Heterologous Aggregates Promote De Novo Prion Appearance via More than One Mechanism

Abstract

Prions are self-perpetuating conformational variants of particular proteins. In yeast, prions cause heritable phenotypic traits. Most known yeast prions contain a glutamine (Q)/asparagine (N)-rich region in their prion domains. [PSI+], the prion form of Sup35, appears de novo at dramatically enhanced rates following transient overproduction of Sup35 in the presence of [PIN+], the prion form of Rnq1. Here, we establish the temporal de novo appearance of Sup35 aggregates during such overexpression in relation to other cellular proteins. Fluorescently-labeled Sup35 initially forms one or a few dots when overexpressed in [PIN+] cells. One of the dots is perivacuolar, colocalizes with the aggregated Rnq1 dot and grows into peripheral rings/lines, some of which also colocalize with Rnq1. Sup35 dots that are not near the vacuole do not always colocalize with Rnq1 and disappear by the time rings start to grow. Bimolecular fluorescence complementation failed to detect any interaction between Sup35-VN and Rnq1-VC in [PSI +][PIN +] cells. In contrast, all Sup35 aggregates, whether newly induced or in established [PSI +], completely colocalize with the molecular chaperones Hsp104, Sis1, Ssa1 and eukaryotic release factor Sup45. In the absence of [PIN+], overexpressed aggregating proteins such as the Q/N-rich Pin4C or the non-Q/N-rich Mod5 can also promote the de novo appearance of [PSI +]. Similar to Rnq1, overexpressed Pin4C transiently colocalizes with newly appearing Sup35 aggregates. However, no interaction was detected between Mod5 and Sup35 during [PSI+] induction in the absence of [PIN +]. While the colocalization of Sup35 and aggregates of Rnq1 or Pin4C are consistent with the model that the heterologous aggregates cross-seed the de novo appearance of [PSI +], the lack of interaction between Mod5 and Sup35 leaves open the possibility of other mechanisms. We also show that Hsp104 is required in the de novo appearance of [PSI+] aggregates in a [PIN +]-independent pathway.

Quantifying nucleationin vivoreveals the physical basis of prion-like phase behavior

“…It has no restrictions on subcellular localization, solubility, or size of the assemblies, thereby mitigating false-negatives and false-positives. In contrast, existing high-throughput assays for protein aggregation report on large puncta (Narayanaswamy et al, 2009;Noree et al, 2010;Pereira et al, 2018;Ramdzan et al, 2012) , inactivation of fusion partners (Alberti et al, 2009;Morell et al, 2011;Newby et al, 2017;Waldo et al, 1999;Zhao et al, 2016) , require restrictive subcellular localization (Sivanathan and Hochschild, 2013) , and/or necessitate expression from dual constructs (Arslan et al, 2015;Blakeley et al, 2012;Cabantous et al, 2013;Shyu and Hu, 2008) . Most importantly, DAmFRET simultaneously reports total protein concentration, and thereby the concentration-dependence of self-assembly, in every single sample.…”

“…Rnq1 prions, for example, enable the formation of Sup35 and Ure2 prions (Derkatch et al, 2001) . The extent to which other prion proteins share this dependence, and its molecular underpinnings, remain unclear (Arslan et al, 2015;Keefer et al, 2017;Suzuki et al, 2012) .…”

Actin impacts the late stages of prion formation and prion propagation

“…Once we confirmed that the variability observed in act1-122 mutants was not due to unforeseen suppression defects, we postulated that the [PIN + ] prion could be changed in these mutants. The cross-seeding model suggests that [PIN + ] increases [PSI + ] induction through heterologous cross seeding of new Sup35p aggregates (Derkatch et al, 2004;Arslan et al, 2015). Therefore, changes in [PIN + ] could result in changes in [PSI + ] induction in act1-122 mutants.…”

“…The visualization of newly formed aggregates during the prion induction process is mediated through the fusion of a fluorescent marker to the Sup35p prion domain (Sup35PrD-GFP; Zhou et al, 2001). Overexpression of Sup35PrD-GFP leads to the appearance of small, transient "early foci" that assemble into cytosolic ring or dot aggregates (Arslan et al, 2015;Sharma et al, 2017). Micromanipulation of either ring or dot containing cells gives rise to future generations that contain [PSI + ] (Ganusova et al, 2006;Sharma et al, 2017), indicating that cells containing these newly formed aggregates are precursors to [PSI + ].…”

Strain conformation controls the specificity of cross-species prion transmission in the yeast model

“…25,52,57 However, an interaction between these proteins is of transient nature and is observed only at early stages of the induction process, while persistent coaggregates are not formed and stable colocalization is not reported. [58][59][60] It was shown that different amylodogenic proteins can be assembled in the form of ordered aggregates in the yeast quality control deposits, such as insoluble protein deposit, IPOD, 61 or aggresome 62,63 (that may represent a version of IPOD with a somewhat different intracellular location). However, the very distantly related Sup35 protein from the other yeast genera, Pichia does not colocalize with the preexisting S. cerevisiae Sup35 prion, 50 suggesting that in case of more closely related proteins, colocalization might be not simply a result of co-sequestration into IPOD.…”