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dc.contributor.authorWijayanti, Indah
dc.contributor.authorTakagi, Hirosi
dc.date.accessioned2017-03-27T08:59:02Z
dc.date.available2017-03-27T08:59:02Z
dc.date.issued2013-06
dc.identifier.issn1336-4839
dc.identifier.urihttp://repository.ipb.ac.id/handle/123456789/83825
dc.description.abstractRsp5 is an essential HECT-type ubiquitin ligase that is the only Saccharomyces cerevisiae member of the Nedd4 family. We previously isolated an interesting mutant of Rsp5, which has the Ala401Glu substitution in the substraterecognizing WW3 domain. The rsp5A401E cells were more sensitive to stresses that induce protein misfolding or denaturation, such as toxic amino acid analogues, high growth temperature, ethanol, and heat shock treatment than the wild-type cells (1). This result suggests that Rsp5 is a key enzyme involved in the degradation and repair of stress-induced abnormal proteins for yeast cell growth under stress conditions (2,3). To improve its enzymatic function, we attempt to isolate new Rsp5 variant(s) using error-prone PCR random mutagenesis on its WW domains and introduce the mutagenized library into rsp5A401E cells. We used α -synuclein as a model stress, because Rsp5 catalyzes α-synuclein ubiquitination and degradation and protects cells from inclusion formation and toxicity (4). α-Synuclein is associated with early onset Parkinson’s disease, in which α -synuclein accumulates in intraneuronal inclusion containing ubiquitin chains. This small lipid-binding protein is prone to misfolding and aggregation, and overexpression of human α-synuclein in S. cerevisiae leads to ER stress, disruption of ER-Golgi vesicle trafficking, accumulation of lipid droplets, mitochondrial dysfunction, and ultimately cell death (5). Here, we successfully obtained Thr255Ala, Asp295Gly, Pro343Ser, and Asn427Asp as new Rsp5 variants that confer tolerance to α -synuclein on yeast cells. We will show the growth phenotype, the localization and ubiquitination of α -synuclein. This approach is promising for breeding of novel stress-tolerant yeast with improved ubiquitin systemid
dc.description.sponsorshipEmbo, Olympus, Trigonid
dc.language.isoenid
dc.publisherInternational Commission on Yeasts (ICY)id
dc.titleEngineering of yeast essential ubiquitin ligase Rsp5 by introduction of random mutagenesis into its WW domains to improve enzymatic functionsid
dc.typeArticleid
dc.subject.keywordEngineering, essential ubiquitin ligaseid


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