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Molecular basis for transmission barrier and interference between closely related prion proteins in yeast.

Afanasieva EG, Kushnirov VV, Tuite MF, Ter-Avanesyan MD - J. Biol. Chem. (2011)

Bottom Line: Replicating amyloids, called prions, are responsible for transmissible neurodegenerative diseases in mammals and some heritable phenotypes in fungi.The observed loss of S. cerevisiae [PSI(+)] can be related to inhibition of prion polymerization of S. cerevisiae Sup35 and formation of a non-heritable form of amyloid.We have therefore identified two distinct molecular origins of prion transmission barriers between closely sequence-related prion proteins: first, the inability of heterologous proteins to co-aggregate with host prion polymers, and second, acquisition by these proteins of a non-heritable amyloid fold.

View Article: PubMed Central - PubMed

Affiliation: Cardiology Research Center, Moscow, Russia.

ABSTRACT
Replicating amyloids, called prions, are responsible for transmissible neurodegenerative diseases in mammals and some heritable phenotypes in fungi. The transmission of prions between species is usually inhibited, being highly sensitive to small differences in amino acid sequence of the prion-forming proteins. To understand the molecular basis of this prion interspecies barrier, we studied the transmission of the [PSI(+)] prion state from Sup35 of Saccharomyces cerevisiae to hybrid Sup35 proteins with prion-forming domains from four other closely related Saccharomyces species. Whereas all the hybrid Sup35 proteins could adopt a prion form in S. cerevisiae, they could not readily acquire the prion form from the [PSI(+)] prion of S. cerevisiae. Expression of the hybrid Sup35 proteins in S. cerevisiae [PSI(+)] cells often resulted in frequent loss of the native [PSI(+)] prion. Furthermore, all hybrid Sup35 proteins showed different patterns of interaction with the native [PSI(+)] prion in terms of co-polymerization, acquisition of the prion state, and induced prion loss, all of which were also dependent on the [PSI(+)] variant. The observed loss of S. cerevisiae [PSI(+)] can be related to inhibition of prion polymerization of S. cerevisiae Sup35 and formation of a non-heritable form of amyloid. We have therefore identified two distinct molecular origins of prion transmission barriers between closely sequence-related prion proteins: first, the inability of heterologous proteins to co-aggregate with host prion polymers, and second, acquisition by these proteins of a non-heritable amyloid fold.

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Electrophoretic analysis of co-polymerization of hybrid                                        Sup35 proteins. Yeast cells with the indicated                                        [PSI+]                                    variants were transformed with centromeric plasmids producing                                    hybrid Sup35-3-HA proteins. Cell lysates were loaded onto gels                                    without boiling and run for half a distance. The whole gels were                                    then boiled, and the electrophoretic separation was continued.                                    The gels were blotted, and the blots were stained with antibody                                    to the Sup35-cer N-terminal and middle domains.                                        Sx-3HA, hybrid Sup35-3-HA proteins;                                        Sc, Sup35-cer lacking the tag. For ease of                                    comparison, the values for                                        [PSI+] loss                                    and transmission are given below the gels. The W1                                        panel also shows lysates of the cells having lost                                    the plasmid encoding Sup35-cer (asterisk).
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Figure 4: Electrophoretic analysis of co-polymerization of hybrid Sup35 proteins. Yeast cells with the indicated [PSI+] variants were transformed with centromeric plasmids producing hybrid Sup35-3-HA proteins. Cell lysates were loaded onto gels without boiling and run for half a distance. The whole gels were then boiled, and the electrophoretic separation was continued. The gels were blotted, and the blots were stained with antibody to the Sup35-cer N-terminal and middle domains. Sx-3HA, hybrid Sup35-3-HA proteins; Sc, Sup35-cer lacking the tag. For ease of comparison, the values for [PSI+] loss and transmission are given below the gels. The W1 panel also shows lysates of the cells having lost the plasmid encoding Sup35-cer (asterisk).

Mentions: To differentiate between the different Sup35 proteins, we created an additional set of Sup35 constructs by inserting the 3-HA tag after amino acid residue 251, i.e. just before the C-terminal domain (Sup35-3-HA); addition of the 3-HA tag reduced the electrophoretic mobility of the Sup35 protein. All hybrid Sup35 proteins, including those with the 3-HA tag, supported viability of the otherwise Sup35-deficient 22V-H63-ΔS35 [psi−] strain. Cells producing Sup35 without a 3-HA tag showed the expected non-suppressed phenotype, although the red colony color was less pronounced than in the [psi−] control (Fig. 2, left panel). This phenotypic effect may be related to a minor change in the 5′-untranslated region, which led to a small decrease in Sup35 levels (see Fig. 4, cer lane, and supplemental Fig. S1). Cells producing Sup35 with the 3-HA tag showed an Ade+ phenotype and white colony color, suggesting that the 3-HA tag reduced activity of the Sup35-3-HA proteins in translation termination.


Molecular basis for transmission barrier and interference between closely related prion proteins in yeast.

Afanasieva EG, Kushnirov VV, Tuite MF, Ter-Avanesyan MD - J. Biol. Chem. (2011)

Electrophoretic analysis of co-polymerization of hybrid                                        Sup35 proteins. Yeast cells with the indicated                                        [PSI+]                                    variants were transformed with centromeric plasmids producing                                    hybrid Sup35-3-HA proteins. Cell lysates were loaded onto gels                                    without boiling and run for half a distance. The whole gels were                                    then boiled, and the electrophoretic separation was continued.                                    The gels were blotted, and the blots were stained with antibody                                    to the Sup35-cer N-terminal and middle domains.                                        Sx-3HA, hybrid Sup35-3-HA proteins;                                        Sc, Sup35-cer lacking the tag. For ease of                                    comparison, the values for                                        [PSI+] loss                                    and transmission are given below the gels. The W1                                        panel also shows lysates of the cells having lost                                    the plasmid encoding Sup35-cer (asterisk).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3091186&req=5

Figure 4: Electrophoretic analysis of co-polymerization of hybrid Sup35 proteins. Yeast cells with the indicated [PSI+] variants were transformed with centromeric plasmids producing hybrid Sup35-3-HA proteins. Cell lysates were loaded onto gels without boiling and run for half a distance. The whole gels were then boiled, and the electrophoretic separation was continued. The gels were blotted, and the blots were stained with antibody to the Sup35-cer N-terminal and middle domains. Sx-3HA, hybrid Sup35-3-HA proteins; Sc, Sup35-cer lacking the tag. For ease of comparison, the values for [PSI+] loss and transmission are given below the gels. The W1 panel also shows lysates of the cells having lost the plasmid encoding Sup35-cer (asterisk).
Mentions: To differentiate between the different Sup35 proteins, we created an additional set of Sup35 constructs by inserting the 3-HA tag after amino acid residue 251, i.e. just before the C-terminal domain (Sup35-3-HA); addition of the 3-HA tag reduced the electrophoretic mobility of the Sup35 protein. All hybrid Sup35 proteins, including those with the 3-HA tag, supported viability of the otherwise Sup35-deficient 22V-H63-ΔS35 [psi−] strain. Cells producing Sup35 without a 3-HA tag showed the expected non-suppressed phenotype, although the red colony color was less pronounced than in the [psi−] control (Fig. 2, left panel). This phenotypic effect may be related to a minor change in the 5′-untranslated region, which led to a small decrease in Sup35 levels (see Fig. 4, cer lane, and supplemental Fig. S1). Cells producing Sup35 with the 3-HA tag showed an Ade+ phenotype and white colony color, suggesting that the 3-HA tag reduced activity of the Sup35-3-HA proteins in translation termination.

Bottom Line: Replicating amyloids, called prions, are responsible for transmissible neurodegenerative diseases in mammals and some heritable phenotypes in fungi.The observed loss of S. cerevisiae [PSI(+)] can be related to inhibition of prion polymerization of S. cerevisiae Sup35 and formation of a non-heritable form of amyloid.We have therefore identified two distinct molecular origins of prion transmission barriers between closely sequence-related prion proteins: first, the inability of heterologous proteins to co-aggregate with host prion polymers, and second, acquisition by these proteins of a non-heritable amyloid fold.

View Article: PubMed Central - PubMed

Affiliation: Cardiology Research Center, Moscow, Russia.

ABSTRACT
Replicating amyloids, called prions, are responsible for transmissible neurodegenerative diseases in mammals and some heritable phenotypes in fungi. The transmission of prions between species is usually inhibited, being highly sensitive to small differences in amino acid sequence of the prion-forming proteins. To understand the molecular basis of this prion interspecies barrier, we studied the transmission of the [PSI(+)] prion state from Sup35 of Saccharomyces cerevisiae to hybrid Sup35 proteins with prion-forming domains from four other closely related Saccharomyces species. Whereas all the hybrid Sup35 proteins could adopt a prion form in S. cerevisiae, they could not readily acquire the prion form from the [PSI(+)] prion of S. cerevisiae. Expression of the hybrid Sup35 proteins in S. cerevisiae [PSI(+)] cells often resulted in frequent loss of the native [PSI(+)] prion. Furthermore, all hybrid Sup35 proteins showed different patterns of interaction with the native [PSI(+)] prion in terms of co-polymerization, acquisition of the prion state, and induced prion loss, all of which were also dependent on the [PSI(+)] variant. The observed loss of S. cerevisiae [PSI(+)] can be related to inhibition of prion polymerization of S. cerevisiae Sup35 and formation of a non-heritable form of amyloid. We have therefore identified two distinct molecular origins of prion transmission barriers between closely sequence-related prion proteins: first, the inability of heterologous proteins to co-aggregate with host prion polymers, and second, acquisition by these proteins of a non-heritable amyloid fold.

Show MeSH
Related in: MedlinePlus