Z Du

The Role of Sse1 in the de Novo Formation and Variant Determination of the [PSI+] Prion

Yeast prions are a group of non-Mendelian genetic elements transmitted as altered and self-propagating conformations. Extensive studies in the last decade have provided valuable information on the mechanisms responsible for yeast prion propagation. How yeast prions are formed de novo and what cellular factors are required for determining prion “strains” or variants—a single polypeptide capable of existing in multiple conformations to result in distinct heritable phenotypes—continue to defy our understanding. We report here that Sse1, the yeast ortholog of the mammalian heat-shock protein 110 (Hsp110) and a nucleotide exchange factor for Hsp70 proteins, plays an important role in regulating [PSI+] de novo formation and variant determination. Overproduction of the Sse1 chaperone dramatically enhanced [PSI+] formation whereas deletion of SSE1 severely inhibited it. Only an unstable weak [PSI+] variant was formed in SSE1 disrupted cells whereas [PSI+] variants ranging from very strong to very weak were formed in isogenic wild-type cells under identical conditions. Thus, Sse1 is essential for the generation of multiple [PSI+] variants. Mutational analysis further demonstrated that the physical association of Sse1 with Hsp70 but not the ATP hydrolysis activity of Sse1 is required for the formation of multiple [PSI+] variants. Our findings establish a novel role for Sse1 in [PSI+] de novo formation and variant determination, implying that the mammalian Hsp110 may likewise be involved in the etiology of protein-folding diseases.

Distinct subregions of Swi1 manifest striking differences in prion transmission and SWI/SNF function.

ABSTRACT We have recently reported that the yeast chromatin-remodeling factor Swi1 can exist as a prion, [SWI+], demonstrating a link between prionogenesis and global transcriptional regulation. To shed light on how the Swi1 conformational switch influences Swi1 function and to define the sequence and structural requirements for [SWI+] formation and propagation, we functionally dissected the Swi1 molecule. We show here that the [SWI+] prion features are solely attributable to the first 327 amino acid residues (N), a region that is asparagine rich. N was aggregated in [SWI+] cells but diffuse in [swi−] cells; chromosomal deletion of the N-coding region resulted in [SWI+] loss, and recombinant N peptide was able to form infectious amyloid fibers in vitro, enabling [SWI+] de novo formation through a simple transformation. Although the glutamine-rich middle region (Q) was not sufficient to aggregate in [SWI+] cells or essential for SWI/SNF function, it significantly modified the Swi1 aggregation pattern and Swi1 function. We also show that excessive Swi1 incurred Li+/Na+ sensitivity and that the N/Q regions are important for this gain of sensitivity. Taken together, our results provide the final proof of “protein-only” transmission of [SWI+] and demonstrate that the widely distributed “dispensable” glutamine/asparagine-rich regions/motifs might have important and divergent biological functions.

The complexity and implications of yeast prion domains

Prions are infectious proteins with altered conformations converted from otherwise normal host proteins. While there is only one known mammalian prion protein, PrP, a handful of prion proteins have been identified in the yeast Saccharomyces cerevisiae. Yeast prion proteins usually have a defined region called prion domain (PrD) essential for prion properties, which are typically rich in glutamine (Q) and asparagine (N). Despite sharing several common features, individual yeast PrDs are generally intricate and divergent in their compositional characteristics, which potentially implicates their prion phenotypes, such as prion-mediated transcriptional regulations.

Hypersensitive response and acyl‐homoserine lactone production of the fire blight antagonists Erwinia tasmaniensis and Erwinia billingiae

Fire blight caused by the Gram‐negative bacterium Erwinia amylovora can be controlled by antagonistic microorganisms. We characterized epiphytic bacteria isolated from healthy apple and pear trees in Australia, named Erwinia tasmaniensis, and the epiphytic bacterium Erwinia billingiae from England for physiological properties, interaction with plants and interference with growth of E. amylovora. They reduced symptom formation by the fire blight pathogen on immature pears and the colonization of apple flowers. In contrast to E. billingiae, E. tasmaniensis strains induced a hypersensitive response in tobacco leaves and synthesized levan in the presence of sucrose. With consensus primers deduced from lsc as well as hrpL, hrcC and hrcR of the hrp region of E. amylovora and of related bacteria, these genes were successfully amplified from E. tasmaniensis DNA and alignment of the encoded proteins to other Erwinia species supported a role for environmental fitness of the epiphytic bacterium. Unlike E. tasmaniensis, the epiphytic bacterium E. billingiae produced an acyl‐homoserine lactone for bacterial cell‐to‐cell communication. Their competition with the growth of E. amylovora may be involved in controlling fire blight.

A Small, Glutamine-Free Domain Propagates the (SWI) Prion in Budding Yeast

ABSTRACT Yeast prions are self-propagating protein conformations that transmit heritable phenotypes in an epigenetic manner. The recently identified yeast prion [SWI+] is an alternative conformation of Swi1, a component of the evolutionarily conserved SWI/SNF chromatin-remodeling complex. Formation of the [SWI+] prion results in a partial loss-of-function phenotype for Swi1. The amino-terminal region of Swi1 is dispensable for its normal function but is required for [SWI+] formation and propagation; however, the precise prion domain (PrD) of Swi1 has not been elucidated. Here, we define the minimal Swi1 PrD as the first 37 amino acids of the protein. This region is extremely asparagine rich but, unexpectedly, contains no glutamine residues. This unusually small prion domain is sufficient for aggregation, propagation, and transmission of the [SWI+] prion. Because of its unusual size and composition, the Swi1 prion domain defined here has important implications for describing and identifying novel prions.

New insights into prion biology from the novel [SWI+] system

Our laboratory recently reported a novel prion [SWI+], in the budding yeast Saccharomyces cerevisiae. [SWI+] is the prion form of Swi1, a component of the SWI/SNF chromatin-remodeling complex. Cells harboring [SWI+] exhibit a partial loss-of-function phenotype for SWI/SNF, which can be easily assayed by poor growth on some non-fermentable carbon sources such as raffinose. Swi1 is unique among yeast prion proteins for its nuclear localization and the fact that it comprises part of a large, multi-subunit protein complex. The discovery of [SWI+] demonstrates for the first time a link between prion function and chromatin remodeling, implying a possible role for prions in gene regulation. We believe that the unique features of this novel yeast prion will provide new insight into prion biology.

A potential nuclear envelope-targeting domain and an arginine-rich RNA binding element identified in the putative movement protein of the GAV strain of Barley yellow dwarf virus

In this study, the structural elements in the putative movement protein (MP) of the GAV strain of Barley yellow dwarf virus (BYDV-GAV) were investigated. The GFP fusion protein of BYDV-GAV MP was found to be associated with the nuclear envelope (NE) in transgenic Arabidopsis thaliana (L.) cells. Serial deletion mapping demonstrated that the predicted α-helical domain located at the N-terminus of BYDV-GAV MP was required and sufficient for NE targeting in onion epidermal cells. This α-helical domain does not contain any sequence elements similar to known nuclear localisation signals or bear any significant resemblance to previously characterised NE-targeting structure, indicating that it may represent a novel NE-targeting domain in plant cells. Deletion mutagenesis showed that the C-terminal end of BYDV-GAV MP possessed an element required for its RNA binding activity in vitro. Further analysis revealed that the arginine amino acids within the last 11 residues of the C-terminal end were crucial for the binding of BYDV-GAV MP to RNA. This C-terminal element enriched in basic residues was also present in the MPs of other BYDV strains and the polerovirus Potato leaf roll virus (PLRV), suggesting the conservation of a RNA binding element in the MPs from both luteoviruses and poleroviruses. The data in this work present an initial characterisation of a novel plant NE-targeting domain and a RNA binding element on BYDV-GAV MP. Further studies are underway to investigate the function of these elements in the biology of natural BYDV-GAV infection.

Analysis of small critical regions of Swi1 conferring prion formation, maintenance, and transmission

ABSTRACT Saccharomyces cerevisiae contains several prion elements, which are epigenetically transmitted as self-perpetuating protein conformations. One such prion is [SWI+], whose protein determinant is Swi1, a subunit of the SWI/SNF chromatin-remodeling complex. We previously reported that [SWI+] formation results in a partial loss-of-function phenotype of poor growth in nonglucose medium and abolishment of multicellular features. We also showed that the first 38 amino acids of Swi1 propagated [SWI+]. We show here that a region as small as the first 32 amino acids of Swi1 (Swi11–32) can decorate [SWI+] aggregation and stably maintain and transmit [SWI+] independently of full-length Swi1. Regions smaller than Swi11–32 are either incapable of aggregation or unstably propagate [SWI+]. When fused to Sup35MC, the [PSI+] determinant lacking its PrD, Swi11–31 and Swi11–32 can act as transferable prion domains (PrDs). The resulting fusions give rise to a novel chimeric prion, [SPS+], exhibiting [PSI+]-like nonsense suppression. Thus, an NH2-terminal region of ∼30 amino acids of Swi1 contains all the necessary information for in vivo prion formation, maintenance, and transmission. This PrD is unique in size and composition: glutamine free, asparagine rich, and the smallest defined to date. Our findings broaden our understanding of what features allow a protein region to serve as a PrD.

An insight into the complex prion-prion interaction network in the budding yeast Saccharomyces cerevisiae

The budding yeast Saccharomyces cerevisiae is a valuable model system for studying prion-prion interactions as it contains multiple prion proteins. A recent study from our laboratory showed that the existence of Swi1 prion ([SWI+]) and overproduction of Swi1 can have strong impacts on the formation of 2 other extensively studied yeast prions, [PSI+] and [PIN+] ([RNQ+]) (Genetics, Vol. 197, 685–700). We showed that a single yeast cell is capable of harboring at least 3 heterologous prion elements and these prions can influence each others appearance positively and/or negatively. We also showed that during the de novo [PSI+] formation process upon Sup35 overproduction, the aggregation patterns of a preexisting inducer ([RNQ+] or [SWI+]) can undergo significant remodeling from stably transmitted dot-shaped aggregates to aggregates that co-localize with the newly formed Sup35 aggregates that are ring/ribbon/rod- shaped. Such co-localization disappears once the newly formed [PSI+] prion stabilizes. Our finding provides strong evidence supporting the “cross-seeding” model for prion-prion interactions and confirms earlier reports that the interactions among different prions and their prion proteins mostly occur at the initiation stages of prionogenesis. Our results also highlight a complex prion interaction network in yeast. We believe that elucidating the mechanism underlying the yeast prion-prion interaction network will not only provide insight into the process of prion de novo generation and propagation in yeast but also shed light on the mechanisms that govern protein misfolding, aggregation, and amyloidogenesis in higher eukaryotes.