Selective degradation of misfolded proteins in quiescent yeast Saccharomyces cerevisiae

Abstract

Kako bi se zaštitile od nakupljanja pogrešno smotanih proteina, stanice su razvile mehanizme za kontrolu kvalitete proteina, uključujući put selektivne razgradnje sustavom ubikvitin-proteasoma. Prethodna istraživanja kontrole kvalitete proteina uglavnom su provedena u stanicama koje se aktivno dijele, međutim mnoge stanice, poput neurona i matičnih stanica u mirovanju, se ne dijele. U ovoj disertaciji istražili smo puteve selektivne razgradnje proteina u stanicama u mirovanju, koristeći modelni organizam, kvasac Saccharomyces cerevisiae. Po ulasku stanica kvasca u mirovanje aktivira se autofagija, a veliki dio proteasoma reorganizira se u granule u kojima proteasomi vjerojatno nisu aktivni, stoga nije bilo jasno oslanjaju li se stanice u mirovanju na sustav ubikvitin-proteasoma za eliminaciju pogrešno smotanih proteina. U ovom radu uspostavili smo ekspresiju modelnih pogrešno smotanih proteina tGnd1, stGnd1 i Ubc9ts u stanicama u mirovanju, koristeći PIR3- i Z-promotore. Pokazali smo da stanice u mirovanju prepoznaju pogrešno smotane proteine te ih usmjeravaju u selektivnu razgradnju, čak i u kasnijim fazama mirovanja. Nadalje, razgradnja je ovisila o aktivnosti E3 ligaza ubikvitina Ubr1 i San1, te proteasomu, što upućuje na sličan put razgradnje kao u proliferirajućim stanicama. Sveukupno rezultati pokazuju da stanice kvasca u mirovanju zadržavaju funkcionalnu kontrolu kvalitete proteina, te da se ona primarno temelji na selektivnoj razgradnji proteina.To prevent the negative impact of misfolded protein accumulation, cells have developed protein quality control pathways, including selective degradation by the ubiquitin-proteasome system. Previous research on protein quality control has mostly been conducted in actively dividing cells. However, many cells, such as neurons and quiescent stem cells, are non-dividing. In this thesis, we investigated selective protein degradation in quiescent cells, using yeast Saccharomyces cerevisiae as a model organism. Upon entry into quiescence, yeast cells induce autophagy, and a large portion of the proteasomes relocalizes into cytoplasmic granules, presumably in an inactive form. Therefore, it has been unclear whether quiescent cells rely on the ubiquitin-proteasome system for the elimination of misfolded proteins. In this study, we established an expression system of model misfolded proteins tGnd1, stGnd1, and Ubc9ts in quiescent cells using PIR3- and Z-promoters. We show that quiescent cells recognize misfolded proteins and direct them to selective degradation, even in the later stages of quiescence. Furthermore, degradation was dependent on the E3-ubiquitin ligases Ubr1 and San1 and the proteasome, suggesting a similar pathway as in proliferating cells. Overall, the results show that quiescent yeast cells maintain functional protein quality control, which is primarily based on selective protein degradation