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

Ольга Михайловна Землянко, Евгения Михайловна Максютенко, Нина Павловна Трубицина, Татьяна Михайловна Рогоза, Екатерина Игоревна Порфирьева, Галина Анатольевна Журавлева

Результат исследований: Материалы конференцийтезисы

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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»
Язык оригиналаанглийский
СостояниеОпубликовано - 2019

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Землянко, Ольга Михайловна ; Максютенко, Евгения Михайловна ; Трубицина, Нина Павловна ; Рогоза, Татьяна Михайловна ; Порфирьева, Екатерина Игоревна ; Журавлева, Галина Анатольевна. / MUTATIONS IN C-PART OF SUP35 INFLUENCE PROPERTIES OF [PSI+] FACTOR IN YEASTS.
@conference{73d1e000b66545e3b8349ef16a8e05a6,
title = "MUTATIONS IN C-PART OF SUP35 INFLUENCE PROPERTIES OF [PSI+] FACTOR IN YEASTS",
abstract = "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»",
author = "Землянко, {Ольга Михайловна} and Максютенко, {Евгения Михайловна} and Трубицина, {Нина Павловна} and Рогоза, {Татьяна Михайловна} and Порфирьева, {Екатерина Игоревна} and Журавлева, {Галина Анатольевна}",
year = "2019",
language = "English",

}

MUTATIONS IN C-PART OF SUP35 INFLUENCE PROPERTIES OF [PSI+] FACTOR IN YEASTS. / Землянко, Ольга Михайловна; Максютенко, Евгения Михайловна; Трубицина, Нина Павловна; Рогоза, Татьяна Михайловна; Порфирьева, Екатерина Игоревна; Журавлева, Галина Анатольевна.

2019.

Результат исследований: Материалы конференцийтезисы

TY - CONF

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

AU - Землянко, Ольга Михайловна

AU - Максютенко, Евгения Михайловна

AU - Трубицина, Нина Павловна

AU - Рогоза, Татьяна Михайловна

AU - Порфирьева, Екатерина Игоревна

AU - Журавлева, Галина Анатольевна

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»

M3 - Abstract

ER -