Proteome-scale movements and compartment connectivity during the eukaryotic cell cycle.

Cell cycle progression relies on coordinated changes in the composition and subcellular localization of the proteome. By applying two distinct convolutional neural networks on images of millions of live yeast cells, we resolved proteome-level dynamics in both concentration and localization during the cell cycle, with resolution of ∼20 subcellular localization classes. We show that a quarter of the proteome displays cell cycle periodicity, with proteins tending to be controlled either at the level of localization or concentration, but not both. Distinct levels of protein regulation are preferentially utilized for different aspects of the cell cycle, with changes in protein concentration being mostly involved in cell cycle control and changes in protein localization in the biophysical implementation of the cell cycle program. We present a resource for exploring global proteome dynamics during the cell cycle, which will aid in understanding a fundamental biological process at a systems level.

Stress-dependent condensate formation regulated by the ubiquitin-related modifier Urm1.

The ability of proteins and RNA to coalesce into phase-separated assemblies, such as the nucleolus and stress granules, is a basic principle in organizing membraneless cellular compartments. While the constituents of biomolecular condensates are generally well documented, the mechanisms underlying their formation under stress are only partially understood. Here, we show in yeast that covalent modification with the ubiquitin-like modifier Urm1 promotes the phase separation of a wide range of proteins. We find that the drop in cellular pH induced by stress triggers Urm1 self-association and its interaction with both target proteins and the Urm1-conjugating enzyme Uba4. Urmylation of stress-sensitive proteins promotes their deposition into stress granules and nuclear condensates. Yeast cells lacking Urm1 exhibit condensate defects that manifest in reduced stress resilience. We propose that Urm1 acts as a reversible molecular "adhesive" to drive protective phase separation of functionally critical proteins under cellular stress.

The proteomic landscape of genome-wide genetic perturbations.

Functional genomic strategies have become fundamental for annotating gene function and regulatory networks. Here, we combined functional genomics with proteomics by quantifying protein abundances in a genome-scale knockout library in Saccharomyces cerevisiae, using data-independent acquisition mass spectrometry. We find that global protein expression is driven by a complex interplay of (1) general biological properties, including translation rate, protein turnover, the formation of protein complexes, growth rate, and genome architecture, followed by (2) functional properties, such as the connectivity of a protein in genetic, metabolic, and physical interaction networks. Moreover, we show that functional proteomics complements current gene annotation strategies through the assessment of proteome profile similarity, protein covariation, and reverse proteome profiling. Thus, our study reveals principles that govern protein expression and provides a genome-spanning resource for functional annotation.

Engineering a Model Cell for Rational Tuning of GPCR Signaling.

G protein-coupled receptor (GPCR) signaling is the primary method eukaryotes use to respond to specific cues in their environment. However, the relationship between stimulus and response for each GPCR is difficult to predict due to diversity in natural signal transduction architecture and expression. Using genome engineering in yeast, we constructed an insulated, modular GPCR signal transduction system to study how the response to stimuli can be predictably tuned using synthetic tools. We delineated the contributions of a minimal set of key components via computational and experimental refactoring, identifying simple design principles for rationally tuning the dose response. Using five different GPCRs, we demonstrate how this enables cells and consortia to be engineered to respond to desired concentrations of peptides, metabolites, and hormones relevant to human health. This work enables rational tuning of cell sensing while providing a framework to guide reprogramming of GPCR-based signaling in other systems.

Modulation of fungal phosphate homeostasis by the plant hormone strigolactone.

Inter-kingdom communication through small molecules is essential to the coexistence of organisms in an ecosystem. In soil communities, the plant root is a nexus of interactions for a remarkable number of fungi and is a source of small-molecule plant hormones that shape fungal compositions. Although hormone signaling pathways are established in plants, how fungi perceive and respond to molecules is unclear because many plant-associated fungi are recalcitrant to experimentation. Here, we develop an approach using the model fungus, Saccharomyces cerevisiae, to elucidate mechanisms of fungal response to plant hormones. Two plant hormones, strigolactone and methyl jasmonate, produce unique transcript profiles in yeast, affecting phosphate and sugar metabolism, respectively. Genetic analysis in combination with structural studies suggests that SLs require the high-affinity transporter Pho84 to modulate phosphate homeostasis. The ability to study small-molecule plant hormones in a tractable genetic system should have utility in understanding fungal-plant interactions.

Genomic Nucleosome Organization Reconstituted with Pure Proteins.

Chromatin remodelers regulate genes by organizing nucleosomes around promoters, but their individual contributions are obfuscated by the complex in vivo milieu of factor redundancy and indirect effects. Genome-wide reconstitution of promoter nucleosome organization with purified proteins resolves this problem and is therefore a critical goal. Here, we reconstitute four stages of nucleosome architecture using purified components: yeast genomic DNA, histones, sequence-specific Abf1/Reb1, and remodelers RSC, ISW2, INO80, and ISW1a. We identify direct, specific, and sufficient contributions that in vivo observations validate. First, RSC clears promoters by translating poly(dA:dT) into directional nucleosome removal. Second, partial redundancy is recapitulated where INO80 alone, or ISW2 at Abf1/Reb1sites, positions +1 nucleosomes. Third, INO80 and ISW2 each align downstream nucleosomal arrays. Fourth, ISW1a tightens the spacing to canonical repeat lengths. Such a minimal set of rules and proteins establishes core mechanisms by which promoter chromatin architecture arises through a blend of redundancy and specialization.

Manipulating the 3D organization of the largest synthetic yeast chromosome.

Whether synthetic genomes can power life has attracted broad interest in the synthetic biology field. Here, we report de novo synthesis of the largest eukaryotic chromosome thus far, synIV, a 1,454,621-bp yeast chromosome resulting from extensive genome streamlining and modification. We developed megachunk assembly combined with a hierarchical integration strategy, which significantly increased the accuracy and flexibility of synthetic chromosome construction. Besides the drastic sequence changes, we further manipulated the 3D structure of synIV to explore spatial gene regulation. Surprisingly, we found few gene expression changes, suggesting that positioning inside the yeast nucleoplasm plays a minor role in gene regulation. Lastly, we tethered synIV to the inner nuclear membrane via its hundreds of loxPsym sites and observed transcriptional repression of the entire chromosome, demonstrating chromosome-wide transcription manipulation without changing the DNA sequences. Our manipulation of the spatial structure of synIV sheds light on higher-order architectural design of the synthetic genomes.

Complementary strategies for directing in vivo transcription factor binding through DNA binding domains and intrinsically disordered regions.

DNA binding domains (DBDs) of transcription factors (TFs) recognize DNA sequence motifs that are highly abundant in genomes. Within cells, TFs bind a subset of motif-containing sites as directed by either their DBDs or DBD-external (nonDBD) sequences. To define the relative roles of DBDs and nonDBDs in directing binding preferences, we compared the genome-wide binding of 48 (∼30%) budding yeast TFs with their DBD-only, nonDBD-truncated, and nonDBD-only mutants. With a few exceptions, binding locations differed between DBDs and TFs, resulting from the cumulative action of multiple determinants mapped mostly to disordered nonDBD regions. Furthermore, TFs' preferences for promoters of the fuzzy nucleosome architecture were lost in DBD-only mutants, whose binding spread across promoters, implicating nonDBDs' preferences in this hallmark of budding yeast regulatory design. We conclude that DBDs and nonDBDs employ complementary DNA-targeting strategies, whose balance defines TF binding specificity along genomes.

Structural basis of a transcription pre-initiation complex on a divergent promoter.

Most eukaryotic promoter regions are divergently transcribed. As the RNA polymerase II pre-initiation complex (PIC) is intrinsically asymmetric and responsible for transcription in a single direction, it is unknown how divergent transcription arises. Here, the Saccharomyces cerevisiae Mediator complexed with a PIC (Med-PIC) was assembled on a divergent promoter and analyzed by cryoelectron microscopy. The structure reveals two distinct Med-PICs forming a dimer through the Mediator tail module, induced by a homodimeric activator protein localized near the dimerization interface. The tail dimer is associated with ∼80-bp upstream DNA, such that two flanking core promoter regions are positioned and oriented in a suitable form for PIC assembly in opposite directions. Also, cryoelectron tomography visualized the progress of the PIC assembly on the two core promoter regions, providing direct evidence for the role of the Med-PIC dimer in divergent transcription.

Quiescence in Saccharomyces cerevisiae.

Most cells live in environments that are permissive for proliferation only a small fraction of the time. Entering quiescence enables cells to survive long periods of nondivision and reenter the cell cycle when signaled to do so. Here, we describe what is known about the molecular basis for quiescence in Saccharomyces cerevisiae, with emphasis on the progress made in the last decade. Quiescence is triggered by depletion of an essential nutrient. It begins well before nutrient exhaustion, and there is extensive crosstalk between signaling pathways to ensure that all proliferation-specific activities are stopped when any one essential nutrient is limiting. Every aspect of gene expression is modified to redirect and conserve resources. Chromatin structure and composition change on a global scale, from histone modifications to three-dimensional chromatin structure. Thousands of proteins and RNAs aggregate, forming unique structures with unique fates, and the cytoplasm transitions to a glass-like state.

An integrated SAGA and TFIID PIC assembly pathway selective for poised and induced promoters.

Genome-wide, little is understood about how proteins organize at inducible promoters before and after induction and to what extent inducible and constitutive architectures depend on cofactors. We report that sequence-specific transcription factors and their tethered cofactors (e.g., SAGA [Spt-Ada-Gcn5-acetyltransferase], Mediator, TUP, NuA4, SWI/SNF, and RPD3-L) are generally bound to promoters prior to induction ("poised"), rather than recruited upon induction, whereas induction recruits the preinitiation complex (PIC) to DNA. Through depletion and/or deletion experiments, we show that SAGA does not function at constitutive promoters, although a SAGA-independent Gcn5 acetylates +1 nucleosomes there. When inducible promoters are poised, SAGA catalyzes +1 nucleosome acetylation but not PIC assembly. When induced, SAGA catalyzes acetylation, deubiquitylation, and PIC assembly. Surprisingly, SAGA mediates induction by creating a PIC that allows TFIID (transcription factor II-D) to stably associate, rather than creating a completely TFIID-independent PIC, as generally thought. These findings suggest that inducible systems, where present, are integrated with constitutive systems.

The Complex Genetic Basis and Multilayered Regulatory Control of Yeast Pseudohyphal Growth.

Eukaryotic cells are exquisitely responsive to external and internal cues, achieving precise control of seemingly diverse growth processes through a complex interplay of regulatory mechanisms. The budding yeast Saccharomyces cerevisiae provides a fascinating model of cell growth in its stress-responsive transition from planktonic single cells to a filamentous pseudohyphal growth form. During pseudohyphal growth, yeast cells undergo changes in morphology, polarity, and adhesion to form extended and invasive multicellular filaments. This pseudohyphal transition has been studied extensively as a model of conserved signaling pathways regulating cell growth and for its relevance in understanding the pathogenicity of the related opportunistic fungus Candida albicans, wherein filamentous growth is required for virulence. This review highlights the broad gene set enabling yeast pseudohyphal growth, signaling pathways that regulate this process, the role and regulation of proteins conferring cell adhesion, and interesting regulatory mechanisms enabling the pseudohyphal transition.

Motors, anchors, and connectors: orchestrators of organelle inheritance.

Organelle inheritance is a process whereby organelles are actively distributed between dividing cells at cytokinesis. Much valuable insight into the molecular mechanisms of organelle inheritance has come from the analysis of asymmetrically dividing cells, which transport a portion of their organelles to the bud while retaining another portion in the mother cell. Common principles apply to the inheritance of all organelles, although individual organelles use specific factors for their partitioning. Inheritance factors can be classified as motors, which are required for organelle transport; anchors, which immobilize organelles at distinct cell structures; or connectors, which mediate the attachment of organelles to motors and anchors. Here, we provide an overview of recent advances in the field of organelle inheritance and highlight how motor, anchor, and connector molecules choreograph the segregation of a multicopy organelle, the peroxisome. We also discuss the role of organelle population control in the generation of cellular diversity.

Repeated strand invasion and extensive branch migration are hallmarks of meiotic recombination.

Currently favored models for meiotic recombination posit that both noncrossover and crossover recombination are initiated by DNA double-strand breaks but form by different mechanisms: noncrossovers by synthesis-dependent strand annealing and crossovers by formation and resolution of double Holliday junctions centered around the break. This dual mechanism hypothesis predicts different hybrid DNA patterns in noncrossover and crossover recombinants. We show that these predictions are not upheld, by mapping with unprecedented resolution parental strand contributions to recombinants at a model locus. Instead, break repair in both noncrossovers and crossovers involves synthesis-dependent strand annealing, often with multiple rounds of strand invasion. Crossover-specific double Holliday junction formation occurs via processes involving branch migration as an integral feature, one that can be separated from repair of the break itself. These findings reveal meiotic recombination to be a highly dynamic process and prompt a new view of the relationship between crossover and noncrossover recombination.

RNA degradation triggered by decapping is largely independent of initial deadenylation.

RNA stability, important for eukaryotic gene expression, is thought to depend on deadenylation rates, with shortened poly(A) tails triggering decapping and 5' to 3' degradation. In contrast to this view, recent large-scale studies indicate that the most unstable mRNAs have, on average, long poly(A) tails. To clarify the role of deadenylation in mRNA decay, we first modeled mRNA poly(A) tail kinetics and mRNA stability in yeast. Independent of deadenylation rates, differences in mRNA decapping rates alone were sufficient to explain current large-scale results. To test the hypothesis that deadenylation and decapping are uncoupled, we used rapid depletion of decapping and deadenylation enzymes and measured changes in mRNA levels, poly(A) length and stability, both transcriptome-wide and with individual reporters. These experiments revealed that perturbations in poly(A) tail length did not correlate with variations in mRNA stability. Thus, while deadenylation may be critical for specific regulatory mechanisms, our results suggest that for most yeast mRNAs, it is not critical for mRNA decapping and degradation.

Receptor-mediated cargo hitchhiking on bulk autophagy.

While the molecular mechanism of autophagy is well studied, the cargoes delivered by autophagy remain incompletely characterized. To examine the selectivity of autophagy cargo, we conducted proteomics on isolated yeast autophagic bodies, which are intermediate structures in the autophagy process. We identify a protein, Hab1, that is highly preferentially delivered to vacuoles. The N-terminal 42 amino acid region of Hab1 contains an amphipathic helix and an Atg8-family interacting motif, both of which are necessary and sufficient for the preferential delivery of Hab1 by autophagy. We find that fusion of this region with a cytosolic protein results in preferential delivery of this protein to the vacuole. Furthermore, attachment of this region to an organelle allows for autophagic delivery in a manner independent of canonical autophagy receptor or scaffold proteins. We propose a novel mode of selective autophagy in which a receptor, in this case Hab1, binds directly to forming isolation membranes during bulk autophagy.

Processive Activity of Replicative DNA Polymerases in the Replisome of Live Eukaryotic Cells.

DNA replication is carried out by a multi-protein machine called the replisome. In Saccharomyces cerevisiae, the replisome is composed of over 30 different proteins arranged into multiple subassemblies, each performing distinct activities. Synchrony of these activities is required for efficient replication and preservation of genomic integrity. How this is achieved is particularly puzzling at the lagging strand, where current models of the replisome architecture propose turnover of the canonical lagging strand polymerase, Pol δ, at every cycle of Okazaki fragment synthesis. Here, we established single-molecule fluorescence microscopy protocols to study the binding kinetics of individual replisome subunits in live S. cerevisiae. Our results show long residence times for most subunits at the active replisome, supporting a model where all subassemblies bind tightly and work in a coordinated manner for extended periods, including Pol δ, redefining the architecture of the active eukaryotic replisome.

Regulation of Cohesin-Mediated Chromosome Folding by Eco1 and Other Partners.

Cohesin, a member of the SMC complex family, holds sister chromatids together but also shapes chromosomes by promoting the formation of long-range intra-chromatid loops, a process proposed to be mediated by DNA loop extrusion. Here we describe the roles of three cohesin partners, Pds5, Wpl1, and Eco1, in loop formation along either unreplicated or mitotic Saccharomyces cerevisiae chromosomes. Pds5 limits the size of DNA loops via two different pathways: the canonical Wpl1-mediated releasing activity and an Eco1-dependent mechanism. In the absence of Pds5, the main barrier to DNA loop expansion appears to be the centromere. Our data also show that Eco1 acetyl-transferase inhibits the translocase activity that powers loop formation and contributes to the positioning of loops through a mechanism that is distinguishable from its role in cohesion establishment. This study reveals that the mechanisms regulating cohesin-dependent chromatin loops are conserved among eukaryotes while promoting different functions.

DASH/Dam1 complex mutants stabilize ploidy in histone-humanized yeast by weakening kinetochore-microtubule attachments.

Forcing budding yeast to chromatinize their DNA with human histones manifests an abrupt fitness cost. We previously proposed chromosomal aneuploidy and missense mutations as two potential modes of adaptation to histone humanization. Here, we show that aneuploidy in histone-humanized yeasts is specific to a subset of chromosomes that are defined by their centromeric evolutionary origins but that these aneuploidies are not adaptive. Instead, we find that a set of missense mutations in outer kinetochore proteins drives adaptation to human histones. Furthermore, we characterize the molecular mechanism underlying adaptation in two mutants of the outer kinetochore DASH/Dam1 complex, which reduce aneuploidy by suppression of chromosome instability. Molecular modeling and biochemical experiments show that these two mutants likely disrupt a conserved oligomerization interface thereby weakening microtubule attachments. We propose a model through which weakened microtubule attachments promote increased kinetochore-microtubule turnover and thus suppress chromosome instability. In sum, our data show how a set of point mutations evolved in histone-humanized yeasts to counterbalance human histone-induced chromosomal instability through weakening microtubule interactions, eventually promoting a return to euploidy.

When yeast cells change their mind: cell cycle "Start" is reversible under starvation.

Eukaryotic cells decide in late G1 phase of the cell cycle whether to commit to another round of division. This point of cell cycle commitment is termed "Restriction Point" in mammals and "Start" in the budding yeast Saccharomyces cerevisiae. At Start, yeast cells integrate multiple signals such as pheromones and nutrients, and will not pass Start if nutrients are lacking. However, how cells respond to nutrient depletion after the Start decision remains poorly understood. Here, we analyze how post-Start cells respond to nutrient depletion, by monitoring Whi5, the cell cycle inhibitor whose export from the nucleus determines Start. Surprisingly, we find that cells that have passed Start can re-import Whi5 into the nucleus. In these cells, the positive feedback loop activating G1/S transcription is interrupted, and the Whi5 repressor re-binds DNA. Cells which re-import Whi5 become again sensitive to mating pheromone, like pre-Start cells, and CDK activation can occur a second time upon replenishment of nutrients. These results demonstrate that upon starvation, the commitment decision at Start can be reversed. We therefore propose that cell cycle commitment in yeast is a multi-step process, similar to what has been suggested for mammalian cells.