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

PLoS Genetics, Jan 2015

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.

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

Liebman SW (2015) Heterologous Aggregates Promote De Novo Prion Appearance via More than One Mechanism. PLoS Genet 11(1): e1004814. doi:10.1371/journal.pgen.1004814 Heterologous Aggregates Promote De Novo Prion Appearance via More than One Mechanism Fatih Arslan 0 Joo Y. Hong 0 Vydehi Kanneganti 0 Sei-Kyoung Park 0 Susan W. Liebman 0 Heather L. True-Krob, Washington University School of Medicine, United States of America 0 1 Department of Biological Sciences, University of Illinois at Chicago , Chicago , Illinois, United States of America, 2 Department of Biochemistry and Molecular Biology, University of Nevada , Reno, Nevada , United States of America 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. - Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper and its Supporting Information files. Funding: This work was supported by the National Institutes of Health (NIH) Grant R01GM056350 to SWL and an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under grant number P20GM103554. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Prions were first described as self-perpetuating infectious agents devoid of nucleic acids that cause several fatal neurodegenerative diseases. Prion diseases, also known as transmissible spongiform encephalopathies (TSEs), were shown to infect a variety of mammals [1]. All known mammalian prion diseases are caused by conversion of largely a-helical cellular prion protein PrPC into fibrous b-sheet-rich ordered aggregates (amyloids) called PrPSc (associated with scrapie) [2]. Curiously, PrPSc can exist in different heritable forms, called strains, which cause neurodegenerative diseases with different characteristics and pathologies [35]. A number of other neurodegenerative diseases are also associated with conversion of a soluble protein to amyloid. For example, amyloid-like forms of Ab and Tau, a-synuclein, huntingtin, FUS/TLS, TDP-43 or SOD1 are linked respectively to Alzheimers (AD) [6], Parkinsons (PD) [7,8], Huntingtons (Htt) [9] and Amyotrophic Lateral Sclerosis (ALS) diseases [1015]. Factors that influence the spontaneous conversion to amyloid are of considerable interest as possible disease risk factors. One important finding is that heterologous amyloid can promote the de novo conversion of a protein to amyloid. For example, Ab accelerated the in vivo aggregation of tau [16], and Ab and asynuclein seeded each others aggregation in vitro [17]. Indeed, recently distinct conformational variants of a-synuclein aggregates were shown to differentially promote the aggregation of tau in neurons [18]. Several proteins in the simple eukaryote yeast have been shown to convert from soluble to amyloid. The amyloid forms of these prote (...truncated)


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Fatih Arslan, Joo Y. Hong, Vydehi Kanneganti, Sei-Kyoung Park, Susan W. Liebman. Heterologous Aggregates Promote De Novo Prion Appearance via More than One Mechanism, PLoS Genetics, 2015, Volume 11, Issue 1, DOI: 10.1371/journal.pgen.1004814