Downloads: 144
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Autophagy is an evolutionarily conserved process by which portions of cytosol and organelles are sequestered into double-membrane vesicles and degraded upon fusion with lysosomal compartments. As autophagy is involved in cell growth, survival, development and death, the levels of autophagy must be properly regulated, as indicated by the fact that dysregulated autophagy has been linked to many human pathophysiologies, such as cancer, myopathies, neurodegeneration, heart and liver diseases, and gastrointestinal disorders. Therapeutic exploitation of this process requires a clear understanding of its regulation. Although substantial progress has recently been made in understanding the molecular mechanisms of the autophagy machinery, there is limited information on how cellular signaling pathways regulate this complex process and many unanswered questions remain in the regulation of autophagy.To figure out new regulators of autophagy, we screened a yeast knockout library (covering100%of the yeast nonessential genes) and a conditional temperature-sensitive mutants collection (covering~65%of the yeast essential genes) for strains that are deficient in autophagy using the GFP-Atg8assay, and identity the ubiquitin-activating enzyme Ubal temperature-sensitive mutant (ubal-ol) cells with a block in autophagy. Laser scanning confocal microscope (LSCM) and western blot analysis showed that the ubal-ol cells had severe defects in the nitrogen or glucose starvation induced autophagy, which can be rescued by a plasmid-borne wild type UBA1. The fact that the ubal-ol cells showed normal autophagy activity during rapamycin treatment indicated that Ubal probably act through the upstream of TORC1to regulate autophagy. Therefore we move on to detect the TORC1activity in the Ubal wild type (WT) and ubal-ol cells during starvation or rapamycin treatment, and found that ubal-ol delayed the nitrogen starvation induced TORC1inactivation but not the glucose starvation or rapamycin treatment induced TORC1inactivation. Furthermore, ubal-ol doesn’t affect TORCl’s reactivation after its inactivation, suggesting that Ubal probably act on some upstream negative regulator of TORC1or directly affect the TORC1activity to regulate the nitrogen starvation induced autophagy. Notably, TORC1was quickly inactivated during the glucose starvation in the uba1-o1cells but it still showed a severe block in autophagy, which means that Ubal might also act through the downstream of TORC1to regulate the glucose starvation induced autophagy. So we tried to check whether the Atg1complex could be recruited to the pre-autophagosomal structure (PAS) normally in the uba1-o1cells. The results showed that the Atg1complex in the uba1-o1cells could not be properly recruited to the PAS during the nitrogen or glucose starvation, but not that case during the rapamycin treatment. Together with the TORC1activity results, we hypothesized that the uba1-o1cells had defects in some step between TORC1inactivation and the Atg1complex being recruited to the PAS during the glucose starvation. Then we analyzed the phosphorylation states of Atg13and its interaction with Atg1in the uba1-o1cells during the nitrogen or glucose starvation, and found that Atg13were hyperphosphorylated and there were no interaction between Atg1and Atg13in both cases. Therefore, Ubal probably act on the phosphates responsible for Atg13dephosphorylation to regulate the glucose starvation induced autophagy.Overall, we successfully identified Uba1as a new regulator of autophagy and dissected the probable step at which Uba1regulated autophagy during the nitrogen or glucose starvation respectively. However, the direct targets through which Uba1regulates autophagy needs further study.

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CLC: > Agricultural Sciences > Livestock, animal medicine,hunting,silkworm,bee > Animal Medicine ( Veterinary Medicine) > Basic Veterinary Science > Animal physiology, biophysics,biochemistry
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