Deep mutational scanning of Pneumocystis jirovecii dihydrofolate reductase reveals allosteric mechanism of resistance to an antifolate.

Pneumocystis jirovecii is a fungal pathogen that causes pneumocystis pneumonia, a disease that mainly affects immunocompromised individuals. This fungus has historically been hard to study because of our inability to grow it in vitro. One of the main drug targets in P. jirovecii is its dihydrofolate reductase (PjDHFR). Here, by using functional complementation of the baker's yeast ortholog, we show that PjDHFR can be inhibited by the antifolate methotrexate in a dose-dependent manner. Using deep mutational scanning of PjDHFR, we identify mutations conferring resistance to methotrexate. Thirty-one sites spanning the protein have at least one mutation that leads to resistance, for a total of 355 high-confidence resistance mutations. Most resistance-inducing mutations are found inside the active site, and many are structurally equivalent to mutations known to lead to resistance to different antifolates in other organisms. Some sites show specific resistance mutations, where only a single substitution confers resistance, whereas others are more permissive, as several substitutions at these sites confer resistance. Surprisingly, one of the permissive sites (F199) is without direct contact to either ligand or cofactor, suggesting that it acts through an allosteric mechanism. Modeling changes in binding energy between F199 mutants and drug shows that most mutations destabilize interactions between the protein and the drug. This evidence points towards a more important role of this position in resistance than previously estimated and highlights potential unknown allosteric mechanisms of resistance to antifolate in DHFRs. Our results offer unprecedented resources for the interpretation of mutation effects in the main drug target of an uncultivable fungal pathogen.

Cross-feeding affects the target of resistance evolution to an antifungal drug.

Pathogenic fungi are a cause of growing concern. Developing an efficient and safe antifungal is challenging because of the similar biological properties of fungal and host cells. Consequently, there is an urgent need to better understand the mechanisms underlying antifungal resistance to prolong the efficacy of current molecules. A major step in this direction would be to be able to predict or even prevent the acquisition of resistance. We leverage the power of experimental evolution to quantify the diversity of paths to resistance to the antifungal 5-fluorocytosine (5-FC), commercially known as flucytosine. We generated hundreds of independent 5-FC resistant mutants derived from two genetic backgrounds from wild isolates of Saccharomyces cerevisiae. Through automated pin-spotting, whole-genome and amplicon sequencing, we identified the most likely causes of resistance for most strains. Approximately a third of all resistant mutants evolved resistance through a pleiotropic drug response, a potentially novel mechanism in response to 5-FC, marked by cross-resistance to fluconazole. These cross-resistant mutants are characterized by a loss of respiration and a strong tradeoff in drug-free media. For the majority of the remaining two thirds, resistance was acquired through loss-of-function mutations in FUR1, which encodes an important enzyme in the metabolism of 5-FC. We describe conditions in which mutations affecting this particular step of the metabolic pathway are favored over known resistance mutations affecting a step upstream, such as the well-known target cytosine deaminase encoded by FCY1. This observation suggests that ecological interactions may dictate the identity of resistance hotspots.

Parallel Nonfunctionalization of CK1δ/ε Kinase Ohnologs Following a Whole-Genome Duplication Event.

Whole-genome duplication (WGD) followed by speciation allows us to examine the parallel evolution of ohnolog pairs. In the yeast family Saccharomycetaceae, HRR25 is a rare case of repeated ohnolog maintenance. This gene has reverted to a single copy in Saccharomyces cerevisiae where it is now essential, but has been maintained as pairs in at least 7 species post-WGD. In S. cerevisiae, HRR25 encodes the casein kinase 1δ/ε and plays a role in a variety of functions through its kinase activity and protein-protein interactions (PPIs). We hypothesized that the maintenance of duplicated HRR25 ohnologs could be a result of repeated subfunctionalization. We tested this hypothesis through a functional complementation assay in S. cerevisiae, testing all pairwise combinations of 25 orthologs (including 7 ohnolog pairs). Contrary to our expectations, we observed no cases of pair-dependent complementation, which would have supported the subfunctionalization hypothesis. Instead, most post-WGD species have one ohnolog that failed to complement, suggesting their nonfunctionalization or neofunctionalization. The ohnologs incapable of complementation have undergone more rapid protein evolution, lost most PPIs that were observed for their functional counterparts and singletons from post-WGD and non-WGD species, and have nonconserved cellular localization, consistent with their ongoing loss of function. The analysis in Naumovozyma castellii shows that the noncomplementing ohnolog is expressed at a lower level and has become nonessential. Taken together, our results indicate that HRR25 orthologs are undergoing gradual nonfunctionalization.

Evolutionary Trajectories are Contingent on Mitonuclear Interactions.

Critical mitochondrial functions, including cellular respiration, rely on frequently interacting components expressed from both the mitochondrial and nuclear genomes. The fitness of eukaryotic organisms depends on a tight collaboration between both genomes. In the face of an elevated rate of evolution in mtDNA, current models predict that the maintenance of mitonuclear compatibility relies on compensatory evolution of the nuclear genome. Mitonuclear interactions would therefore exert an influence on evolutionary trajectories. One prediction from this model is that the same nuclear genome evolving with different mitochondrial haplotypes would follow distinct molecular paths toward higher fitness. To test this prediction, we submitted 1,344 populations derived from 7 mitonuclear genotypes of Saccharomyces cerevisiae to >300 generations of experimental evolution in conditions that either select for a mitochondrial function or do not strictly require respiration for survival. Performing high-throughput phenotyping and whole-genome sequencing on independently evolved individuals, we identified numerous examples of gene-level evolutionary convergence among populations with the same mitonuclear background. Phenotypic and genotypic data on strains derived from this evolution experiment identify the nuclear genome and the environment as the main determinants of evolutionary divergence, but also show a modulating role for the mitochondrial genome exerted both directly and via interactions with the two other components. We finally recapitulated a subset of prominent loss-of-function alleles in the ancestral backgrounds and confirmed a generalized pattern of mitonuclear-specific and highly epistatic fitness effects. Together, these results demonstrate how mitonuclear interactions can dictate evolutionary divergence of populations with identical starting nuclear genotypes.

Barcode fusion genetics-protein-fragment complementation assay (BFG-PCA): tools and resources that expand the potential for binary protein interaction discovery.

Barcode fusion genetics (BFG) utilizes deep sequencing to improve the throughput of protein-protein interaction (PPI) screening in pools. BFG has been implemented in Yeast two-hybrid (Y2H) screens (BFG-Y2H). While Y2H requires test protein pairs to localize in the nucleus for reporter reconstruction, dihydrofolate reductase protein-fragment complementation assay (DHFR-PCA) allows proteins to localize in broader subcellular contexts and proves to be largely orthogonal to Y2H. Here, we implemented BFG to DHFR-PCA (BFG-PCA). This plasmid-based system can leverage ORF collections across model organisms to perform comparative analysis, unlike the original DHFR-PCA that requires yeast genomic integration. The scalability and quality of BFG-PCA were demonstrated by screening human and yeast interactions for >11 000 bait-prey pairs. BFG-PCA showed high-sensitivity and high-specificity for capturing known interactions for both species. BFG-Y2H and BFG-PCA capture distinct sets of PPIs, which can partially be explained based on the domain orientation of the reporter tags. BFG-PCA is a high-throughput protein interaction technology to interrogate binary PPIs that exploits clone collections from any species of interest, expanding the scope of PPI assays.

Expression attenuation as a mechanism of robustness against gene duplication.

Gene duplication is ubiquitous and a major driver of phenotypic diversity across the tree of life, but its immediate consequences are not fully understood. Deleterious effects would decrease the probability of retention of duplicates and prevent their contribution to long-term evolution. One possible detrimental effect of duplication is the perturbation of the stoichiometry of protein complexes. Here, we measured the fitness effects of the duplication of 899 essential genes in the budding yeast using high-resolution competition assays. At least 10% of genes caused a fitness disadvantage when duplicated. Intriguingly, the duplication of most protein complex subunits had small to nondetectable effects on fitness, with few exceptions. We selected four complexes with subunits that had an impact on fitness when duplicated and measured the impact of individual gene duplications on their protein-protein interactions. We found that very few duplications affect both fitness and interactions. Furthermore, large complexes such as the 26S proteasome are protected from gene duplication by attenuation of protein abundance. Regulatory mechanisms that maintain the stoichiometric balance of protein complexes may protect from the immediate effects of gene duplication. Our results show that a better understanding of protein regulation and assembly in complexes is required for the refinement of current models of gene duplication.

Turnover of ribosome-associated transcripts from de novo ORFs produces gene-like characteristics available for de novo gene emergence in wild yeast populations.

Little is known about the rate of emergence of de novo genes, what their initial properties are, and how they spread in populations. We examined wild yeast populations (Saccharomyces paradoxus) to characterize the diversity and turnover of intergenic ORFs over short evolutionary timescales. We find that hundreds of intergenic ORFs show translation signatures similar to canonical genes, and we experimentally confirmed the translation of many of these ORFs in laboratory conditions using a reporter assay. Compared with canonical genes, intergenic ORFs have lower translation efficiency, which could imply a lack of optimization for translation or a mechanism to reduce their production cost. Translated intergenic ORFs also tend to have sequence properties that are generally close to those of random intergenic sequences. However, some of the very recent translated intergenic ORFs, which appeared <110 kya, already show gene-like characteristics, suggesting that the raw material for functional innovations could appear over short evolutionary timescales.

Extended Linkers Improve the Detection of Protein-protein Interactions (PPIs) by Dihydrofolate Reductase Protein-fragment Complementation Assay (DHFR PCA) in Living Cells.

Understanding the function of cellular systems requires describing how proteins assemble with each other into transient and stable complexes and to determine their spatial relationships. Among the tools available to perform these analyses on a large scale is Protein-fragment Complementation Assay based on the dihydrofolate reductase (DHFR PCA). Here we test how longer linkers between the fusion proteins and the reporter fragments affect the performance of this assay. We investigate the architecture of the RNA polymerases, the proteasome and the conserved oligomeric Golgi (COG) complexes in living cells and performed large-scale screens with these extended linkers. We show that longer linkers significantly improve the detection of protein-protein interactions and allow to measure interactions further in space than the standard ones. We identify new interactions, for instance between the retromer complex and proteins related to autophagy and endocytosis. Longer linkers thus contribute an enhanced additional tool to the existing toolsets for the detection and measurements of protein-protein interactions and protein proximity in living cells.

The Rapid Evolution of an Ohnolog Contributes to the Ecological Specialization of Incipient Yeast Species.

Identifying the molecular changes that lead to ecological specialization during speciation is one of the major goals of molecular evolution. One question that remains to be thoroughly investigated is whether ecological specialization derives strictly from adaptive changes and their associated trade-offs, or from conditionally neutral mutations that accumulate under relaxed selection. We used whole-genome sequencing, genome annotation and computational analyses to identify genes that have rapidly diverged between two incipient species of Saccharomyces paradoxus that occupy different climatic regions along a south-west to north-east gradient. As candidate loci for ecological specialization, we identified genes that show signatures of adaptation and accelerated rates of amino acid substitutions, causing asymmetric evolution between lineages. This set of genes includes a glycyl-tRNA-synthetase, GRS2, which is known to be transcriptionally induced under heat stress in the model and sister species S. cerevisiae. Molecular modelling, expression analysis and fitness assays suggest that the accelerated evolution of this gene in the Northern lineage may be caused by relaxed selection. GRS2 arose during the whole-genome duplication (WGD) that occurred 100 million years ago in the yeast lineage. While its ohnolog GRS1 has been preserved in all post-WGD species, GRS2 has frequently been lost and is evolving rapidly, suggesting that the fate of this ohnolog is still to be resolved. Our results suggest that the asymmetric evolution of GRS2 between the two incipient S. paradoxus species contributes to their restricted climatic distributions and thus that ecological specialization derives at least partly from relaxed selection rather than a molecular trade-off resulting from adaptive evolution.

Systematic identification of signal integration by protein kinase A.

Cellular processes and homeostasis control in eukaryotic cells is achieved by the action of regulatory proteins such as protein kinase A (PKA). Although the outbound signals from PKA directed to processes such as metabolism, growth, and aging have been well charted, what regulates this conserved regulator remains to be systematically identified to understand how it coordinates biological processes. Using a yeast PKA reporter assay, we identified genes that influence PKA activity by measuring protein-protein interactions between the regulatory and the two catalytic subunits of the PKA complex in 3,726 yeast genetic-deletion backgrounds grown on two carbon sources. Overall, nearly 500 genes were found to be connected directly or indirectly to PKA regulation, including 80 core regulators, denoting a wide diversity of signals regulating PKA, within and beyond the described upstream linear pathways. PKA regulators span multiple processes, including the antagonistic autophagy and methionine biosynthesis pathways. Our results converge toward mechanisms of PKA posttranslational regulation by lysine acetylation, which is conserved between yeast and humans and that, we show, regulates protein complex formation in mammals and carbohydrate storage and aging in yeast. Taken together, these results show that the extent of PKA input matches with its output, because this kinase receives information from upstream and downstream processes, and highlight how biological processes are interconnected and coordinated by PKA.

Evolutionary rescue by compensatory mutations is constrained by genomic and environmental backgrounds.

Since deleterious mutations may be rescued by secondary mutations during evolution, compensatory evolution could identify genetic solutions leading to therapeutic targets. Here, we tested this hypothesis and examined whether these solutions would be universal or would need to be adapted to one's genetic and environmental make-ups. We performed experimental evolutionary rescue in a yeast disease model for the Wiskott-Aldrich syndrome in two genetic backgrounds and carbon sources. We found that multiple aspects of the evolutionary rescue outcome depend on the genotype, the environment, or a combination thereof. Specifically, the compensatory mutation rate and type, the molecular rescue mechanism, the genetic target, and the associated fitness cost varied across contexts. The course of compensatory evolution is therefore highly contingent on the initial conditions in which the deleterious mutation occurs. In addition, these results reveal biologically favored therapeutic targets for the Wiskott-Aldrich syndrome, including the target of an unrelated clinically approved drug. Our results experimentally illustrate the importance of epistasis and environmental evolutionary constraints that shape the adaptive landscape and evolutionary rate of molecular networks.

N-terminal entrance loop of yeast Yps1 and O-glycosylation of substrates are determinant factors controlling the shedding activity of this GPI-anchored endopeptidase.

BACKGROUND: S. cerevisiae Yps1 is the prototypical aspartic endopeptidase of the fungal yapsin family. This glycosylphosphatidylinositol (GPI) anchored enzyme was recently shown to be involved in the shedding of the GPI proteins Utr2, Gas1 and itself. It was also proposed to be part of a novel quality control mechanism that eliminates excess and/or misfolded GPI proteins. What regulates its shedding activity at the cell surface is however poorly understood. Yps1 is initially synthesized as a zymogen requiring proteolytic activation to remove a pro-peptide and further processing within a large insertion loop (N-entrance loop) generates a two-subunit endopeptidase. To investigate the role of this loop on its shedding activity, which typically takes place within Ser/Thr-rich domains, it was replaced with the short peptide found at the analogous position in Yps3. We also tested whether O-glycosylation might protect against proteolytic processing by Yps1. RESULTS: We show here that replacement of the N-entrance loop (N-ent loop) of Yps1 generates a single chain endopeptidase that undergoes partial (pH 6.0) or complete (pH 3.0) pro-peptide removal. At both pH, the shedding activity of the chimeric endopeptidase (Yps1-DL) toward Gas1 and itself is strongly and drastically increased, respectively. A direct correlation between endoproteolytic cleavage of this loop in native Yps1 and its shedding is observed. The Yps1-dependent shedding of two model GPI proteins (Gas1 and Yps1) is also stimulated by the absence of the O-mannosyltransferases, Pmt4 and Pmt2 respectively, involved in O-glycosylation of their Ser/Thr-rich domains. Under these conditions, some Yps1-independent shedding is also observed. CONCLUSIONS: Partial pro-peptide removal is essential to produce a functional Yps1 endopeptidase. The Yps1 N-ent loop plays a major role in regulating the shedding activity of the endopeptidase, most likely by limiting access to the active site, and its cleavage in native Yps1 is associated with its shedding. O-glycosylation protects against Yps1-dependent and -independent shedding of GPI proteins. It is postulated that hypoglycosylation of cell surface proteins, which may occur for misfolded proteins that escaped the ER-associated degradation, might target their elimination through shedding by Yps1 and possibly other yapsin members.

Evidence for the robustness of protein complexes to inter-species hybridization.

Despite the tremendous efforts devoted to the identification of genetic incompatibilities underlying hybrid sterility and inviability, little is known about the effect of inter-species hybridization at the protein interactome level. Here, we develop a screening platform for the comparison of protein-protein interactions (PPIs) among closely related species and their hybrids. We examine in vivo the architecture of protein complexes in two yeast species (Saccharomyces cerevisiae and Saccharomyces kudriavzevii) that diverged 5-20 million years ago and in their F1 hybrids. We focus on 24 proteins of two large complexes: the RNA polymerase II and the nuclear pore complex (NPC), which show contrasting patterns of molecular evolution. We found that, with the exception of one PPI in the NPC sub-complex, PPIs were highly conserved between species, regardless of protein divergence. Unexpectedly, we found that the architecture of the complexes in F1 hybrids could not be distinguished from that of the parental species. Our results suggest that the conservation of PPIs in hybrids likely results from the slow evolution taking place on the very few protein residues involved in the interaction or that protein complexes are inherently robust and may accommodate protein divergence up to the level that is observed among closely related species.

Local climatic adaptation in a widespread microorganism.

Exploring the ability of organisms to locally adapt is critical for determining the outcome of rapid climate changes, yet few studies have addressed this question in microorganisms. We investigated the role of a heterogeneous climate on adaptation of North American populations of the wild yeast Saccharomyces paradoxus. We found abundant among-strain variation for fitness components across a range of temperatures, but this variation was only partially explained by climatic variation in the distribution area. Most of fitness variation was explained by the divergence of genetically distinct groups, distributed along a north-south cline, suggesting that these groups have adapted to distinct climatic conditions. Within-group fitness components were correlated with climatic conditions, illustrating that even ubiquitous microorganisms locally adapt and harbour standing genetic variation for climate-related traits. Our results suggest that global climatic changes could lead to adaptation to new conditions within groups, or changes in their geographical distributions.

Exploring the northern limit of the distribution of Saccharomyces cerevisiae and Saccharomyces paradoxus in North America.

We examined the northern limit of Saccharomyces cerevisiae and Saccharomyces paradoxus in northeast America. We collected 876 natural samples at 29 sites and applied enrichment methods for the isolation of mesophilic yeasts. We uncovered a large diversity of yeasts, in some cases, associated with specific substrates. Sequencing of the ITS1, 5.8S and ITS2 loci allowed to assign 226 yeast strains at the species level, including 41 S. paradoxus strains. Our intensive sampling suggests that if present, S. cerevisiae is rare at these northern latitudes. Our sampling efforts spread across several months of the year revealed that successful sampling increases throughout the summer and diminishes significantly at the beginning of the fall. The data obtained on the ecological context of yeasts corroborate what was previously reported on Pichiaceae, Saccharomycodaceae, Debaryomycetaceae and Phaffomycetaceae yeast families. We identified 24 yeast isolates that could not be assigned to any known species and that may be of taxonomic, medical, or biotechnological importance. Our study reports new data on the taxonomic diversity of yeasts and new resources for studying the evolution and ecology of S. paradoxus.

Dissection of the role of a Src homology 3 domain in the evolution of binding preference of paralogous proteins.

Protein-protein interactions (PPIs) drive many cellular processes. Some interactions are directed by Src homology 3 (SH3) domains that bind proline-rich motifs on other proteins. The evolution of the binding specificity of SH3 domains is not completely understood, particularly following gene duplication. Paralogous genes accumulate mutations that can modify protein functions and, for SH3 domains, their binding preferences. Here, we examined how the binding of the SH3 domains of 2 paralogous yeast type I myosins, Myo3 and Myo5, evolved following duplication. We found that the paralogs have subtly different SH3-dependent interaction profiles. However, by swapping SH3 domains between the paralogs and characterizing the SH3 domains freed from their protein context, we find that very few of the differences in interactions, if any, depend on the SH3 domains themselves. We used ancestral sequence reconstruction to resurrect the preduplication SH3 domains and examined, moving back in time, how the binding preference changed. Although the most recent ancestor of the 2 domains had a very similar binding preference as the extant ones, older ancestral domains displayed a gradual loss of interaction with the modern interaction partners when inserted in the extant paralogs. Molecular docking and experimental characterization of the free ancestral domains showed that their affinity with the proline motifs is likely not the cause for this loss of binding. Taken together, our results suggest that a SH3 and its host protein could create intramolecular or allosteric interactions essential for the SH3-dependent PPIs, making domains not functionally equivalent even when they have the same binding specificity.

Cryptic genetic variation shapes the fate of gene duplicates in a protein interaction network.

Paralogous genes are often redundant for long periods of time before they diverge in function. While their functions are preserved, paralogous proteins can accumulate mutations that, through epistasis, could impact their fate in the future. By quantifying the impact of all single-amino acid substitutions on the binding of two myosin proteins to their interaction partners, we find that the future evolution of these proteins is highly contingent on their regulatory divergence and the mutations that have silently accumulated in their protein binding domains. Differences in the promoter strength of the two paralogs amplify the impact of mutations on binding in the lowly expressed one. While some mutations would be sufficient to non-functionalize one paralog, they would have minimal impact on the other. Our results reveal how functionally equivalent protein domains could be destined to specific fates by regulatory and cryptic coding sequence changes that currently have little to no functional impact.

Compensatory mutations potentiate constructive neutral evolution by gene duplication.

Protein functions generally depend on their assembly into complexes. During evolution, some complexes have transitioned from homomers encoded by a single gene to heteromers encoded by duplicate genes. This transition could occur without adaptive evolution through intermolecular compensatory mutations. Here, we experimentally duplicate and evolve an homodimeric enzyme to examine if and how this could happen. We identify hundreds of deleterious mutations that inactivate individual homodimers but produce functional enzymes when co-expressed as duplicated proteins that heterodimerize. The structure of one such heteromer reveals how both losses of function are buffered through the introduction of asymmetry in the complex that allows them to subfunctionalize. Constructive neutral evolution can thus occur by gene duplication followed by only one deleterious mutation per duplicate.

Epistasis between promoter activity and coding mutations shapes gene evolvability.

The evolution of protein-coding genes proceeds as mutations act on two main dimensions: regulation of transcription level and the coding sequence. The extent and impact of the connection between these two dimensions are largely unknown because they have generally been studied independently. By measuring the fitness effects of all possible mutations on a protein complex at various levels of promoter activity, we show that promoter activity at the optimal level for the wild-type protein masks the effects of both deleterious and beneficial coding mutations. Mutations that are deleterious at low activity but masked at optimal activity are slightly destabilizing for individual subunits and binding interfaces. Coding mutations that increase protein abundance are beneficial at low expression but could potentially incur a cost at high promoter activity. We thereby demonstrate that promoter activity in interaction with protein properties can dictate which coding mutations are beneficial, neutral, or deleterious.