The yeast Saccharomyces cerevisiae is a tractable model organism in which both to explore the molecular mechanisms underlying the generation of disease-associated protein misfolding and to map the cellular responses to potentially toxic misfolded proteins. Specific targets have included proteins which in certain disease states form amyloids and lead to neurodegeneration. Such studies are greatly facilitated by the extensive ‘toolbox’ available to the yeast researcher that provides a range of cell engineering options. Consequently, a number of assays at the cell and molecular level have been set up to report on specific protein misfolding events associated with endogenous or heterologous proteins. One major target is the mammalian prion protein PrP because we know little about what specific sequence and/or structural feature(s) of PrP are important for its conversion to the infectious prion form, PrPSc. Here, using a study of the expression in yeast of fusion proteins comprising the yeast prion protein Sup35 fused to various regions of mouse PrP protein, we show how PrP sequences can direct the formation of non-transmissible amyloids and focus in particular on the role of the mouse octarepeat region. Through this study we illustrate the benefits and limitations of yeast-based models for protein misfolding disorders.
PrP; Sup35 fusions; nonsense suppression; prion; yeast (Saccharomyces cerevisiae)
The rate of spontaneous change from ψ to the ψ+ condition, determined in yeast by states of the Sup35p protein, is briefly discussed together with the conditions necessary for such change to occur. Conditions that promote and which affect the rate of induction of ψ+ in Sup35p and of other prion-forming proteins to their respective prion forms are also discussed. These include the influence of the amount of non-prion protein, the presence of other prions, the activity of chaperones, and brief descriptions of the role of native sequences in the proteins and how alteration of sequences in prion-forming proteins influences the rate of induction of [prion+] and amyloid forms.
The second part of this article discusses the conditions which affect the reversion of ψ+ to ψ-, including factors which affect the copy-number of prion “seeds” or propagons and their partition. The principal factor discussed is the activity of the chaperone Hsp104, but the existence of other factors, such protein sequence and of other, less well-studied agents is touched upon and comparisons are made, as appropriate, with studies with other yeast prions.
We conclude with a discussion of models of maintenance, in particular that of Tanaka et al. published in Nature (2006),6 which provides much insight into the phenotypic and genetic parameters of the numerous “variants” of prions increasingly being described in the literature.
Saccharomyces cerevisiae; [PSI]; [URE3]; [PIN]; prion induction; prion curing; prion inheritance; Hsp104
Although intensively researched, the fundamental mechanism of protein misfolding that leads to protein aggregation and associated diseases remains somewhat enigmatic. The failure of a protein to correctly fold de novo or to remain correctly folded can have profound consequences on a living system especially when the cellular quality control processes fail to eliminate the rogue proteins. Over 20 different human diseases have now been designated as ‘conformational diseases’ and include neurodegenerative diseases such as Alzheimer's disease (AD), Huntington's disease (HD) and Creutzfeldt Jakob disease (CJD) that are becoming increasingly prevalent in an ageing human population. Such diseases are usually characterised by the deposition of specific misfolded proteins as amyloid fibrils and hence are often referred to as the amyloidoses.
conformational disease; folding intermediates; conformational changes; prion; amyloid; fibrils; toxicity; aggregation propensity; molecular chaperones; therapeutics
It is over 40 years since it was first reported that the yeast Saccahromyces cerevisiae contains two unusual cytoplasmic ‘genetic’ elements: [PSI+] and [URE3]. Remarkably the underlying determinants are protein-based rather than nucleic acid-based, i.e., that they are prions, and we have already learnt much about their inheritance and phenotypic effects from the application of ‘classical’ genetic studies alongside the more modern molecular, cellular and biochemical approaches. Of particular value has been the exploitation of chemical mutagens and ‘antagonistic’ mutants which directly affect the replication and/or transmission of yeast prions. In this Chapter we describe what has emerged from the application of classical and molecular genetic studies, to the most intensively studied of the three native yeast prions, the [PSI+] prion.
yeast; [PSI]; prion; SUP35/eRF3; SUP45/eRF1; antisuppressor; [PSI+] maintenance genes; Hsp104; prion antagonists
Prions are propagated in Saccharomyces cerevisiae with remarkable efficiency, yet we know little about the structural basis of sequence variations in the prion protein that support or prohibit propagation of the prion conformation. We show that certain single-amino-acid substitutions in the prion protein Sup35 impact negatively on the maintenance of the associated prion-based [PSI+] trait by combining in vivo phenotypic analysis with solution NMR structural studies. A clear correlation is observed between mutationally induced conformational differences in one of the oligopeptide repeats (R2) in the N terminus of Sup35 and the relative ability to propagate [PSI+]. Strikingly, substitution of one of a Gly-Gly pair with highly charged residues that significantly increase structural definition of R2 lead to a severe [PSI+] propagation defect. These findings offer a molecular explanation for the dominant-negative effects of such psi-no-more (PNM) mutations and demonstrate directly the importance of localized structural definition in prion propagation.
•Mutations in a Gly58-Gly59 pair in Sup35 inhibit propagation of the [PSI+] prion•Biochemistry of the amino acid introduced dictates the resulting phenotype•Prion propagation defects correlate with local changes in Sup35 structure•Increased local structural definition is associated with impaired prion propagation
There is an increasing call for the absolute quantification of time-resolved metabolite data. However, a number of technical issues exist, such as metabolites being modified/degraded either chemically or enzymatically during the extraction process. Additionally, capillary electrophoresis mass spectrometry (CE-MS) is incompatible with high salt concentrations often used in extraction protocols. In microbial systems, metabolite yield is influenced by the extraction protocol used and the cell disruption rate. Here we present a method that rapidly quenches metabolism using dry-ice ethanol bath and methanol N-ethylmaleimide solution (thus stabilising thiols), disrupts cells efficiently using bead-beating and avoids artefacts created by live-cell pelleting. Rapid sample processing minimised metabolite leaching. Cell weight, number and size distribution was used to calculate metabolites to an attomol/cell level. We apply this method to samples obtained from the respiratory oscillation that occurs when yeast are grown continuously.
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.
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.
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.
In S. pombe, about 5% of genes are meiosis-specific and accumulate little or no mRNA during vegetative growth. Here we use Affymetrix tiling arrays to characterize transcripts in vegetative and meiotic cells. In vegetative cells, many meiotic genes, especially those induced in mid-meiosis, have abundant antisense transcripts. Disruption of the antisense transcription of three of these mid-meiotic genes allowed vegetative sense transcription. These results suggest that antisense transcription represses sense transcription of meiotic genes in vegetative cells. Although the mechanism(s) of antisense mediated transcription repression need to be further explored, our data indicates that RNAi machinery is not required for repression. Previously, we and others used non-strand specific methods to study splicing regulation of meiotic genes and concluded that 28 mid-meiotic genes are spliced only in meiosis. We now demonstrate that the “unspliced” signal in vegetative cells comes from the antisense RNA, not from unspliced sense RNA, and we argue against the idea that splicing regulates these mid-meiotic genes. Most of these mid-meiotic genes are induced in mid-meiosis by the forkhead transcription factor Mei4. Interestingly, deletion of a different forkhead transcription factor, Fkh2, allows low levels of sense expression of some mid-meiotic genes in vegetative cells. We propose that vegetative expression of mid-meiotic genes is repressed at least two independent ways: antisense transcription and Fkh2 repression.
The frequency with which the yeast [PSI+] prion form of Sup35 arises de novo is controlled by a number of genetic and environmental factors. We have previously shown that in cells lacking the antioxidant peroxiredoxin proteins Tsa1 and Tsa2, the frequency of de novo formation of [PSI+] is greatly elevated. We show here that Tsa1/Tsa2 also function to suppress the formation of the [PIN+] prion form of Rnq1. However, although oxidative stress increases the de novo formation of both [PIN+] and [PSI+], it does not overcome the requirement of cells being [PIN+] to form the [PSI+] prion. We use an anti-methionine sulfoxide antibody to show that methionine oxidation is elevated in Sup35 during oxidative stress conditions. Abrogating Sup35 methionine oxidation by overexpressing methionine sulfoxide reductase (MSRA) prevents [PSI+] formation, indicating that Sup35 oxidation may underlie the switch from a soluble to an aggregated form of Sup35. In contrast, we were unable to detect methionine oxidation of Rnq1, and MSRA overexpression did not affect [PIN+] formation in a tsa1 tsa2 mutant. The molecular basis of how yeast and mammalian prions form infectious amyloid-like structures de novo is poorly understood. Our data suggest a causal link between Sup35 protein oxidation and de novo [PSI+] prion formation.
Methionine; Oxidative Stress; Peroxiredoxin; Prions; Protein Aggregation; Translation; Translation Release Factors; Yeast
Prions are unusual proteinaceous infectious agents that are typically associated with a class of fatal degenerative diseases of the mammalian brain. However, the discovery of fungal prions, which are not associated with disease, suggests that we must now consider the impact of these factors on basic cellular physiology in a different light. Fungal prions are epigenetic determinants that can alter a range of cellular processes, including metabolism and gene expression pathways, and these changes can lead to a range of prion-associated phenotypes. The mechanistic similarities between prion propagation in mammals and fungi suggest that prions are not a biological anomaly but instead are a new appreciated and perhaps ubiquitous regulatory mechanism.
Replicating amyloids, called prions, are responsible for transmissible
neurodegenerative diseases in mammals and some heritable phenotypes in fungi.
The transmission of prions between species is usually inhibited, being highly
sensitive to small differences in amino acid sequence of the prion-forming
proteins. To understand the molecular basis of this prion interspecies barrier,
we studied the transmission of the
[PSI+] prion state from
Sup35 of Saccharomyces cerevisiae to hybrid Sup35 proteins with
prion-forming domains from four other closely related
Saccharomyces species. Whereas all the hybrid Sup35
proteins could adopt a prion form in S. cerevisiae, they could
not readily acquire the prion form from the
[PSI+] prion of S.
cerevisiae. Expression of the hybrid Sup35 proteins in S.
cells often resulted in frequent loss of the native
[PSI+] prion. Furthermore,
all hybrid Sup35 proteins showed different patterns of interaction with the
native [PSI+] prion in terms of
co-polymerization, acquisition of the prion state, and induced prion loss, all
of which were also dependent on the
[PSI+] variant. The
observed loss of S. cerevisiae
[PSI+] can be related to
inhibition of prion polymerization of S. cerevisiae Sup35 and
formation of a non-heritable form of amyloid. We have therefore identified two
distinct molecular origins of prion transmission barriers between closely
sequence-related prion proteins: first, the inability of heterologous proteins
to co-aggregate with host prion polymers, and second, acquisition by these
proteins of a non-heritable amyloid fold.
Amyloid; Prions; Protein Folding; Translation Release Factors; Yeast; Saccharomyces; Sup35; Prion Interference; Prion Species Barrier
Translation termination in eukaryotes typically requires the decoding of one of three stop codons UAA, UAG or UGA by the eukaryotic release factor eRF1. The molecular mechanisms that allow eRF1 to decode either A or G in the second nucleotide, but to exclude UGG as a stop codon, are currently not well understood. Several models of stop codon recognition have been developed on the basis of evidence from mutagenesis studies, as well as studies on the evolutionary sequence conservation of eRF1. We show here that point mutants of Saccharomyces cerevisiae eRF1 display significant variability in their stop codon read-through phenotypes depending on the background genotype of the strain used, and that evolutionary conservation of amino acids in eRF1 is only a poor indicator of the functional importance of individual residues in translation termination. We further show that many phenotypes associated with eRF1 mutants are quantitatively unlinked with translation termination defects, suggesting that the evolutionary history of eRF1 was shaped by a complex set of molecular functions in addition to translation termination. We reassess current models of stop-codon recognition by eRF1 in the light of these new data.
The aging related decline of heat shock factor-1 (HSF1) signaling may be causally related to protein aggregation diseases. To model such disease, we tried to cripple HSF1 signaling in the Xenopus tadpole.
Over-expression of heat shock factor binding protein-1 did not inhibit the heat shock response in Xenopus. RNAi against HSF1 mRNA inhibited the heat shock response by 70% in Xenopus A6 cells, but failed in transgenic tadpoles. Expression of XHSF380, a dominant-negative HSF1 mutant, was embryonic lethal, which could be circumvented by delaying expression via a tetracycline inducible promoter. HSF1 signaling is thus essential for embryonic Xenopus development. Surprisingly, transgenic expression of the XHSF380 or of full length HSF1, whether driven by a ubiquitous or a neural specific promoter, was not detectable in the larval brain.
Our finding that the majority of neurons, which have little endogenous HSF1, refused to accept transgene-driven expression of HSF1 or its mutant suggests that HSF1 levels are strictly controlled in neuronal tissue.
The metacaspase Mca1 from Saccharomyces cerevisiae displays a Q/N-rich region at its N-terminus reminiscent of yeast prion proteins. In this study, we show that the ability of Mca1 to form insoluble aggregates is modulated by a peptide stretch preceding its putative prion-forming domain. Based on its genomic locus, three potential translational start sites of Mca1 can give rise to two slightly different long Mca1 proteins or a short version, Mca1451/453 and Mca1432, respectively, although under normal physiological conditions Mca1432 is the predominant form expressed. All Mca1 variants exhibit the Q/N-rich regions, while only the long variants Mca1451/453 share an extra stretch of 19 amino acids at their N-terminal end. Strikingly, only long versions of Mca1 but not Mca1432 revealed pronounced aggregation in vivo and displayed prion-like properties when fused to the C-terminal domain of Sup35 suggesting that the N-terminal peptide element promotes the conformational switch of Mca1 protein into an insoluble state. Transfer of the 19 N-terminal amino acid stretch of Mca1451 to the N-terminus of firefly luciferase resulted in increased aggregation of luciferase, suggesting a protein destabilizing function of the peptide element. We conclude that the aggregation propensity of the potential yeast prion protein Mca1 in vivo is strongly accelerated by a short peptide segment preceding its Q/N-rich region and we speculate that such a conformational switch might occur in vivo via the usage of alternative translational start sites.
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 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.
The molecular chaperone Hsp104 is not only a key component of the cellular machinery induced to disassemble aggregated proteins in stressed cells of Saccharomyces cerevisiae but also plays an essential role in the propagation of the [PSI+], [URE3], and [RNQ/PIN+] prions in this organism. Here we demonstrate that the fungal pathogen Candida albicans carries an 899-residue stress-inducible orthologue of Hsp104 (CaHsp104) that shows a high degree of amino acid identity to S. cerevisiae Hsp104 (ScHsp104). This identity is significantly lower in the N- and C-terminal regions implicated in substrate recognition and cofactor binding, respectively. CaHsp104 is able to provide all known functions of ScHsp104 in an S. cerevisiae hsp104 null mutant, i.e., tolerance to high-temperature stress, reactivation of heat-denatured proteins, and propagation of the [PSI+] prion. As also observed for ScHsp104, overexpression of CaHsp104 leads to a loss of the [PSI+] prion. However, unlike that of ScHsp104, CaHsp104 function is resistant to guanidine hydrochloride (GdnHCl), an inhibitor of the ATPase activity of this chaperone. These findings have implications both in terms of the mechanism of inhibition of Hsp104 by GdnHCl and in the evolution of the ability of fungal species to propagate prions.
[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.
Many proteins can misfold into β-sheet-rich, self-seeding polymers (amyloids). Prions are exceptional among such aggregates in that they are also infectious. In fungi, prions are not pathogenic but rather act as epigenetic regulators of cell physiology, providing a powerful model for studying the mechanism of prion replication. We used prion-forming domains from two budding yeast proteins (Sup35p and New1p) to examine the requirements for prion formation and inheritance. In both proteins, a glutamine/asparagine-rich (Q/N-rich) tract mediates sequence-specific aggregation, while an adjacent motif, the oligopeptide repeat, is required for the replication and stable inheritance of these aggregates. Our findings help to explain why although Q/N-rich proteins are relatively common, few form heritable aggregates: prion inheritance requires both an aggregation sequence responsible for self-seeded growth and an element that permits chaperone-dependent replication of the aggregate. Using this knowledge, we have designed novel artificial prions by fusing the replication element of Sup35p to aggregation-prone sequences from other proteins, including pathogenically expanded polyglutamine.
Artificial prions - infectious, misfolded proteins - can be created by fusing the replication element of one prion to aggregation sequences from another
Evidence is now accumulating that sub-populations of ribosomes - so-called specialized ribosomes - can favour the translation of subsets of mRNAs. Here we use a large collection of diploid yeast strains, each deficient in one or other copy of the set of ribosomal protein (RP) genes, to generate eukaryotic cells carrying distinct populations of altered ‘specialized’ ribosomes. We show by comparative protein synthesis assays that different heterologous mRNA reporters based on luciferase are preferentially translated by distinct populations of specialized ribosomes. These mRNAs include reporters carrying premature termination codons (PTC) thus allowing us to identify specialized ribosomes that alter the efficiency of translation termination leading to enhanced synthesis of the wild-type protein. This finding suggests that these strains can be used to identify novel therapeutic targets in the ribosome. To explore this further we examined the translation of the mRNA encoding the extracellular matrix protein laminin β3 (LAMB3) since a LAMB3-PTC mutant is implicated in the blistering skin disease Epidermolysis bullosa (EB). This screen identified specialized ribosomes with reduced levels of RP L35B as showing enhanced synthesis of full-length LAMB3 in cells expressing the LAMB3-PTC mutant. Importantly, the RP L35B sub-population of specialized ribosomes leave both translation of a reporter luciferase carrying a different PTC and bulk mRNA translation largely unaltered.
In response to severe environmental stresses eukaryotic cells shut down translation and accumulate components of the translational machinery in stress granules (SGs). Since they contain mainly mRNA, translation initiation factors and 40S ribosomal subunits, they have been referred to as dominant accumulations of stalled translation preinitiation complexes. Here we present evidence that the robust heat shock-induced SGs of S. cerevisiae also contain translation elongation factors eEF3 (Yef3p) and eEF1Bγ2 (Tef4p) as well as translation termination factors eRF1 (Sup45p) and eRF3 (Sup35p). Despite the presence of the yeast prion protein Sup35 in heat shock-induced SGs, we found out that its prion-like domain is not involved in the SGs assembly. Factors eEF3, eEF1Bγ2 and eRF1 were accumulated and co-localized with Dcp2 foci even upon a milder heat shock at 42°C independently of P-bodies scaffolding proteins. We also show that eEF3 accumulations at 42°C determine sites of the genuine SGs assembly at 46°C. We suggest that identification of translation elongation and termination factors in SGs might help to understand the mechanism of the eIF2α factor phosphorylation-independent repression of translation and SGs assembly.
Ubiquitin (Ub) and ubiquitin-like (UBL) proteins regulate a diverse array of cellular pathways through covalent as well as non-covalent interactions with target proteins. Yeast protein Mdy2 (Get5) and its human homolog GdX (Ubl4a) belong to the class of UBL proteins which do not form conjugates with other proteins. Mdy2 is required for cell survival under heat stress and for efficient mating. As part of a complex with Sgt2 and Get4 it has been implicated in the biogenesis of tail-anchored proteins. Interestingly, in response to heat stress, Mdy2 protein that is predominantly localized in the nucleus co-localized with poly(A)-binding protein Pab1 to cytoplasmic stress granules suggesting that nucleocytoplasmic shuttling is of functional importance. Here we investigate the nuclear import of Mdy2, a process that is independent of the Get4/Sgt2 complex but required for stress response. Nuclear import is mediated by an N-terminal nuclear localization signal (NLS) and this process is essential for the heat stress response. In contrast, cells expressing Mdy2 lacking a nuclear export signal (NES) behave like wild type. Importantly, both Mdy2 and Mdy2-ΔNES, but not Mdy2-ΔNLS, physically interact with Pab1 and this interaction correlates with the accumulation in cytoplasmic stress granules. Thus, the nuclear history of the UBL Mdy2 appears to be essential for its function in cytoplasmic stress granules during the rapid cellular response to heat stress.
Protein misfolding disorders such as Alzheimer, Parkinson and transthyretin amyloidosis are characterized by the formation of protein amyloid deposits. Although the nature and location of the aggregated proteins varies between different diseases, they all share similar molecular pathways of protein unfolding, aggregation and amyloid deposition. Most effects of these proteins are likely to occur at the proteome level, a virtually unexplored reality. To investigate the effects of an amyloid protein expression on the cellular proteome, we created a yeast expression system using human transthyretin (TTR) as a model amyloidogenic protein. We used Saccharomyces cerevisiae, a living test tube, to express native TTR (non-amyloidogenic) and the amyloidogenic TTR variant L55P, the later forming aggregates when expressed in yeast. Differential proteome changes were quantitatively analyzed by 2D-differential in gel electrophoresis (2D-DIGE). We show that the expression of the amyloidogenic TTR-L55P causes a metabolic shift towards energy production, increased superoxide dismutase expression as well as of several molecular chaperones involved in protein refolding. Among these chaperones, members of the HSP70 family and the peptidyl-prolyl-cis-trans isomerase (PPIase) were identified. The latter is highly relevant considering that it was previously found to be a TTR interacting partner in the plasma of ATTR patients but not in healthy or asymptomatic subjects. The small ubiquitin-like modifier (SUMO) expression is also increased. Our findings suggest that refolding and degradation pathways are activated, causing an increased demand of energetic resources, thus the metabolic shift. Additionally, oxidative stress appears to be a consequence of the amyloidogenic process, posing an enhanced threat to cell survival.
A major limitation to yeast aging study has been the inability to track mother cells and observe molecular markers during the aging process. The traditional lifespan assay relies on manual micro-manipulation to remove daughter cells from the mother, which is laborious, time consuming, and does not allow long term tracking with high resolution microscopy. Recently, we have developed a microfluidic system capable of retaining mother cells in the microfluidic chambers while removing daughter cells automatically, making it possible to observe fluorescent reporters in single cells throughout their lifespan. Here we report the development of a new generation of microfluidic device that overcomes several limitations of the previous system, making it easier to fabricate and operate, and allowing functions not possible with the previous design. The basic unit of the device consists of microfluidic channels with pensile columns that can physically trap the mother cells while allowing the removal of daughter cells automatically by the flow of the fresh media. The whole microfluidic device contains multiple independent units operating in parallel, allowing simultaneous analysis of multiple strains. Using this system, we have reproduced the lifespan curves for the known long and short-lived mutants, demonstrating the power of the device for automated lifespan measurement. Following fluorescent reporters in single mother cells throughout their lifespan, we discovered a surprising change of expression of the translation elongation factor TEF2 during aging, suggesting altered translational control in aged mother cells. Utilizing the capability of the new device to trap mother-daughter pairs, we analyzed mother-daughter inheritance and found age dependent asymmetric partitioning of a general stress response reporter between mother and daughter cells.
Maintenance of an optimal redox environment is critical for appropriate functioning of cellular processes and cell survival. Despite the importance of maintaining redox homeostasis, it is not clear how the optimal redox potential is sensed and set, and the processes that impact redox on a cellular/organellar level are poorly understood. The genetic bases of cellular redox homeostasis were investigated using a green fluorescent protein (GFP) based redox probe, roGFP2 and a pH sensitive GFP-based probe, pHluorin. The use of roGFP2, in conjunction with pHluorin, enabled determination of pH-adjusted sub-cellular redox potential in a non-invasive and real-time manner. A genome-wide screen using both the non-essential and essential gene collections was carried out in Saccharomyces cerevisiae using cytosolic-roGFP2 to identify factors essential for maintenance of cytosolic redox state under steady-state conditions. 102 genes of diverse function were identified that are required for maintenance of cytosolic redox state. Mutations in these genes led to shifts in the half-cell glutathione redox potential by 75-10 mV. Interestingly, some specific oxidative stress-response processes were identified as over-represented in the data set. Further investigation of the role of oxidative stress-responsive systems in sub-cellular redox homeostasis was conducted using roGFP2 constructs targeted to the mitochondrial matrix and peroxisome and EGSH was measured in cells in exponential and stationary phase. Analyses allowed for the identification of key redox systems on a sub-cellular level and the identification of novel genes involved in the regulation of cellular redox homeostasis.
The correct folding of a protein is a pre-requirement for its proper posttranslational modification. The Escherichia coli Sec pathway, in which preproteins, in an unfolded, translocation-competent state, are rapidly secreted across the cytoplasmic membrane, is commonly assumed to be unfavorable for their modification in the cytosol. Whether posttranslationally modified recombinant preproteins can be efficiently transported via the Sec pathway, however, remains unclear. ACP and BCCP domain (BCCP87) are carrier proteins that can be converted into active phosphopantetheinylated ACP (holo-ACP) and biotinylated-BCCP (holo-BCCP) by AcpS and BirA, respectively. In the present study, we show that, when ACP or BCCP87 is fused to the C-terminus of secretory protein YebF or MBP, the resulting fusion protein preYebF-ACP, preYebF-BCCP87, preMBP-ACP or preMBP-BCCP87 can be modified and then secreted. Our data demonstrate that posttranslational modification of preYebF-ACP, preYebF-BCCP87 preMBP-ACP and preMBP-BCCP87 can take place in the cytosol prior to translocation, and the Sec machinery accommodates these previously modified fusion proteins. High levels of active holo-ACP and holo-BCCP87 are achieved when AcpS or BirA is co-expressed, especially when sodium azide is used to retard their translocation across the inner membrane. Our results also provide an alternative to achieve a high level of modified recombinant proteins expressed extracellularly.