Increasing Prion Propensity by Hydrophobic Insertion

Abstract

Prion formation involves the conversion of proteins from a soluble form into an infectious amyloid form. Most yeast prion proteins contain glutamine/asparagine-rich regions that are responsible for prion aggregation. Prion formation by these domains is driven primarily by amino acid composition, not primary sequence, yet there is a surprising disconnect between the amino acids thought to have the highest aggregation propensity and those that are actually found in yeast prion domains. Specifically, a recent mutagenic screen suggested that both aromatic and non-aromatic hydrophobic residues strongly promote prion formation. However, while aromatic residues are common in yeast prion domains, non-aromatic hydrophobic residues are strongly under-represented. Here, we directly test the effects of hydrophobic and aromatic residues on prion formation. Remarkably, we found that insertion of as few as two hydrophobic residues resulted in a multiple orders-of-magnitude increase in prion formation, and significant acceleration of in vitro amyloid formation. Thus, insertion or deletion of hydrophobic residues provides a simple tool to control the prion activity of a protein. These data, combined with bioinformatics analysis, suggest a limit on the number of strongly prion-promoting residues tolerated in glutamine/asparagine-rich domains. This limit may explain the under-representation of non-aromatic hydrophobic residues in yeast prion domains. Prion activity requires not only that a protein be able to form prion fibers, but also that these fibers be cleaved to generate new independently-segregating aggregates to offset dilution by cell division. Recent studies suggest that aromatic residues, but not non-aromatic hydrophobic residues, support the fiber cleavage step. Therefore, we propose that while both aromatic and non-aromatic hydrophobic residues promote prion formation, aromatic residues are favored in yeast prion domains because they serve a dual function, promoting both prion formation and chaperone-dependent prion propagation.

Distinct Amino Acid Compositional Requirements for Formation and Maintenance of the [PSI+] Prion in Yeast

“…5-FOA was used to select for loss of pJ526. Plasmids for transient overexpression of each PFD from the GAL1 promoter were constructed, and prion formation assays were performed as previously described (42).…”

“…Plasmids expressing PFD-green fluorescent protein (GFP) fusions were constructed as previously described (42). To test for focus formation, these plasmids were transformed into 780-1D/pJ533 and YER632/ pJ533.…”

“…The Sup35 PFD contains 20 Tyr residues, one Phe, and no Trp, Ile, or Val. Five of the Tyr residues are located in the ND, and nonaromatic hydrophobic residues can replace these ND tyrosines in supporting prion activity (42). Here, we examined the effects of replacing different residues for Tyr in the ORD.…”

A new player in X identification: the CLAMP protein is a key factor in Drosophila dosage compensation

“…Clearly, a physical interaction between CLAMP and MSL complex would be required for this. This would likely be mediated through the glutamine-rich domain of CLAMP because glutamine-rich domains are known to be involved in protein–protein interactions (Wang et al 2013; Gonzalez Nelson et al 2014). …”

Amyloids or prions? That is the question

“…It has been proposed that this might respond to the fact that aromatic residues might facilitate both prion formation and chaperone dependent prion propagation. 33 However, F is indeed under-represented in PFDs with an odds ratio of 0.72 and the Y/F relationship between odds ratios in PFDs is 2.4, suggesting that the additional hydroxyl group in Y should provide a certain advantage, which in our opinion is allowing a better balance of amyloid propensity and intrinsic disorder. Despite its aromatic character, W is one of the most under-represented residues in prion domains with and odd ratio of 0.091, only C, which is able to crosslink covalently polypeptide chains, being less frequent.…”