Distinct Type of Transmission Barrier Revealed by Study of Multiple Prion Determinants of Rnq1

Prions are self-propagating protein conformations. Transmission of the prion state between non-identical proteins, e.g. between homologous proteins from different species, is frequently inefficient. Transmission barriers are attributed to sequence differences in prion proteins, but their underlying mechanisms are not clear. Here we use a yeast Rnq1/[PIN+]-based experimental system to explore the nature of transmission barriers. [PIN+], the prion form of Rnq1, is common in wild and laboratory yeast strains, where it facilitates the appearance of other prions. Rnq1's prion domain carries four discrete QN-rich regions. We start by showing that Rnq1 encompasses multiple prion determinants that can independently drive amyloid formation in vitro and transmit the [PIN+] prion state in vivo. Subsequent analysis of [PIN+] transmission between Rnq1 fragments with different sets of prion determinants established that (i) one common QN-rich region is required and usually sufficient for the transmission; (ii) despite identical sequences of the common QNs, such transmissions are impeded by barriers of different strength. Existence of transmission barriers in the absence of amino acid mismatches in transmitting regions indicates that in complex prion domains multiple prion determinants act cooperatively to attain the final prion conformation, and reveals transmission barriers determined by this cooperative fold

[PSI+] Maintenance Is Dependent on the Composition, Not Primary Sequence, of the Oligopeptide Repeat Domain

[PSI+], the prion form of the yeast Sup35 protein, results from the structural conversion of Sup35 from a soluble form into an infectious amyloid form. The infectivity of prions is thought to result from chaperone-dependent fiber cleavage that breaks large prion fibers into smaller, inheritable propagons. Like the mammalian prion protein PrP, Sup35 contains an oligopeptide repeat domain. Deletion analysis indicates that the oligopeptide repeat domain is critical for [PSI+] propagation, while a distinct region of the prion domain is responsible for prion nucleation. The PrP oligopeptide repeat domain can substitute for the Sup35 oligopeptide repeat domain in supporting [PSI+] propagation, suggesting a common role for repeats in supporting prion maintenance. However, randomizing the order of the amino acids in the Sup35 prion domain does not block prion formation or propagation, suggesting that amino acid composition is the primary determinant of Sup35's prion propensity. Thus, it is unclear what role the oligopeptide repeats play in [PSI+] propagation: the repeats could simply act as a non-specific spacer separating the prion nucleation domain from the rest of the protein; the repeats could contain specific compositional elements that promote prion propagation; or the repeats, while not essential for prion propagation, might explain some unique features of [PSI+]. Here, we test these three hypotheses and show that the ability of the Sup35 and PrP repeats to support [PSI+] propagation stems from their amino acid composition, not their primary sequences. Furthermore, we demonstrate that compositional requirements for the repeat domain are distinct from those of the nucleation domain, indicating that prion nucleation and propagation are driven by distinct compositional features