scholarly journals [URE3] Prion Propagation in Saccharomyces cerevisiae: Requirement for Chaperone Hsp104 and Curing by Overexpressed Chaperone Ydj1p

2000 ◽  
Vol 20 (23) ◽  
pp. 8916-8922 ◽  
Author(s):  
Hiromitsu Moriyama ◽  
Herman K. Edskes ◽  
Reed B. Wickner
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Conformation ◽  
Common Factor ◽  
Complete Loss ◽  
Insertion Mutation ◽  
Genetic Element ◽  
Yeast Prions ◽  
Prion Propagation ◽  
The Stability ◽  
Infectious Form

ABSTRACT The [URE3] nonchromosomal genetic element is an infectious form (prion) of the Ure2 protein, apparently a self-propagating amyloidosis. We find that an insertion mutation or deletion of HSP104results in inability to propagate the [URE3] prion. Our results indicate that Hsp104 is a common factor in the maintenance of two independent yeast prions. However, overproduction of Hsp104 does not affect the stability of [URE3], in contrast to what is found for the [PSI+] prion, which is known to be cured by either overproduction or deficiency of Hsp104. Like Hsp104, the Hsp40 class chaperone Ydj1p, with the Hsp70 class Ssa1p, can renature proteins. We find that overproduction of Ydj1p results in a gradual complete loss of [URE3]. The involvement of protein chaperones in the propagation of [URE3] indicates a role for protein conformation in inheritance.

scholarly journals Schizosaccharomyces pombe Disaggregation Machinery Chaperones Support Saccharomyces cerevisiae Growth and Prion Propagation

2013 ◽  
Vol 12 (5) ◽  
pp. 739-745 ◽  
Author(s):  
Michael Reidy ◽  
Ruchika Sharma ◽  
Daniel C. Masison
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Schizosaccharomyces Pombe ◽  
Similar Species ◽  
Nucleotide Exchange ◽  
Content Type ◽  
E Coli ◽  
Yeast Prions ◽  
Nucleotide Exchange Factor ◽  
Prion Propagation ◽  
Species Specific

ABSTRACT Hsp100 chaperones protect microorganisms and plants from environmental stress by cooperating with Hsp70 and its nucleotide exchange factor (NEF) and Hsp40 cochaperones to resolubilize proteins from aggregates. The Saccharomyces cerevisiae Hsp104 (Sc-Hsp104)-based disaggregation machinery also is essential for replication of amyloid-based prions. Escherichia coli ClpB can substitute for Hsp104 to propagate [ PSI + ] prions in yeast, but only if E. coli DnaK and GrpE (Hsp70 and NEF) are coexpressed. Here, we tested if the reported inability of Schizosaccharomyces pombe Hsp104 (Sp-Hsp104) to support [ PSI + ] propagation was due to similar species-specific chaperone requirements and find that Sp-Hsp104 alone supported propagation of three different yeast prions. Sp-Hsp70 and Sp-Fes1p (NEF) likewise functioned in place of their Sa. cerevisiae counterparts. Thus, chaperones of these long-diverged species possess conserved activities that function in processes essential for both cell growth and prion propagation, suggesting Sc. pombe can propagate its own prions. We show that curing by Hsp104 overexpression and inactivation can be distinguished and confirm the observation that, unlike Sc-Hsp104, Sp-Hsp104 cannot cure yeast of [ PSI + ] when it is overexpressed. These results are consistent with a view that mechanisms underlying prion replication and elimination are distinct.

scholarly journals Development and Characterization of Two Types of Surface Displayed Levansucrases for Levan Biosynthesis

Catalysts ◽  
2021 ◽  
Vol 11 (7) ◽  
pp. 757
Author(s):  
Huiyi Shang ◽  
Danni Yang ◽  
Dairong Qiao ◽  
Hui Xu ◽  
Yi Cao
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Molecular Weight ◽  
Large Scale ◽  
Surface Display ◽  
Yeast Surface Display ◽  
Fusion Partner ◽  
Whole Cell ◽  
Food Industries ◽  
Yeast Surface ◽  
The Stability

Levan has wide applications in chemical, cosmetic, pharmaceutical and food industries. The free levansucrase is usually used in the biosynthesis of levan, but the poor reusability and low stability of free levansucrase have limited its large-scale use. To address this problem, the surface-displayed levansucrase in Saccharomyces cerevisiae were generated and evaluated in this study. The levansucrase from Zymomonas mobilis was displayed on the cell surface of Saccharomyces cerevisiae EBY100 using a various yeast surface display platform. The N-terminal fusion partner is based on a-agglutinin, and the C-terminal one is Flo1p. The yield of levan produced by these two whole-cell biocatalysts reaches 26 g/L and 34 g/L in 24 h, respectively. Meanwhile, the stability of the surface-displayed levansucrases is significantly enhanced. After six reuses, these two biocatalysts retained over 50% and 60% of their initial activities, respectively. Furthermore, the molecular weight and polydispersity test of the products suggested that the whole-cell biocatalyst of levansucrase displayed by Flo1p has more potentials in the production of levan with low molecular weight which is critical in certain applications. In conclusion, our method not only enable the possibility to reuse the enzyme, but also improves the stability of the enzyme.

Effect of limited homology on gene conversion in a Saccharomyces cerevisiae plasmid recombination system

1988 ◽  
Vol 8 (6) ◽  
pp. 2442-2448 ◽  
Author(s):  
B Y Ahn ◽  
K J Dornfeld ◽  
T J Fagrelius ◽  
D M Livingston
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Gene Conversion ◽  
Dna Sequences ◽  
Homologous Sequence ◽  
Insertion Mutation ◽  
Base Pairs ◽  
Recombination System ◽  
Plasmid Recombination ◽  
His3 Gene ◽  
Conversion Tract

Plasmids containing heteroallelic copies of the Saccharomyces cerevisiae HIS3 gene undergo intramolecular gene conversion in mitotically dividing S. cerevisiae cells. We have used this plasmid system to determine the minimum amount of homology required for gene conversion, to examine how conversion tract lengths are affected by limited homology, and to analyze the role of flanking DNA sequences on the pattern of exchange. Plasmids with homologous sequences greater than 2 kilobases have mitotic exchange rates as high as 2 x 10(-3) events per cell per generation. As the homology is reduced, the exchange rate decreases dramatically. A plasmid with 26 base pairs (bp) of homology undergoes gene conversion at a rate of approximately 1 x 10(-10) events per cell per generation. These studies have also shown that an 8-bp insertion mutation 13 bp from a border between homologous and nonhomologous sequences undergoes conversion, but that a similar 8-bp insertion 5 bp from a border does not. Examination of independent conversion events which occurred in plasmids with heteroallelic copies of the HIS3 gene shows that markers within 280 bp of a border between homologous and nonhomologous sequences undergo conversion less frequently than the same markers within a more extensive homologous sequence. Thus, proximity to a border between homologous and nonhomologous sequences shortens the conversion tract length.

The GCR1 gene encodes a positive transcriptional regulator of the enolase and glyceraldehyde-3-phosphate dehydrogenase gene families in Saccharomyces cerevisiae

1987 ◽  
Vol 7 (2) ◽  
pp. 813-820
Author(s):  
M J Holland ◽  
T Yokoi ◽  
J P Holland ◽  
K Myambo ◽  
M A Innis
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mutant Strain ◽  
Gene Families ◽  
Gene Replacement ◽  
Insertion Mutation ◽  
Gene Insertion ◽  
Glycolytic Gene ◽  
Dehydrogenase Gene ◽  
Multiple Copies ◽  
State Concentration

The intracellular concentrations of the polypeptides encoded by the two enolase (ENO1 and ENO2) and three glyceraldehyde-3-phosphate dehydrogenase (TDH1, TDH2, and TDH3) genes were coordinately reduced more than 20-fold in a Saccharomyces cerevisiae strain carrying the gcr1-1 mutation. The steady-state concentration of glyceraldehyde-3-phosphate dehydrogenase mRNA was shown to be approximately 50-fold reduced in the mutant strain. Overexpression of enolase and glyceraldehyde-3-phosphate dehydrogenase in strains carrying multiple copies of either ENO1 or TDH3 was reduced more than 50-fold in strains carrying the gcr1-1 mutation. These results demonstrated that the GCR1 gene encodes a trans-acting factor which is required for efficient and coordinate expression of these glycolytic gene families. The GCR1 gene and the gcr1-1 mutant allele were cloned and sequenced. GCR1 encodes a predicted 844-amino-acid polypeptide; the gcr1-1 allele contains a 1-base-pair insertion mutation at codon 304. A null mutant carrying a deletion of 90% of the GCR1 coding sequence and a URA3 gene insertion was constructed by gene replacement. The phenotype of a strain carrying this null mutation was identical to that of the gcr1-1 mutant strain.

Observation of Amyloid-Like Fibers of the Sup35 Protein from Saccharomyces Cerevisiae

2000 ◽  
Vol 6 (S2) ◽  
pp. 664-665
Author(s):  
Anthony S. Kowal ◽  
Thomas Scheibel ◽  
Susan L. Lindquist
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Conformation ◽  
Prion Proteins ◽  
Translation Termination ◽  
Polarized Light ◽  
Yeast Cells ◽  
Yeast Prion ◽  
Epigenetic Modifier ◽  
Insoluble Form

In the yeast Saccharomyces cerevisiae, [PST] acts as an epigenetic modifier of translation termination efficiency. [PSI+] can be passed through generations of yeast cells via changes in protein conformation rather than changes in DNA or RNA, and has thus been referred to as a yeast prion. The [PSI+] determinant is the Sup35 protein. Sup35 can exist in two states - soluble and insoluble. Soluble Sup35 functions in translation termination, but when insoluble, stop codons are read through, resulting in incorrect protein products.Sup35 is composed of three distinct domains, N, M, and C. The N region is rich in glutamine and asparagine and is required for the [PST] phenotype to exist. M is a highly charged domain, and no specific function has been assigned to it. C is essential in yeast, as it is responsible for translation termination. The insoluble form of Sup35 has characteristics reminiscent of other prion proteins - in vitro it binds to the dye Congo Red and it exhibits apple green birefringence in polarized light.

scholarly journals Difference in Stability of the N-domain Underlies Distinct Intracellular Properties of the E1064A and H1069Q Mutants of Copper-transporting ATPase ATP7B

2011 ◽  
Vol 286 (18) ◽  
pp. 16355-16362 ◽  
Author(s):  
Oleg Y. Dmitriev ◽  
Ashima Bhattacharjee ◽  
Sergiy Nokhrin ◽  
Eva-Maria E. Uhlemann ◽  
Svetlana Lutsenko
Keyword(s):  
Phenotypic Variability ◽  
Copper Metabolism ◽  
Complete Loss ◽  
Atp Binding ◽  
Wild Type ◽  
Helical Hairpin ◽  
The Difference ◽  
The Stability ◽  
Golgi Network

Wilson disease (WD) is a disorder of copper metabolism caused by mutations in the Cu-transporting ATPase ATP7B. WD is characterized by significant phenotypic variability, the molecular basis of which is poorly understood. The E1064A mutation in the N-domain of ATP7B was previously shown to disrupt ATP binding. We have now determined, by NMR, the structure of the N-domain containing this mutation and compared properties of E1064A and H1069Q, another mutant with impaired ATP binding. The E1064A mutation does not change the overall fold of the N-domain. However, the position of the α1,α2-helical hairpin (α-HH) that houses Glu1064 and His1069 is altered. The α-HH movement produces a more open structure compared with the wild-type ATP-bound form and misaligns ATP coordinating residues, thus explaining complete loss of ATP binding. In the cell, neither the stability nor targeting of ATP7B-E1064A to the trans-Golgi network differs significantly from the wild type. This is in a contrast to the H1069Q mutation within the same α-HH, which greatly destabilizes protein both in vitro and in cells. The difference between two mutants can be linked to a lower stability of the α-HH in the H1069Q variant at the physiological temperature. We conclude that the structural stability of the N-domain rather than the loss of ATP binding plays a defining role in the ability of ATP7B to reach the trans-Golgi network, thus contributing to phenotypic variability in WD.

scholarly journals Guanidine Hydrochloride Inhibits the Generation of Prion “Seeds” but Not Prion Protein Aggregation in Yeast

2002 ◽  
Vol 22 (15) ◽  
pp. 5593-5605 ◽  
Author(s):  
Frédérique Ness ◽  
Paulo Ferreira ◽  
Brian S. Cox ◽  
Mick F. Tuite
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Molecular Chaperone ◽  
Guanidine Hydrochloride ◽  
Free Medium ◽  
Seed Number ◽  
Cycle Model ◽  
Yeast Prions ◽  
Independent Process ◽  
Kinetics Of ◽  
Number Of Seeds

ABSTRACT [PSI +] strains of the yeast Saccharomyces cerevisiae replicate and transmit the prion form of the Sup35p protein but can be permanently cured of this property when grown in millimolar concentrations of guanidine hydrochloride (GdnHCl). GdnHCl treatment leads to the inhibition of the replication of the [PSI +] seeds necessary for continued [PSI +] propagation. Here we demonstrate that the rate of incorporation of newly synthesized Sup35p into the high-molecular-weight aggregates, diagnostic of [PSI +] strains, is proportional to the number of seeds in the cell, with seed number declining (and the levels of soluble Sup35p increasing) in the presence of GdnHCl. GdnHCl does not cause breakdown of preexisting Sup35p aggregates in [PSI +] cells. Transfer of GdnHCl-treated cells to GdnHCl-free medium reverses GdnHCl inhibition of [PSI +] seed replication and allows new prion seeds to be generated exponentially in the absence of ongoing protein synthesis. Following such release the [PSI +] seed numbers double every 20 to 22 min. Recent evidence (P. C. Ferreira, F. Ness, S. R. Edwards, B. S. Cox, and M. F. Tuite, Mol. Microbiol. 40:1357-1369, 2001; G. Jung and D. C. Masison, Curr. Microbiol. 43:7-10, 2001), together with data presented here, suggests that curing yeast prions by GdnHCl is a consequence of GdnHCl inhibition of the activity of molecular chaperone Hsp104, which in turn is essential for [PSI +] propagation. The kinetics of elimination of [PSI +] by coexpression of a dominant, ATPase-negative allele of HSP104 were similar to those observed for GdnHCl-induced elimination. Based on these and other data, we propose a two-cycle model for “prionization” of Sup35p in [PSI +] cells: cycle A is the GdnHCl-sensitive (Hsp104-dependent) replication of the prion seeds, while cycle B is a GdnHCl-insensitive (Hsp104-independent) process that converts these seeds to pelletable aggregates.

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