Elsevier

Methods

Volume 39, Issue 1, May 2006, Pages 3-8
Methods

How to find a prion: [URE3], [PSI+] and [β]

https://doi.org/10.1016/j.ymeth.2006.04.009Get rights and content

Abstract

Infectious proteins (prions) in yeast or other microorganisms can be identified by genetic methods of rather general applicability. Infection in yeast means transfer by cytoplasmic mixing (cytoduction), a property of all non-chromosomal genetic elements whether plasmids, viruses, or prions. Prions can be diagnosed by reversible curability, increased occurrence when the corresponding protein is overproduced, a requirement for the gene for the corresponding protein for propagation, and, in some cases, similarity of phenotype of: (a) mutations in the gene for the protein and (b) the presence of the prion. This approach is illustrated with [URE3], an amyloid-based prion of the regulator of nitrogen catabolism, Ure2p and [PSI+] as a prion of the translation termination factor Sup35p. The prion concept is not limited to infectious amyloids, but includes proteins whose active form is necessary for the activation of the inactive precursor. We detail methods used in studies of [URE3] and [β], a self-activating protease, some of which are of broad application.

Introduction

If a protein alone is sufficient to pass an infection, without a required nucleic acid, then that protein is called a prion. This concept originated from studies of the transmissible spongiform encephalopathies (TSEs) of mammals, but applies as well to other organisms. The known prions are chromosomally encoded proteins which have changed in such a way that they have acquired the ability to transmit this change to the unchanged form of the protein. In yeast and filamentous fungi, infectious elements, such as viruses, are passed from cell to cell by the mating process, and do not take an extracellular route (reviewed in Ref. [1]). Like yeast viruses, yeast prions must behave as non-chromosomal genetic elements [2].

Section snippets

Three genetic criteria for a yeast prion

Yeast prions have several genetic properties which serve as a means of identifying them as prions, independent of their mechanisms of propagation and relatively independent of the means by which they produce a phenotype [3] (Fig. 1).

[URE3] and [PSI+] as prions of Ure2p and Sup35p, respectively

The normal function of Ure2p is to block transcription of the DAL5 gene (and many others) by keeping the transcription factor Gln3p in the cytoplasm [4]. The [URE3] non-chromosomal gene abrogates the repression by a good nitrogen source of enzymes and transporters needed to utilize poor nitrogen sources [5], the same as the phenotype of mutants in the chromosomal URE2 gene [6], [7]. [URE3] is cured by growth in 3 mM guanidine [8], but cured strains can again give rise to [URE3] derivatives [3],

Assay of [URE3] by ureidosuccinate (USA) uptake

Either ure2 mutants or [URE3] strains show derepressed transcription of DAL5 encoding the allantoate transporter, which can also transport the structurally similar ureidosuccinate (USA) [5], [6], [7], [17] (Fig. 2). USA is an intermediate in uracil biosynthesis, the product of aspartate transcarbamylase encoded by URA2. A ura2 strain is plated on or replica plated to minimal medium lacking uracil and containing 20–30 μg/ml of ureidosuccinate. Either ure2 or [URE3] strains will grow. USA uptake

Cytoduction

Prions of yeast should be non-chromosomal genetic elements. The best way to demonstrate this is by transmission by cytoplasmic mixing (cytoduction) using the kar1-1 mutation that prevents nuclear fusion [25] (Fig. 3). If one of the strains in a mating has the kar1-1 mutation, then cells fuse, but nuclei do not fuse. At the next cell division, the parental nuclei separate into the two progeny cells. However, there is a mixture of cytoplasmic contents which is distributed to both daughter cells.

Curing with guanidine

Guanidine is a specific inhibitor of the disaggregating chaperone Hsp104 [28], [29], [30]. Efficient guanidine curing of [URE3] strains is done as was described first for [PSI+] [31]. Cells are streaked for single colonies on YPAD containing 3–5 mM guanidine HCl. Some strains grow poorly on 5 mM guanidine, but all strains we have used can be cured of [URE3] at 3 mM guanidine HCl. The cured strain is then purified by subcloning in the absence of guanidine. If one is trying to prove one is dealing

Protein overproduction increases prion generation

Prion formation can be induced by a protein overproduced constitutively, or from an induced promoter, such as the GAL1 promoter turned on by galactose. The latter is preferable since cells can be tested for the putative prion phenotype on repressing media (containing glucose). This avoids a possible phenotype due to overproduction of the protein that is not due to prion generation. We use YEp351G, a high copy 2 μm DNA-based LEU2 plasmid with the GAL1,10 promoter [3]. For example, strain 3469 (

Phenotype relation when the prion form is inactive

This approach requires no special methods. Its applicability will be particularly evident if a gene required to maintain the putative prion has a similar mutant phenotype as the presence of the prion, as was the case for [URE3] and ure2 mutants and [PSI+] and sup35 mutants (see above).

Visualization of [URE3] using Ure2-GFP fusions

When a Ure2-GFP fusion protein is expressed in a [ure-o] strain from a centromeric plasmid using the native URE2 promoter, the fluorescence signal is homogeneously distributed throughout the cytoplasm. However, when this same plasmid is introduced into a [URE3] strain the fluorescence signal has a punctate appearance [34]. The same fluorescence pattern is observed when instead of full-length Ure2p the first 65 amino acids of Ure2p, comprising a large part of the prion domain, are fused to GFP.

Problems in working with [URE3]

[URE3] is unstable in many laboratory strains. This problem may relate to the destabilization of [URE3] by [PSI+] demonstrated by Schwimmer and Masison [36]. It is also doubtless related to the slow growth phenotype of [URE3] strains, resulting in a constant selection for cells that have lost the prion. This requires care in determining that strains known to carry [URE3] have not lost it during storage. Keeping strains under selection for ability to take up USA is not a solution, since this

Different manifestations of a prion domain

A part of a prion protein can manifest its prion-related activity in several different ways.

Purification of Ure2p

In vitro Ure2p can spontaneously form ordered filaments [43], which are in turn infectious upon introduction to yeast cells that express Ure2p [20]. The filaments formed by Ure2p have all of the traits of amyloid [43] (Fig. 4). The characterization of structural properties and filament formation using homogeneous protein preparations of Ure2p are facilitated by purification of Ure2p from Escherichia coli using a polyhistidine tag [43], [44]. Moreover, the addition of the amino-terminal tag has

[β], the prion of vacuolar protease B

Vacuolar protease A (Pep4p) plays a major role in activating protease B (Prb1p) and carboxypeptidase Y (Prc1p) by cleaving their inactive precursor proteins (reviewed by [46]). However, the spore clones from a pep4/+heterozygous diploid do not immediately lose Prb1p or Prc1p activity, a phenomenon called “phenotypic lag” and presumed due to some self-activation by Prb1p [47]. If spores are germinated and grown on glycerol or ethanol as a carbon source in place of dextrose, the activity of Prb1

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