Gateway vectors for efficient artificial gene assembly in vitro and expression in yeast Saccharomyces cerevisiae

Peer reviewedPublisher PD

[SWI+], the Prion Formed by the Chromatin Remodeling Factor Swi1, Is Highly Sensitive to Alterations in Hsp70 Chaperone System Activity

The yeast prion [SWI+], formed of heritable amyloid aggregates of the Swi1 protein, results in a partial loss of function of the SWI/SNF chromatin-remodeling complex, required for the regulation of a diverse set of genes. Our genetic analysis revealed that [SWI+] propagation is highly dependent upon the action of members of the Hsp70 molecular chaperone system, specifically the Hsp70 Ssa, two of its J-protein co-chaperones, Sis1 and Ydj1, and the nucleotide exchange factors of the Hsp110 family (Sse1/2). Notably, while all yeast prions tested thus far require Sis1, [SWI+] is the only one known to require the activity of Ydj1, the most abundant J-protein in yeast. The C-terminal region of Ydj1, which contains the client protein interaction domain, is required for [SWI+] propagation. However, Ydj1 is not unique in this regard, as another, closely related J-protein, Apj1, can substitute for it when expressed at a level approaching that of Ydj1. While dependent upon Ydj1 and Sis1 for propagation, [SWI+] is also highly sensitive to overexpression of both J-proteins. However, this increased prion-loss requires only the highly conserved 70 amino acid J-domain, which serves to stimulate the ATPase activity of Hsp70 and thus to stabilize its interaction with client protein. Overexpression of the J-domain from Sis1, Ydj1, or Apj1 is sufficient to destabilize [SWI+]. In addition, [SWI+] is lost upon overexpression of Sse nucleotide exchange factors, which act to destabilize Hsp70's interaction with client proteins. Given the plethora of genes affected by the activity of the SWI/SNF chromatin-remodeling complex, it is possible that this sensitivity of [SWI+] to the activity of Hsp70 chaperone machinery may serve a regulatory role, keeping this prion in an easily-lost, meta-stable state. Such sensitivity may provide a means to reach an optimal balance of phenotypic diversity within a cell population to better adapt to stressful environments

Amyloid-Mediated Sequestration of Essential Proteins Contributes to Mutant Huntingtin Toxicity in Yeast

BACKGROUND: Polyglutamine expansion is responsible for several neurodegenerative disorders, among which Huntington disease is the most well-known. Studies in the yeast model demonstrated that both aggregation and toxicity of a huntingtin (htt) protein with an expanded polyglutamine region strictly depend on the presence of the prion form of Rnq1 protein ([PIN+]), which has a glutamine/asparagine-rich domain. PRINCIPAL FINDINGS: Here, we showed that aggregation and toxicity of mutant htt depended on [PIN+] only quantitatively: the presence of [PIN+] elevated the toxicity and the levels of htt detergent-insoluble polymers. In cells lacking [PIN+], toxicity of mutant htt was due to the polymerization and inactivation of the essential glutamine/asparagine-rich Sup35 protein and related inactivation of another essential protein, Sup45, most probably via its sequestration into Sup35 aggregates. However, inhibition of growth of [PIN+] cells depended on Sup35/Sup45 depletion only partially, suggesting that there are other sources of mutant htt toxicity in yeast. CONCLUSIONS: The obtained data suggest that induced polymerization of essential glutamine/asparagine-rich proteins and related sequestration of other proteins which interact with these polymers represent an essential source of htt toxicity

Complex Adaptations Can Drive the Evolution of the Capacitor [PSI+], Even with Realistic Rates of Yeast Sex

The [PSI+] prion may enhance evolvability by revealing previously cryptic genetic variation, but it is unclear whether such evolvability properties could be favored by natural selection. Sex inhibits the evolution of other putative evolvability mechanisms, such as mutator alleles. This paper explores whether sex also prevents natural selection from favoring modifier alleles that facilitate [PSI+] formation. Sex may permit the spread of “cheater” alleles that acquire the benefits of [PSI+] through mating without incurring the cost of producing [PSI+] at times when it is not adaptive. Using recent quantitative estimates of the frequency of sex in Saccharomyces paradoxus, we calculate that natural selection for evolvability can drive the evolution of the [PSI+] system, so long as yeast populations occasionally require complex adaptations involving synergistic epistasis between two loci. If adaptations are always simple and require substitution at only a single locus, then the [PSI+] system is not favored by natural selection. Obligate sex might inhibit the evolution of [PSI+]-like systems in other species

The Ankyrin Repeats and DHHC S-acyl Transferase Domain of AKR1 Act Independently to Regulate Switching from Vegetative to Mating States in Yeast

Signal transduction from G-protein coupled receptors to MAPK cascades through heterotrimeric G-proteins has been described for many eukaryotic systems. One of the best-characterised examples is the yeast pheromone response pathway, which is negatively regulated by AKR1. AKR1-like proteins are present in all eukaryotes and contain a DHHC domain and six ankyrin repeats. Whilst the DHHC domain dependant S-acyl transferase (palmitoyl transferase) function of AKR1 is well documented it is not known whether the ankyrin repeats are also required for this activity. Here we show that the ankyrin repeats of AKR1 are required for full suppression of the yeast pheromone response pathway, by sequestration of the Gβγ dimer, and act independently of AKR1 S-acylation function. Importantly, the functions provided by the AKR1 ankyrin repeats and DHHC domain are not required on the same molecule to fully restore WT phenotypes and function. We also show that AKR1 molecules are S-acylated at locations other than the DHHC cysteine, increasing the abundance of AKR1 in the cell. Our results have important consequences for studies of AKR1 function, including recent attempts to characterise S-acylation enzymology and kinetics. Proteins similar to AKR1 are found in all eukaryotes and our results have broad implications for future work on these proteins and the control of switching between Gβγ regulated pathways

The Number and Transmission of [PSI+] Prion Seeds (Propagons) in the Yeast Saccharomyces cerevisiae

Yeast (Saccharomyces cerevisiae) prions are efficiently propagated and the on-going generation and transmission of prion seeds (propagons) to daughter cells during cell division ensures a high degree of mitotic stability. The reversible inhibition of the molecular chaperone Hsp104p by guanidine hydrochloride (GdnHCl) results in cell division-dependent elimination of yeast prions due to a block in propagon generation and the subsequent dilution out of propagons by cell division.Analysing the kinetics of the GdnHCl-induced elimination of the yeast [PSI+] prion has allowed us to develop novel statistical models that aid our understanding of prion propagation in yeast cells. Here we describe the application of a new stochastic model that allows us to estimate more accurately the mean number of propagons in a [PSI+] cell. To achieve this accuracy we also experimentally determine key cell reproduction parameters and show that the presence of the [PSI+] prion has no impact on these key processes. Additionally, we experimentally determine the proportion of propagons transmitted to a daughter cell and show this reflects the relative cell volume of mother and daughter cells at cell division.While propagon generation is an ATP-driven process, the partition of propagons to daughter cells occurs by passive transfer via the distribution of cytoplasm. Furthermore, our new estimates of n(0), the number of propagons per cell (500-1000), are some five times higher than our previous estimates and this has important implications for our understanding of the inheritance of the [PSI+] and the spontaneous formation of prion-free cells

Conversion of deoxynivalenol to 3-acetyldeoxynivalenol in barley-derived fuel ethanol co-products with yeast expressing trichothecene 3-O-acetyltransferases

<p>Abstract</p> <p>Background</p> <p>The trichothecene mycotoxin deoxynivalenol (DON) may be concentrated in distillers dried grains with solubles (DDGS; a co-product of fuel ethanol fermentation) when grain containing DON is used to produce fuel ethanol. Even low levels of DON (≤ 5 ppm) in DDGS sold as feed pose a significant threat to the health of monogastric animals. New and improved strategies to reduce DON in DDGS need to be developed and implemented to address this problem. Enzymes known as trichothecene 3-<it>O-</it>acetyltransferases convert DON to 3-acetyldeoxynivalenol (3ADON), and may reduce its toxicity in plants and animals.</p> <p>Results</p> <p>Two <it>Fusarium </it>trichothecene 3-<it>O-</it>acetyltransferases (FgTRI101 and FfTRI201) were cloned and expressed in yeast (<it>Saccharomyces cerevisiae</it>) during a series of small-scale ethanol fermentations using barley (<it>Hordeum vulgare</it>). DON was concentrated 1.6 to 8.2 times in DDGS compared with the starting ground grain. During the fermentation process, FgTRI101 converted 9.2% to 55.3% of the DON to 3ADON, resulting in DDGS with reductions in DON and increases in 3ADON in the Virginia winter barley cultivars Eve, Thoroughbred and Price, and the experimental line VA06H-25. Analysis of barley mashes prepared from the barley line VA04B-125 showed that yeast expressing FfTRI201 were more effective at acetylating DON than those expressing FgTRI101; DON conversion for FfTRI201 ranged from 26.1% to 28.3%, whereas DON conversion for FgTRI101 ranged from 18.3% to 21.8% in VA04B-125 mashes. Ethanol yields were highest with the industrial yeast strain Ethanol Red^®, which also consumed galactose when present in the mash.</p> <p>Conclusions</p> <p>This study demonstrates the potential of using yeast expressing a trichothecene 3-<it>O</it>-acetyltransferase to modify DON during commercial fuel ethanol fermentation.</p

Substrate specificity of the TRAMP nuclear surveillance complexes

During nuclear surveillance in yeast and human cells, the RNA exosome functions together with the TRAMP complexes. Here, the authors defined the protein composition of the TRAMP complexes and identified specific RNA binding sites for the different TRAMP components