Comprehensive characterization of the Hsp70 interactome reveals novel client proteins and interactions mediated by posttranslational modifications.

Hsp70 interactions are critical for cellular viability and the response to stress. Previous attempts to characterize Hsp70 interactions have been limited by their transient nature and the inability of current technologies to distinguish direct versus bridged interactions. We report the novel use of cross-linking mass spectrometry (XL-MS) to comprehensively characterize the Saccharomyces cerevisiae (budding yeast) Hsp70 protein interactome. Using this approach, we have gained fundamental new insights into Hsp70 function, including definitive evidence of Hsp70 self-association as well as multipoint interaction with its client proteins. In addition to identifying a novel set of direct Hsp70 interactors that can be used to probe chaperone function in cells, we have also identified a suite of posttranslational modification (PTM)-associated Hsp70 interactions. The majority of these PTMs have not been previously reported and appear to be critical in the regulation of client protein function. These data indicate that one of the mechanisms by which PTMs contribute to protein function is by facilitating interaction with chaperones. Taken together, we propose that XL-MS analysis of chaperone complexes may be used as a unique way to identify biologically important PTMs on client proteins.

Unifying the mechanism of mitotic exit control in a spatiotemporal logical model.

The transition from mitosis into the first gap phase of the cell cycle in budding yeast is controlled by the Mitotic Exit Network (MEN). The network interprets spatiotemporal cues about the progression of mitosis and ensures that release of Cdc14 phosphatase occurs only after completion of key mitotic events. The MEN has been studied intensively; however, a unified understanding of how localisation and protein activity function together as a system is lacking. In this paper, we present a compartmental, logical model of the MEN that is capable of representing spatial aspects of regulation in parallel to control of enzymatic activity. We show that our model is capable of correctly predicting the phenotype of the majority of mutants we tested, including mutants that cause proteins to mislocalise. We use a continuous time implementation of the model to demonstrate that Cdc14 Early Anaphase Release (FEAR) ensures robust timing of anaphase, and we verify our findings in living cells. Furthermore, we show that our model can represent measured cell-cell variation in Spindle Position Checkpoint (SPoC) mutants. This work suggests a general approach to incorporate spatial effects into logical models. We anticipate that the model itself will be an important resource to experimental researchers, providing a rigorous platform to test hypotheses about regulation of mitotic exit.

Polo kinase recruitment via the constitutive centromere-associated network at the kinetochore elevates centromeric RNA.

The kinetochore, a multi-protein complex assembled on centromeres, is essential to segregate chromosomes during cell division. Deficiencies in kinetochore function can lead to chromosomal instability and aneuploidy-a hallmark of cancer cells. Kinetochore function is controlled by recruitment of regulatory proteins, many of which have been documented, however their function often remains uncharacterized and many are yet to be identified. To identify candidates of kinetochore regulation we used a proteome-wide protein association strategy in budding yeast and detected many proteins that are involved in post-translational modifications such as kinases, phosphatases and histone modifiers. We focused on the Polo-like kinase, Cdc5, and interrogated which cellular components were sensitive to constitutive Cdc5 localization. The kinetochore is particularly sensitive to constitutive Cdc5 kinase activity. Targeting Cdc5 to different kinetochore subcomplexes produced diverse phenotypes, consistent with multiple distinct functions at the kinetochore. We show that targeting Cdc5 to the inner kinetochore, the constitutive centromere-associated network (CCAN), increases the levels of centromeric RNA via an SPT4 dependent mechanism.

ScreenGarden: a shinyR application for fast and easy analysis of plate-based high-throughput screens.

BACKGROUND: Colony growth on solid media is a simple and effective measure for high-throughput genomic experiments such as yeast two-hybrid, synthetic dosage lethality and Synthetic Physical Interaction screens. The development of robotic pinning tools has facilitated the experimental design of these assays, and different imaging software can be used to automatically measure colony sizes on plates. However, comparison to control plates and statistical data analysis is often laborious and pinning issues or plate specific growth effects can lead to the detection of false-positive growth defects. RESULTS: We have developed ScreenGarden, a shinyR application, to enable easy, quick and robust data analysis of plate-based high throughput assays. The code allows comparisons of different formats of data and different sized arrays of colonies. A comparison of ScreenGarden with previous analysis tools shows that it performs, at least, equivalently. The software can be run either via a website or offline via the RStudio program; the code is available and can be modified by expert uses to customise the analysis. CONCLUSIONS: ScreenGarden provides a simple, fast and effective tool to analyse colony growth data from genomic experiments.

Cell-cycle phospho-regulation of the kinetochore.

The kinetochore is a mega-dalton protein assembly that forms within centromeric regions of chromosomes and directs their segregation during cell division. Here we review cell cycle-mediated phosphorylation events at the kinetochore, with a focus on the budding yeast Saccharomyces cerevisiae and the insight gained from forced associations of kinases and phosphatases. The point centromeres found in the budding yeast S. cerevisiae are one of the simplest such structures found in eukaryotes. The S. cerevisiae kinetochore comprises a single nucleosome, containing a centromere-specific H3 variant Cse4CENP-A, bound to a set of kinetochore proteins that connect to a single microtubule. Despite the simplicity of the budding yeast kinetochore, the proteins are mostly homologous with their mammalian counterparts. In some cases, human proteins can complement their yeast orthologs. Like its mammalian equivalent, the regulation of the budding yeast kinetochore is complex: integrating signals from the cell cycle, checkpoints, error correction, and stress pathways. The regulatory signals from these diverse pathways are integrated at the kinetochore by post-translational modifications, notably phosphorylation and dephosphorylation, to control chromosome segregation. Here we highlight the complex interplay between the activity of the different cell-cycle kinases and phosphatases at the kinetochore, emphasizing how much more we have to understand this essential structure.

CATS: Cas9-assisted tag switching. A high-throughput method for exchanging genomic peptide tags in yeast.

BACKGROUND: The creation of arrays of yeast strains each encoding a different protein with constant tags is a powerful method for understanding how genes and their proteins control cell function. As genetic tools become more sophisticated there is a need to create custom libraries encoding proteins fused with specialised tags to query gene function. These include protein tags that enable a multitude of added functionality, such as conditional degradation, fluorescent labelling, relocalization or activation and also DNA and RNA tags that enable barcoding of genes or their mRNA products. Tools for making new libraries or modifying existing ones are becoming available, but are often limited by the number of strains they can be realistically applied to or by the need for a particular starting library. RESULTS: We present a new recombination-based method, CATS - Cas9-Assisted Tag Switching, that switches tags in any existing library of yeast strains. This method employs the reprogrammable RNA guided nuclease, Cas9, to both introduce endogenous double strand breaks into the genome as well as liberating a linear DNA template molecule from a plasmid. It exploits the relatively high efficiency of homologous recombination in budding yeast compared with non-homologous end joining. CONCLUSIONS: The method takes less than 2 weeks, is cost effective and can simultaneously introduce multiple genetic changes, thus providing a rapid, genome-wide approach to genetic modification.

Synergistic Control of Kinetochore Protein Levels by Psh1 and Ubr2.

The accurate segregation of chromosomes during cell division is achieved by attachment of chromosomes to the mitotic spindle via the kinetochore, a large multi-protein complex that assembles on centromeres. The budding yeast kinetochore comprises more than 60 different proteins. Although the structure and function of many of these proteins has been investigated, we have little understanding of the steady state regulation of kinetochores. The primary model of kinetochore homeostasis suggests that kinetochores assemble hierarchically from the centromeric DNA via the inclusion of a centromere-specific histone into chromatin. We tested this model by trying to perturb kinetochore protein levels by overexpressing an outer kinetochore gene, MTW1. This increase in protein failed to change protein recruitment, consistent with the hierarchical assembly model. However, we find that deletion of Psh1, a key ubiquitin ligase that is known to restrict inner kinetochore protein loading, does not increase levels of outer kinetochore proteins, thus breaking the normal kinetochore stoichiometry. This perturbation leads to chromosome segregation defects, which can be partially suppressed by mutation of Ubr2, a second ubiquitin ligase that normally restricts protein levels at the outer kinetochore. Together these data show that Psh1 and Ubr2 synergistically control the amount of proteins at the kinetochore.

Synthetic physical interactions map kinetochore regulators and regions sensitive to constitutive Cdc14 localization.

The location of proteins within eukaryotic cells is often critical for their function and relocation of proteins forms the mainstay of regulatory pathways. To assess the importance of protein location to cellular homeostasis, we have developed a methodology to systematically create binary physical interactions between a query protein and most other members of the proteome. This method allows us to rapidly assess which of the thousands of possible protein interactions modify a phenotype. As proof of principle we studied the kinetochore, a multiprotein assembly that links centromeres to the microtubules of the spindle during cell division. In budding yeast, the kinetochores from the 16 chromosomes cluster together to a single location within the nucleus. The many proteins that make up the kinetochore are regulated through ubiquitylation and phosphorylation. By systematically associating members of the proteome to the kinetochore, we determine which fusions affect its normal function. We identify a number of candidate kinetochore regulators, including the phosphatase Cdc14. We examine where within the kinetochore Cdc14 can act and show that the effect is limited to regions that correlate with known phosphorylation sites, demonstrating the importance of serine phospho-regulation for normal kinetochore homeostasis.

Misregulation of cell cycle-dependent methylation of budding yeast CENP-A contributes to chromosomal instability.

Centromere (CEN) identity is specified epigenetically by specialized nucleosomes containing evolutionarily conserved CEN-specific histone H3 variant CENP-A (Cse4 in Saccharomyces cerevisiae, CENP-A in humans), which is essential for faithful chromosome segregation. However, the epigenetic mechanisms that regulate Cse4 function have not been fully defined. In this study, we show that cell cycle-dependent methylation of Cse4-R37 regulates kinetochore function and high-fidelity chromosome segregation. We generated a custom antibody that specifically recognizes methylated Cse4-R37 and showed that methylation of Cse4 is cell cycle regulated with maximum levels of methylated Cse4-R37 and its enrichment at the CEN chromatin occur in the mitotic cells. Methyl-mimic cse4-R37F mutant exhibits synthetic lethality with kinetochore mutants, reduced levels of CEN-associated kinetochore proteins and chromosome instability (CIN), suggesting that mimicking the methylation of Cse4-R37 throughout the cell cycle is detrimental to faithful chromosome segregation. Our results showed that SPOUT methyltransferase Upa1 contributes to methylation of Cse4-R37 and overexpression of UPA1 leads to CIN phenotype. In summary, our studies have defined a role for cell cycle-regulated methylation of Cse4 in high-fidelity chromosome segregation and highlight an important role of epigenetic modifications such as methylation of kinetochore proteins in preventing CIN, an important hallmark of human cancers.

Kinetochore asymmetry defines a single yeast lineage.

Asymmetric cell division is of fundamental importance in biology as it allows for the establishment of separate cell lineages during the development of multicellular organisms. Although microbial systems, including the yeast Saccharomyces cerevisiae, are excellent models of asymmetric cell division, this phenotype occurs in all cell divisions; consequently, models of lineage-specific segregation patterns in these systems do not exist. Here, we report the first example of lineage-specific asymmetric division in yeast. We used fluorescent tags to show that components of the yeast kinetochore, the protein complex that anchors chromosomes to the mitotic spindle, divide asymmetrically in a single postmeiotic lineage. This phenotype is not seen in vegetatively dividing haploid or diploid cells. This kinetochore asymmetry suggests a mechanism for the selective segregation of sister centromeres to daughter cells to establish different cell lineages or fates. These results provide a mechanistic link between lineage-defining asymmetry of metazoa with unicellular eukaryotes.

Forced association of SARS-CoV-2 proteins with the yeast proteome perturb vesicle trafficking.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the highly infectious coronavirus disease COVID-19. Extensive research has been performed in recent months to better understand how SARS-CoV-2 infects and manipulates its host to identify potential drug targets and support patient recovery from COVID-19. However, the function of many SARS-CoV-2 proteins remains uncharacterised. Here we used the Synthetic Physical Interactions (SPI) method to recruit SARS-CoV-2 proteins to most of the budding yeast proteome to identify conserved pathways which are affected by SARS-CoV-2 proteins. The set of yeast proteins that result in growth defects when associated with the viral proteins have homologous functions that overlap those identified in studies performed in mammalian cells. Specifically, we were able to show that recruiting the SARS-CoV-2 NSP1 protein to HOPS, a vesicle-docking complex, is sufficient to perturb membrane trafficking in yeast consistent with the hijacking of the endoplasmic-reticulum-Golgi intermediate compartment trafficking pathway during viral infection of mammalian cells. These data demonstrate that the yeast SPI method is a rapid way to identify potential functions of ectopic viral proteins.

Cdc7-mediated phosphorylation of Cse4 regulates high-fidelity chromosome segregation in budding yeast.

Faithful chromosome segregation maintains chromosomal stability as errors in this process contribute to chromosomal instability (CIN), which has been observed in many diseases including cancer. Epigenetic regulation of kinetochore proteins such as Cse4 (CENP-A in humans) plays a critical role in high-fidelity chromosome segregation. Here we show that Cse4 is a substrate of evolutionarily conserved Cdc7 kinase, and that Cdc7-mediated phosphorylation of Cse4 prevents CIN. We determined that Cdc7 phosphorylates Cse4 in vitro and interacts with Cse4 in vivo in a cell cycle-dependent manner. Cdc7 is required for kinetochore integrity as reduced levels of CEN-associated Cse4, a faster exchange of Cse4 at the metaphase kinetochores, and defects in chromosome segregation, are observed in a cdc7-7 strain. Phosphorylation of Cse4 by Cdc7 is important for cell survival as constitutive association of a kinase-dead variant of Cdc7 (cdc7-kd) with Cse4 at the kinetochore leads to growth defects. Moreover, phospho-deficient mutations of Cse4 for consensus Cdc7 target sites contribute to CIN phenotype. In summary, our results have defined a role for Cdc7-mediated phosphorylation of Cse4 in faithful chromosome segregation.

More is not always better: the genetic constraints of polyploidy.

Polyploid cells are a characteristic feature of certain human tissues, and notably many cancers. In a systematic genomic screen in yeast, Storchová and co-workers identified the genetic requirements of tetraploidy. Surprisingly, they showed that only three connected pathways are essential for the viability of tetraploid yeast cells. These data provide exciting new targets that might be essential specifically in polyploid cancer cells.

Asymmetric Transcription Factor Partitioning During Yeast Cell Division Requires the FACT Chromatin Remodeler and Cell Cycle Progression.

The polarized partitioning of proteins in cells underlies asymmetric cell division, which is an important driver of development and cellular diversity. The budding yeast Saccharomyces cerevisiae divides asymmetrically, like many other cells, to generate two distinct progeny cells. A well-known example of an asymmetric protein is the transcription factor Ace2, which localizes specifically to the daughter nucleus, where it drives a daughter-specific transcriptional network. We screened a collection of essential genes to analyze the effects of core cellular processes in asymmetric cell division based on Ace2 localization. This screen identified mutations that affect progression through the cell cycle, suggesting that cell cycle delay is sufficient to disrupt Ace2 asymmetry. To test this model, we blocked cells from progressing through mitosis and found that prolonged metaphase delay is sufficient to disrupt Ace2 asymmetry after release, and that Ace2 asymmetry is restored after cytokinesis. We also demonstrate that members of the evolutionarily conserved facilitates chromatin transcription (FACT) chromatin-reorganizing complex are required for both asymmetric and cell cycle-regulated localization of Ace2, and for localization of the RAM network components.

Cells expressing murine RAD52 splice variants favor sister chromatid repair.

The RAD52 gene is essential for homologous recombination in the yeast Saccharomyces cerevisiae. RAD52 is the archetype in an epistasis group of genes essential for DNA damage repair. By catalyzing the replacement of replication protein A with Rad51 on single-stranded DNA, Rad52 likely promotes strand invasion of a double-stranded DNA molecule by single-stranded DNA. Although the sequence and in vitro functions of mammalian RAD52 are conserved with those of yeast, one difference is the presence of introns and consequent splicing of the mammalian RAD52 pre-mRNA. We identified two novel splice variants from the RAD52 gene that are expressed in adult mouse tissues. Expression of these splice variants in tissue culture cells elevates the frequency of recombination that uses a sister chromatid template. To characterize this dominant phenotype further, the RAD52 gene from the yeast Saccharomyces cerevisiae was truncated to model the mammalian splice variants. The same dominant sister chromatid recombination phenotype seen in mammalian cells was also observed in yeast. Furthermore, repair from a homologous chromatid is reduced in yeast, implying that the choice of alternative repair pathways may be controlled by these variants. In addition, a dominant DNA repair defect induced by one of the variants in yeast is suppressed by overexpression of RAD51, suggesting that the Rad51-Rad52 interaction is impaired.

Synthetic Physical Interactions with the Yeast Centrosome.

The yeast centrosome or Spindle Pole Body (SPB) is an organelle situated in the nuclear membrane, where it nucleates spindle microtubules and acts as a signaling hub. Various studies have explored the effects of forcing individual proteins to interact with the yeast SPB, however no systematic study has been performed. We used synthetic physical interactions to detect proteins that inhibit growth when forced to associate with the SPB. We found the SPB to be especially sensitive to relocalization, necessitating a novel data analysis approach. This novel analysis of SPI screening data shows that regions of the cell are locally more sensitive to forced relocalization than previously thought. Furthermore, we found a set of associations that result in elevated SPB number and, in some cases, multi-polar spindles. Since hyper-proliferation of centrosomes is a hallmark of cancer cells, these associations point the way for the use of yeast models in the study of spindle formation and chromosome segregation in cancer.

Cell cycle-dependent association of polo kinase Cdc5 with CENP-A contributes to faithful chromosome segregation in budding yeast.

Evolutionarily conserved polo-like kinase, Cdc5 (Plk1 in humans), associates with kinetochores during mitosis; however, the role of cell cycle-dependent centromeric ( CEN) association of Cdc5 and its substrates that exclusively localize to the kinetochore have not been characterized. Here we report that evolutionarily conserved CEN histone H3 variant, Cse4 (CENP-A in humans), is a substrate of Cdc5, and that the cell cycle-regulated association of Cse4 with Cdc5 is required for cell growth. Cdc5 contributes to Cse4 phosphorylation in vivo and interacts with Cse4 in mitotic cells. Mass spectrometry analysis of in vitro kinase assays showed that Cdc5 phosphorylates nine serine residues clustered within the N-terminus of Cse4. Strains with cse4-9SA exhibit increased errors in chromosome segregation, reduced levels of CEN-associated Mif2 and Mcd1/Scc1 when combined with a deletion of MCM21. Moreover, the loss of Cdc5 from the CEN chromatin contributes to defects in kinetochore integrity and reduction in CEN-associated Cse4. The cell cycle-regulated association of Cdc5 with Cse4 is essential for cell viability as constitutive association of Cdc5 with Cse4 at the kinetochore leads to growth defects. In summary, our results have defined a role for Cdc5-mediated Cse4 phosphorylation in faithful chromosome segregation.

Rewiring the Budding Yeast Proteome using Synthetic Physical Interactions.

Artificially tethering two proteins or protein fragments together is a powerful method to query molecular mechanisms. However, this approach typically relies upon a prior understanding of which two proteins, when fused, are most likely to provide a specific function and is therefore not readily amenable to large-scale screening. Here, we describe the Synthetic Physical Interaction (SPI) method to create proteome-wide forced protein associations in the budding yeast Saccharomyces cerevisiae. This method allows thousands of protein-protein associations to be screened for those that affect either normal growth or sensitivity to drugs or specific conditions. The method is amenable to proteins, protein domains, or any genetically encoded peptide sequence.

Synthetic Physical Interactions Map Kinetochore-Checkpoint Activation Regions.

The spindle assembly checkpoint (SAC) is a key mechanism to regulate the timing of mitosis and ensure that chromosomes are correctly segregated to daughter cells. The recruitment of the Mad1 and Mad2 proteins to the kinetochore is normally necessary for SAC activation. This recruitment is coordinated by the SAC kinase Mps1, which phosphorylates residues at the kinetochore to facilitate binding of Bub1, Bub3, Mad1, and Mad2. There is evidence that the essential function of Mps1 is to direct recruitment of Mad1/2. To test this model, we have systematically recruited Mad1, Mad2, and Mps1 to most proteins in the yeast kinetochore, and find that, while Mps1 is sufficient for checkpoint activation, recruitment of either Mad1 or Mad2 is not. These data indicate an important role for Mps1 phosphorylation in SAC activation, beyond the direct recruitment of Mad1 and Mad2.

Synthetic protein interactions reveal a functional map of the cell.

To understand the function of eukaryotic cells, it is critical to understand the role of protein-protein interactions and protein localization. Currently, we do not know the importance of global protein localization nor do we understand to what extent the cell is permissive for new protein associations - a key requirement for the evolution of new protein functions. To answer this question, we fused every protein in the yeast Saccharomyces cerevisiae with a partner from each of the major cellular compartments and quantitatively assessed the effects upon growth. This analysis reveals that cells have a remarkable and unanticipated tolerance for forced protein associations, even if these associations lead to a proportion of the protein moving compartments within the cell. Furthermore, the interactions that do perturb growth provide a functional map of spatial protein regulation, identifying key regulatory complexes for the normal homeostasis of eukaryotic cells.