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

Interaction of human laminin receptor with Sup35, the [PSI⁺] prion-forming protein from S. cerevisiae: a yeast model for studies of LamR interactions with amyloidogenic proteins.

The laminin receptor (LamR) is a cell surface receptor for extracellular matrix laminin, whereas the same protein within the cell interacts with ribosomes, nuclear proteins and cytoskeletal fibers. LamR has been shown to be a receptor for several bacteria and viruses. Furthermore, LamR interacts with both cellular and infectious forms of the prion protein, PrP(C) and PrP(Sc). Indeed, LamR is a receptor for PrP(C). Whether LamR interacts with PrP(Sc) exclusively in a capacity of the PrP receptor, or LamR specifically recognizes prion determinants of PrP(Sc), is unclear. In order to explore whether LamR has a propensity to interact with prions and amyloids, we examined LamR interaction with the yeast prion-forming protein, Sup35. Sup35 is a translation termination factor with no homology or functional relationship to PrP. Plasmids expressing LamR or LamR fused with the green fluorescent protein (GFP) were transformed into yeast strain variants differing by the presence or absence of the prion conformation of Sup35, respectively [PSI⁺] and [psi⁻]. Analyses by immunoprecipitation, centrifugal fractionation and fluorescent microscopy reveal interaction between LamR and Sup35 in [PSI⁺] strains. The presence of [PSI⁺] promotes LamR co-precipitation with Sup35 as well as LamR aggregation. In [PSI⁺] cells, LamR tagged with GFP or mCherry forms bright fluorescent aggregates that co-localize with visible [PSI⁺] foci. The yeast prion model will facilitate studying the interaction of LamR with amyloidogenic prions in a safe and easily manipulated system that may lead to a better understanding and treatment of amyloid diseases

Comment on Billant et al. p53, A Victim of the Prion Fashion. <i>Cancers</i> 2021, <i>13</i>, 269

The p53 tumor suppressor is a central protein in the fight against cancer [...

Centrifugation assay demonstrates aggregation of exogenous human LamR in the presence of the [<i>PSI</i>⁺] prion.

<p>(A) Total lysate (T), supernatant (S), and resuspended pellet (P) (20 µg per sample) of pCUP1-LamR::GFP and pCUP1-LamR transformants of yeast [<i>psi</i>⁻][<i>pin</i>⁻] and [<i>PSI⁺</i>][<i>PIN⁺</i>] strains were analyzed by western blot using the indicated antibodies. pCUP1-GFP was expressed in the [<i>PSI</i>⁺] strain as a control (bottom panel), and anti-yeast hexokinase antibody (anti-HK) was used to ensure pellets were free of cytoplasmic proteins (fifth panel from top). Shown are representative experiments out of 3 independent experiments. (B) Corresponding densitometric quantitation of percent distribution between supernatant and pellet fractions was determined from three independent experiments. Bars show standard error of the mean.</p

Human LamR is expressed in yeast cells.

<p>A. LamR protein (right panel) or LamR-GFP fusion (left panel) expressed in the [<i>PSI</i>⁺][<i>PIN⁺</i>] yeast prion strain grown in synthetic media either supplemented with 25 µM CuSO₄ (right panel and lane 2 of left panel) or containing no excess copper (lane1 of left panel). B. Expression of GPF (27 kDa), and LamR-GFP and Sup35-GFP fusion proteins (64 kDa and 104 kDa, respectively) in [<i>psi</i>⁻][<i>PIN⁺</i>] (−) and [<i>PSI⁺</i>][<i>PIN⁺</i>] (+) yeast strains. Anti-LamR (A) and anti-GFP (B) antibodies were used to detect LamR expression in yeast lysates (25 ug). Numbers in the middle (A) and right (B) refer to protein size markers (kDa). Similar expression levels were observed in [<i>pin</i>⁻] strains (not shown).</p

Immunofluorescence of LamR-GFP and Sup35-GFP fusion proteins reveals aggregation of LamR in [<i>PSI</i>⁺] cells.

<p>Late-log cultures of (A) [<i>PSI⁺</i>][<i>PIN⁺</i>], (B) [<i>PSI⁺</i>][<i>pin</i>⁻], (C) [<i>psi</i>⁻][<i>PIN⁺</i>] and (D) [<i>psi</i>⁻ ][<i>pin</i>⁻] were examined with 100× oil immersion lens of a fluorescent microscope using a 488_ex, 507_em filter. Representative images from two independent transformants of each yeast strain are displayed.</p