Lee J. Byrne

The number and transmission of [PSI+] prion seeds (Propagons) in the yeast Saccharomyces cerevisiae

Background 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. Principal Findings 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. Conclusions 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 n0, 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.

Cell division is essential for elimination of the yeast [PSI+] prion by guanidine hydrochloride

Guanidine hydrochloride (Gdn·HCl) blocks the propagation of yeast prions by inhibiting Hsp104, a molecular chaperone that is absolutely required for yeast prion propagation. We had previously proposed that ongoing cell division is required for Gdn·HCl-induced loss of the [PSI+] prion. Subsequently, Wu et al.[Wu Y, Greene LE, Masison DC, Eisenberg E (2005) Proc Natl Acad Sci USA 102:12789–12794] claimed to show that Gdn·HCl can eliminate the [PSI+] prion from α-factor-arrested cells leading them to propose that in Gdn·HCl-treated cells the prion aggregates are degraded by an Hsp104-independent mechanism. Here we demonstrate that the results of Wu et al. can be explained by an unusually high rate of α-factor-induced cell death in the [PSI+] strain (780-1D) used in their studies. What appeared to be no growth in their experiments was actually no increase in total cell number in a dividing culture through a counterbalancing level of cell death. Using media-exchange experiments, we provide further support for our original proposal that elimination of the [PSI+] prion by Gdn·HCl requires ongoing cell division and that prions are not destroyed during or after the evident curing phase.

The ligand-binding b' domain of human protein disulphide-isomerase mediates homodimerization.

Purified preparations of the recombinant b′x domain fragment of human protein‐disulphide isomerase (PDI), which are homogeneous by mass spectrometry and sodium dodecyl sulfate polyacrylamide gel electrophoresis, comprise more than one species when analyzed by ion‐exchange chromatography and nondenaturing polyacrylamide gel electrophoresis. These species were resolved and shown to be monomer and dimer by analytical ultracentrifugation and analytical size‐exclusion chromatography. Spectroscopic properties indicate that the monomeric species corresponds to the “capped” conformation observed in the x‐ray structure of the I272A mutant of b′x (Nguyen, Wallis, Howard, Haapalainen, Salo, Saaranen, Sidhu, Wierenga, Freedman, Ruddock, and Williamson, J Mol Biol 2008;383:1144‐1155) in which the x region binds to a hydrophobic patch on the surface of the b′ domain; conversely, the dimeric species has an “open” or “uncapped” conformation in which the x region does not bind to this surface. The larger bb′x fragment of human PDI shows very similar behavior to b′x and can be resolved into a capped monomeric species and an uncapped dimer. Preparations of recombinant b′ domain of human PDI and of the bb′ domain pair are found exclusively as dimers. Full‐length PDI is known to comprise a mixture of monomeric and dimeric species, whereas the isolated a, b, and a′ domains of PDI are found exclusively as monomers. These results show that the b′ domain of human PDI tends to form homodimers—both in isolation and in other contexts—and that this tendency is moderated by the adjacent x region, which can bind to a surface patch on the b′ domain.

20 Yeast Prions and Their Analysis InVivo

Publisher Summary This chapter discusses the three yeast (Saccharomyces cerevisiae) prions—Ure2p, Sup35p, and , and Rnq1p—in terms of their associated phenotypes and their cell biological and biochemical properties and reviews the experimental approaches that can be taken to study the three native prions in vivo. The chapter also discusses the way a researcher establishes whether a new or novel phenotype is associated with the prion-like behavior of a cellular protein. Yeast prions are transmitted efficiently from cell-to-cell during mitosis and meiosis, indicating that a very effective mechanism must exist that ensures their continued propagation even in rapidly dividing cells. Two different, but not mutually exclusive, models have been put forward to explain the self-propagation of prions: template-directed refolding and seeded polymerization. The more widely accepted seeded polymerization model is based on the prion protein existing in an altered conformational state, which is in a reversible dynamic equilibrium with a soluble form of that protein. The generation and transmission of propagons in yeast appears to be dependent upon a number of different molecular chaperones with one particular chaperone, the heat-shock-inducible protein Hsp104p, being essential for the propagation of all three native prions.

20 Yeast Prions and Their Analysis

Publisher Summary This chapter discusses the three yeast (Saccharomyces cerevisiae) prions—Ure2p, Sup35p, and , and Rnq1p—in terms of their associated phenotypes and their cell biological and biochemical properties and reviews the experimental approaches that can be taken to study the three native prions in vivo. The chapter also discusses the way a researcher establishes whether a new or novel phenotype is associated with the prion-like behavior of a cellular protein. Yeast prions are transmitted efficiently from cell-to-cell during mitosis and meiosis, indicating that a very effective mechanism must exist that ensures their continued propagation even in rapidly dividing cells. Two different, but not mutually exclusive, models have been put forward to explain the self-propagation of prions: template-directed refolding and seeded polymerization. The more widely accepted seeded polymerization model is based on the prion protein existing in an altered conformational state, which is in a reversible dynamic equilibrium with a soluble form of that protein. The generation and transmission of propagons in yeast appears to be dependent upon a number of different molecular chaperones with one particular chaperone, the heat-shock-inducible protein Hsp104p, being essential for the propagation of all three native prions.

8 Reporter Genes and Their Uses in Studying Yeast Gene Expression

Publisher Summary Reporter genes are the tools of ever-increasing importance in the study of yeast gene expression. Their versatility and the ease of use have made them widely available and ongoing developments will undoubtedly continue to increase their usefulness and their potential applications. A few examples of studies on transcriptional, translational, and post-translational regulation of gene expression are used in this chapter to illustrate some general principles of the use of reporter genes. Many of the reporters developed for use in yeast were originally designed to investigate promoter activity. A good example for this is the original description of the chloramphenicol acetyltransferase (CAT) assay in yeast. The authors of this study demonstrated the validity of CAT as a yeast reporter gene by constructing vectors that contained the coding region of the bacterial cat gene, but no promoter. Any essential gene can be used as a reporter in the corresponding knockout yeast strains because in these strains the ability to grow depends on the expression of the respective gene. The growth rate, thus, provides an easy readout for the expression of the gene.

‘Something in the way she moves’: The functional significance of flexibility in the multiple roles of protein disulfide isomerase (PDI)

Protein disulfide isomerase (PDI) has diverse functions in the endoplasmic reticulum as catalyst of redox transfer, disulfide isomerization and oxidative protein folding, as molecular chaperone and in multi-subunit complexes. It interacts with an extraordinarily wide range of substrate and partner proteins, but there is only limited structural information on these interactions. Extensive evidence on the flexibility of PDI in solution is not matched by any detailed picture of the scope of its motion. A new rapid method for simulating the motion of large proteins provides detailed molecular trajectories for PDI demonstrating extensive changes in the relative orientation of its four domains, great variation in the distances between key sites and internal motion within the core ligand-binding domain. The review shows that these simulations are consistent with experimental evidence and provide insight into the functional capabilities conferred by the extensive flexible motion of PDI.