Prions in Saccharomyces and Podospora spp.: protein-based inheritance

Abstract

Genetic evidence showed two non-Mendelian genetic elements of Saccharomyces cerevisiae, called [URE3] and [PSI], to be prions of Ure2p and Sup35p, respectively. [URE3] makes cells derepressed for nitrogen catabolism, while [PSI] elevates the efficiency of weak suppressor tRNAs. The same approach led to identification of the non-Mendelian element [Het-s] of the filamentous fungus Podospora anserina, as a prion of the het-s protein. The prion form of the het-s protein is required for heterokaryon incompatibility, a normal fungal function, suggesting that other normal cellular functions may be controlled by prions. [URE3] and [PSI] involve a self-propagating aggregation of Ure2p and Sup35p, respectively. In vitro, Ure2p and Sup35p form amyloid, a filamentous protein structure, high in beta-sheet with a characteristic green birefringent staining by the dye Congo Red. Amyloid deposits are a cardinal feature of Alzheimer's disease, non-insulin-dependent diabetes mellitus, the transmissible spongiform encephalopathies, and many other diseases. The prion domain of Ure2p consists of Asn-rich residues 1 to 80, but two nonoverlapping fragments of the molecule can, when overproduced, induce the de nova appearance of [URE3]. The prion domain of Sup35 consists of residues 1 to 114, also rich in Asn and Gln residues. While runs of Asn and Gln are important for [URE3] and [PSI], no such structures are found in PrP or the Het-s protein. Either elevated or depressed levels of the chaperone Hsp104 interfere with propagation of [PSI]. Both [URE3] and [PSI] are cured by growth of cells in millimolar guanidine HCl. [URE3] is also cured by overexpression of fragments of Ure2p or fusion proteins including parts of Ure2p.

FIG. 1

Definition of a prion. “Prion” means an infectious protein. Many mechanisms can be imagined for such an entity (48), including a protein that induces its own gene’s transcription (A); a self-propagating covalent protein modification, such as acetylation, in which the modified form of the protein is much better at self-acetylation than the unacetylated form (B); and a self-propagating change in conformation, such as amyloid formation—the likely mechanism for the known prions (C).

FIG. 2

Genetic criteria for a prion illustrated by [URE3]. These genetic properties are expected of a prion but not of a nucleic acid replicon (102). Reversible curability means that a cured strain can again develop the prion de novo. Overproduction of the normal form of the protein increases the probability that the prion will arise. The presence of the prion causes the same phenotype as deletion of the chromosomal gene for the protein, because both lack the normal form, and the chromosomal gene for the protein is necessary for propagation of the prion. The requirement for a chromosomal gene for propagation of a nonchromosomal genetic element or infectious entity is frequently seen and is not an indication that the genetic element is a prion.

FIG. 3

How ureidosuccinate uptake is controlled by the nitrogen source. Yeast cells growing on a rich source of nitrogen (such as ammonia or glutamine) repress the expression of transporters and metabolic enzymes needed for utilization of poor nitrogen sources. This is called nitrogen catabolite repression (19, 66). Ureidosuccinate (USA) is the product of aspartate transcarbamylase (ura2), the first step in uracil biosynthesis. The chance chemical resemblance of USA to the poor nitrogen source allantoate results in the ability of the allantoate transporter, Dal5p, to import USA. The presence of a rich nitrogen source, such as ammonia, is transmitted to Ure2p, which then blocks the action of the positive transcription factor, Gln3p. Thus, ammonia represses USA uptake, preventing ura2 cells from using USA in place of uracil. Modified from reference with permission of the publisher.

FIG. 4

Prion domain(s) of Ure2p. Deletion mutants overexpressing fragments of Ure2p from a plasmid were assayed for the ability to induce the de novo formation of [URE3] in a strain expressing the full-length Ure2p from the chromosomal gene, and, separately, for the ability to complement the nitrogen regulation defect of a ure2Δ mutation. (A) The region from positions 1 to 65 is sufficient for prion induction. (B) A fragment of Ure2p lacking the region from positions 1 to 65 can also induce [URE3]. (C) Regions that promote prion formation (black) and block prion formation (white) are shown. Modified from reference with permission of the publisher.

FIG. 5

Ure2p is aggregated in [URE3] cells. A Ure2p-GFP fusion was expressed from a single-copy plasmid with the URE2 promoter, and the distribution of fluorescence was examined in [ure-o] (wild-type), [URE3], and guanidine-cured strains. Aggregation was detected specifically in [URE3] strains (37).

FIG. 6

Amyloid is formed in vitro by Ure2p. (a) Filaments (45 Å) formed by Ure2p^1–65. (b) Ure2p^1–65/Ure2p 1:1 cofilaments 200 Å in diameter. (c) Ure2p^1–65/Ure2p cofilaments digested with proteinase K, leaving the prion domain protease resistant in the form of narrow filaments. (d) Filaments (280 to 400 Å) of native Ure2p formed by seeding with Ure2p^1–65/Ure2p cofilaments. Modified from reference with permission of the publisher.

FIG. 7

Ure2p filaments show birefringence on staining with Congo red. Filaments composed of Ure2p^1–65, cofilaments of equimolar Ure2p^1–65 and full-length Ure2p, and full-length Ure2p seeded with cofilaments were stained with Congo red and observed under bright-field conditions (left) or in a polarizing microscope (right). The green-yellow birefringence seen here is typical of amyloid. Reprinted from reference with permission of the publisher.

FIG. 8

Model of [URE3] amyloid formation. The protease-resistant core of amyloid formed by Ure2p^1–65 and full-length Ure2p is the N-terminal prion domain. The prion domain alone can form amyloid (left) and promotes amyloid formation by the full-length molecule (right and below).

FIG. 9

A prion in yeast or fungi is a protein acting as a gene. Prions pass information from cell to cell by the transfer of the altered form of the protein. They are, in that sense, proteins acting as genes. w.t., wild type.

FIG. 10

Comparison of prion proteins and prion domains. The prion domains of Ure2p and Sup35p are rich in Asn and Gln residues, and these residues are important in prion generation and propagation. PrP and the het-s protein are not Asn or Gln rich. The prion domain of the het-s protein has not been determined. For Ure2p, Sup35p, and PrP, mutations outside the prion domain may have dramatic effects on prion generation or propagation (see the text). Modified from reference with permission of the publisher.

FIG. 11

Possible roles of Hsp104 chaperone in [PSI] propagation. Hsp104 may be directly involved in the prion propagation reaction (top) or act to ensure the segregation to all cells of some of the altered form by breaking a few large aggregates into many smaller aggregates (bottom).

FIG. 12

The [Het-s] prion of Podospora and heterokaryon incompatibility. Only cells carrying the [Het-s] prion can carry out heterokaryon incompatibility (21). Modified from reference with permission of the publisher.