TY - CHAP

T1 - MUTATIONS IN C-PART OF SUP35 INFLUENCE PROPERTIES OF [PSI+] FACTOR IN YEASTS

AU - Zemlyanko, O.M.

AU - Maksutenko, E.M.

AU - Trubitcina, N.P.

AU - Rogoza, T.M.

AU - Porfirieva, E.I.

AU - Zhouravleva, G.A.

N1 - Conference code: VII

PY - 2019

Y1 - 2019

N2 - The cytoplasmic [PSI+] factor is one of the best characterized yeast prions. [PSI+] is formed by Sup35 protein aggregates. Sup35p is encoded by the SUP35 gene. It consists of the nonessential N-terminal [PSI+] prion forming domain; the charged middle (M) domain, which maintains [PSI+], and the C-terminal domain with GTP-binding sites essential for translation termination activity. Mutations in the SUP35 gene leads to nonsense suppression, [PSI+] have the same effect. Previously it was shown that mutations in N-terminal domain of Sup35p affect [PSI+] appearance and maintenance. However, it has been demonstrated that mutations in Sup35p C-terminal domain can also change [PSI+] properties. In this work, we have studied sup35-228, sup35-10, and sup35-25 mutations located in the GTP-binding region of Sup35p. Using SDD-AGE, we have shown that [PSI+] is lost in the presence of only mutant alleles (sup35-228, sup35-10, sup35-25). Using fluorescent microscopy we additionally demonstrated that sup35-228 do not maintain pre-existing [PSI+]. However, several clones were obtained (less than 10%), which retained the [PSI+] factor, but at the same time replaced the mutant sup35 allele with the wild-type allele. Our results indicate that [PSI+] is incompatible with mutations in the C-terminal domain of Sup35p, but reasons of this effect remained unclear. We supposed that this phenomenon may be caused by the [PSI+] destabilization or inability of mutant Sup35p to form and maintain prion aggregates. In order to test this hypothesis we analyzed properties of missense-mutant proteins in vitro. We have shown that mutated Sup35 proteins form aggregates with high molecular weight and their protein aggregation rate is faster relative to the wild-type Sup35p. However, the amyloid nature of these aggregates requires confirmation. Another reason for incompatibility of sup35 missense-mutations and [PSI+] may be significant translation termination impairment. [PSI+] sequestrate functional Sup35p into prion aggregates, this possibly leads to dramatic reduction in accuracy of translation termination and cell death. Taken together, our data suggest that the non-prion C-domain of Sup35p is involved in the process of [PSI +] appearance and maintenance. This work was supported by grant from RSF 18-14-00050, RFBR 17-54-150002, technical help was provided by Resource Center «Development of Molecular and Cell Technologies»

AB - The cytoplasmic [PSI+] factor is one of the best characterized yeast prions. [PSI+] is formed by Sup35 protein aggregates. Sup35p is encoded by the SUP35 gene. It consists of the nonessential N-terminal [PSI+] prion forming domain; the charged middle (M) domain, which maintains [PSI+], and the C-terminal domain with GTP-binding sites essential for translation termination activity. Mutations in the SUP35 gene leads to nonsense suppression, [PSI+] have the same effect. Previously it was shown that mutations in N-terminal domain of Sup35p affect [PSI+] appearance and maintenance. However, it has been demonstrated that mutations in Sup35p C-terminal domain can also change [PSI+] properties. In this work, we have studied sup35-228, sup35-10, and sup35-25 mutations located in the GTP-binding region of Sup35p. Using SDD-AGE, we have shown that [PSI+] is lost in the presence of only mutant alleles (sup35-228, sup35-10, sup35-25). Using fluorescent microscopy we additionally demonstrated that sup35-228 do not maintain pre-existing [PSI+]. However, several clones were obtained (less than 10%), which retained the [PSI+] factor, but at the same time replaced the mutant sup35 allele with the wild-type allele. Our results indicate that [PSI+] is incompatible with mutations in the C-terminal domain of Sup35p, but reasons of this effect remained unclear. We supposed that this phenomenon may be caused by the [PSI+] destabilization or inability of mutant Sup35p to form and maintain prion aggregates. In order to test this hypothesis we analyzed properties of missense-mutant proteins in vitro. We have shown that mutated Sup35 proteins form aggregates with high molecular weight and their protein aggregation rate is faster relative to the wild-type Sup35p. However, the amyloid nature of these aggregates requires confirmation. Another reason for incompatibility of sup35 missense-mutations and [PSI+] may be significant translation termination impairment. [PSI+] sequestrate functional Sup35p into prion aggregates, this possibly leads to dramatic reduction in accuracy of translation termination and cell death. Taken together, our data suggest that the non-prion C-domain of Sup35p is involved in the process of [PSI +] appearance and maintenance. This work was supported by grant from RSF 18-14-00050, RFBR 17-54-150002, technical help was provided by Resource Center «Development of Molecular and Cell Technologies»

UR - https://events.spbu.ru/eventsContent/events/2018/vogis/VII%20VSGB%20Congress%20Abstracts%202019.pdf

M3 - Other chapter contribution

SN - 9785965112371

SP - 650

BT - VII Съезд Вавиловского общества генетиков и селекционеров (ВОГиС)

PB - Издательство «ВВМ»

CY - СПб.

Y2 - 18 June 2019 through 22 June 2019

ER -