Genome-wide screen identifies new set of genes for improved heterologous laccase expression in Saccharomyces cerevisiae.

The yeast Saccharomyces cerevisiae is widely used as a host cell for recombinant protein production due to its fast growth, cost-effective culturing, and ability to secrete large and complex proteins. However, one major drawback is the relatively low yield of produced proteins compared to other host systems. To address this issue, we developed an overlay assay to screen the yeast knockout collection and identify mutants that enhance recombinant protein production, specifically focusing on the secretion of the Trametes trogii fungal laccase enzyme. Gene ontology analysis of these mutants revealed an enrichment of processes including vacuolar targeting, vesicle trafficking, proteolysis, and glycolipid metabolism. We confirmed that a significant portion of these mutants also showed increased activity of the secreted laccase when grown in liquid culture. Notably, we found that the combination of deletions of OCA6, a tyrosine phosphatase gene, along with PMT1 or PMT2, two genes encoding ER membrane protein-O-mannosyltransferases involved in ER quality control, and SKI3, which encode for a component of the SKI complex responsible for mRNA degradation, further increased secreted laccase activity. Conversely, we also identified over 200 gene deletions that resulted in decreased secreted laccase activity, including many genes that encode for mitochondrial proteins and components of the ER-associated degradation pathway. Intriguingly, the deletion of the ER DNAJ co-chaperone gene SCJ1 led to almost no secreted laccase activity. When we expressed SCJ1 from a low-copy plasmid, laccase secretion was restored. However, overexpression of SCJ1 had a detrimental effect, indicating that precise dosing of key chaperone proteins is crucial for optimal recombinant protein expression. This study offers potential strategies for enhancing the overall yield of recombinant proteins and provides new avenues for further research in optimizing protein production systems.

Clionamines stimulate autophagy, inhibit Mycobacterium tuberculosis survival in macrophages, and target Pik1.

The pathogen Mycobacterium tuberculosis (Mtb) evades the innate immune system by interfering with autophagy and phagosomal maturation in macrophages, and, as a result, small molecule stimulation of autophagy represents a host-directed therapeutics (HDTs) approach for treatment of tuberculosis (TB). Here we show the marine natural product clionamines activate autophagy and inhibit Mtb survival in macrophages. A yeast chemical-genetics approach identified Pik1 as target protein of the clionamines. Biotinylated clionamine B pulled down Pik1 from yeast cell lysates and a clionamine analog inhibited phosphatidyl 4-phosphate (PI4P) production in yeast Golgi membranes. Chemical-genetic profiles of clionamines and cationic amphiphilic drugs (CADs) are closely related, linking the clionamine mode of action to co-localization with PI4P in a vesicular compartment. Small interfering RNA (siRNA) knockdown of PI4KB, a human homolog of Pik1, inhibited the survival of Mtb in macrophages, identifying PI4KB as an unexploited molecular target for efforts to develop HDT drugs for treatment of TB.

A scalable Drosophila assay for clinical interpretation of human PTEN variants in suppression of PI3K/AKT induced cellular proliferation.

Gene variant discovery is becoming routine, but it remains difficult to usefully interpret the functional consequence or disease relevance of most variants. To fill this interpretation gap, experimental assays of variant function are becoming common place. Yet, it remains challenging to make these assays reproducible, scalable to high numbers of variants, and capable of assessing defined gene-disease mechanism for clinical interpretation aligned to the ClinGen Sequence Variant Interpretation (SVI) Working Group guidelines for 'well-established assays'. Drosophila melanogaster offers great potential as an assay platform, but was untested for high numbers of human variants adherent to these guidelines. Here, we wished to test the utility of Drosophila as a platform for scalable well-established assays. We took a genetic interaction approach to test the function of ~100 human PTEN variants in cancer-relevant suppression of PI3K/AKT signaling in cellular growth and proliferation. We validated the assay using biochemically characterized PTEN mutants as well as 23 total known pathogenic and benign PTEN variants, all of which the assay correctly assigned into predicted functional categories. Additionally, function calls for these variants correlated very well with our recent published data from a human cell line. Finally, using these pathogenic and benign variants to calibrate the assay, we could set readout thresholds for clinical interpretation of the pathogenicity of 70 other PTEN variants. Overall, we demonstrate that Drosophila offers a powerful assay platform for clinical variant interpretation, that can be used in conjunction with other well-established assays, to increase confidence in the accurate assessment of variant function and pathogenicity.

Ist2 recruits the lipid transporters Osh6/7 to ER-PM contacts to maintain phospholipid metabolism.

ER-plasma membrane (PM) contacts are proposed to be held together by distinct families of tethering proteins, which in yeast include the VAP homologues Scs2/22, the extended-synaptotagmin homologues Tcb1/2/3, and the TMEM16 homologue Ist2. It is unclear whether these tethers act redundantly or whether individual tethers have specific functions at contacts. Here, we show that Ist2 directly recruits the phosphatidylserine (PS) transport proteins and ORP family members Osh6 and Osh7 to ER-PM contacts through a binding site located in Ist2's disordered C-terminal tethering region. This interaction is required for phosphatidylethanolamine (PE) production by the PS decarboxylase Psd2, whereby PS transported from the ER to the PM by Osh6/7 is endocytosed to the site of Psd2 in endosomes/Golgi/vacuoles. This role for Ist2 and Osh6/7 in nonvesicular PS transport is specific, as other tethers/transport proteins do not compensate. Thus, we identify a molecular link between the ORP and TMEM16 families and a role for endocytosis of PS in PE synthesis.

MAPS: machine-assisted phenotype scoring enables rapid functional assessment of genetic variants by high-content microscopy.

BACKGROUND: Genetic testing is widely used in evaluating a patient's predisposition to hereditary diseases. In the case of cancer, when a functionally impactful mutation (i.e. genetic variant) is identified in a disease-relevant gene, the patient is at elevated risk of developing a lesion in their lifetime. Unfortunately, as the rate and coverage of genetic testing has accelerated, our ability to assess the functional status of new variants has fallen behind. Therefore, there is an urgent need for more practical, streamlined and cost-effective methods for classifying variants. RESULTS: To directly address this issue, we designed a new approach that uses alterations in protein subcellular localization as a key indicator of loss of function. Thus, new variants can be rapidly functionalized using high-content microscopy (HCM). To facilitate the analysis of the large amounts of imaging data, we developed a new software toolkit, named MAPS for machine-assisted phenotype scoring, that utilizes deep learning to extract and classify cell-level features. MAPS helps users leverage cloud-based deep learning services that are easy to train and deploy to fit their specific experimental conditions. Model training is code-free and can be done with limited training images. Thus, MAPS allows cell biologists to easily incorporate deep learning into their image analysis pipeline. We demonstrated an effective variant functionalization workflow that integrates HCM and MAPS to assess missense variants of PTEN, a tumor suppressor that is frequently mutated in hereditary and somatic cancers. CONCLUSIONS: This paper presents a new way to rapidly assess variant function using cloud deep learning. Since most tumor suppressors have well-defined subcellular localizations, our approach could be widely applied to functionalize variants of uncertain significance and help improve the utility of genetic testing.

A Premalignant Cell-Based Model for Functionalization and Classification of PTEN Variants.

As sequencing becomes more economical, we are identifying sequence variations in the population faster than ever. For disease-associated genes, it is imperative that we differentiate a sequence variant as either benign or pathogenic, such that the appropriate therapeutic interventions or surveillance can be implemented. PTEN is a frequently mutated tumor suppressor that has been linked to the PTEN hamartoma tumor syndrome. Although the domain structure of PTEN and the functional impact of a number of its most common tumor-linked mutations have been characterized, there is a lack of information about many recently identified clinical variants. To address this challenge, we developed a cell-based assay that utilizes a premalignant phenotype of normal mammary epithelial cells lacking PTEN. We measured the ability of PTEN variants to rescue the spheroid formation phenotype of PTEN-/- MCF10A cells maintained in suspension. As proof of concept, we functionalized 47 missense variants using this assay, only 19 of which have clear classifications in ClinVar. We utilized a machine learning model trained with annotated genotypic data to classify variants as benign or pathogenic based on our functional scores. Our model predicted with high accuracy that loss of PTEN function was indicative of pathogenicity. We also determined that the pathogenicity of certain variants may have arisen from reduced stability of the protein product. Overall, this assay outperformed computational predictions, was scalable, and had a short run time, serving as an ideal alternative for annotating the clinical significance of cancer-associated PTEN variants. SIGNIFICANCE: Combined three-dimensional tumor spheroid modeling and machine learning classifies PTEN missense variants, over 70% of which are currently listed as variants of uncertain significance. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/13/2775/F1.large.jpg.

Multi-model functionalization of disease-associated PTEN missense mutations identifies multiple molecular mechanisms underlying protein dysfunction.

Functional variomics provides the foundation for personalized medicine by linking genetic variation to disease expression, outcome and treatment, yet its utility is dependent on appropriate assays to evaluate mutation impact on protein function. To fully assess the effects of 106 missense and nonsense variants of PTEN associated with autism spectrum disorder, somatic cancer and PTEN hamartoma syndrome (PHTS), we take a deep phenotypic profiling approach using 18 assays in 5 model systems spanning diverse cellular environments ranging from molecular function to neuronal morphogenesis and behavior. Variants inducing instability occur across the protein, resulting in partial-to-complete loss-of-function (LoF), which is well correlated across models. However, assays are selectively sensitive to variants located in substrate binding and catalytic domains, which exhibit complete LoF or dominant negativity independent of effects on stability. Our results indicate that full characterization of variant impact requires assays sensitive to instability and a range of protein functions.

pH Biosensing by PI4P Regulates Cargo Sorting at the TGN.

Phosphoinositides, diacylglycerolpyrophosphate, ceramide-1-phosphate, and phosphatidic acid belong to a unique class of membrane signaling lipids that contain phosphomonoesters in their headgroups having pKa values in the physiological range. The phosphomonoester headgroup of phosphatidic acid enables this lipid to act as a pH biosensor as changes in its protonation state with intracellular pH regulate binding to effector proteins. Here, we demonstrate that binding of pleckstrin homology (PH) domains to phosphatidylinositol 4-phosphate (PI4P) in the yeast trans-Golgi network (TGN) is dependent on intracellular pH, indicating PI4P is a pH biosensor. pH biosensing by TGN PI4P in response to nutrient availability governs protein sorting at the TGN, likely by regulating sterol transfer to the TGN by Osh1, a member of the conserved oxysterol-binding protein (OSBP) family of lipid transfer proteins. Thus, pH biosensing by TGN PI4P allows for direct metabolic regulation of protein trafficking and cell growth.

Mechanisms of PTPσ-Mediated Presynaptic Differentiation.

Formation of synapses between neurons depends in part on binding between axonal and dendritic cell surface synaptic organizing proteins, which recruit components of the developing presynaptic and postsynaptic specializations. One of these presynaptic organizing molecules is protein tyrosine phosphatase σ (PTPσ). Although the protein domains involved in adhesion between PTPσ and its postsynaptic binding partners are known, the mechanisms by which it signals into the presynaptic neuron to recruit synaptic vesicles and other necessary components for regulated transmitter release are not well understood. One attractive candidate to mediate this function is liprin-α, a scaffolding protein with well-established roles at the synapse. We systematically mutated residues of the PTPσ intracellular region (ICR) and used the yeast dihydrofolate reductase (DHFR) protein complementation assay to screen for disrupted interactions between these mutant forms of PTPσ and its various binding partners. Using a molecular replacement strategy, we show that disrupting the interaction between PTPσ and liprin-α, but not between PTPσ and itself or another binding partner, caskin, abolishes presynaptic differentiation. Furthermore, phosphatase activity of PTPσ and binding to extracellular heparan sulfate (HS) proteoglycans are dispensable for presynaptic induction. Previous reports have suggested that binding between PTPσ and liprin-α is mediated by the PTPσ membrane-distal phosphatase-like domain. However, we provide evidence here that both of the PTPσ phosphatase-like domains mediate binding to liprin-α and are required for PTPσ-mediated presynaptic differentiation. These findings further our understanding of the mechanistic basis by which PTPσ acts as a presynaptic organizer.

Viral proteins as a potential driver of histone depletion in dinoflagellates.

Within canonical eukaryotic nuclei, DNA is packaged with highly conserved histone proteins into nucleosomes, which facilitate DNA condensation and contribute to genomic regulation. Yet the dinoflagellates, a group of unicellular algae, are a striking exception to this otherwise universal feature as they have largely abandoned histones and acquired apparently viral-derived substitutes termed DVNPs (dinoflagellate-viral-nucleoproteins). Despite the magnitude of this transition, its evolutionary drivers remain unknown. Here, using Saccharomyces cerevisiae as a model, we show that DVNP impairs growth and antagonizes chromatin by localizing to histone binding sites, displacing nucleosomes, and impairing transcription. Furthermore, DVNP toxicity can be relieved through histone depletion and cells diminish their histones in response to DVNP expression suggesting that histone reduction could have been an adaptive response to these viral proteins. These findings provide insights into eukaryotic chromatin evolution and highlight the potential for horizontal gene transfer to drive the divergence of cellular systems.

Selective aggregation of the splicing factor Hsh155 suppresses splicing upon genotoxic stress.

Upon genotoxic stress, dynamic relocalization events control DNA repair as well as alterations of the transcriptome and proteome, enabling stress recovery. How these events may influence one another is only partly known. Beginning with a cytological screen of genome stability proteins, we find that the splicing factor Hsh155 disassembles from its partners and localizes to both intranuclear and cytoplasmic protein quality control (PQC) aggregates under alkylation stress. Aggregate sequestration of Hsh155 occurs at nuclear and then cytoplasmic sites in a manner that is regulated by molecular chaperones and requires TORC1 activity signaling through the Sfp1 transcription factor. This dynamic behavior is associated with intron retention in ribosomal protein gene transcripts, a decrease in splicing efficiency, and more rapid recovery from stress. Collectively, our analyses suggest a model in which some proteins evicted from chromatin and undergoing transcriptional remodeling during stress are targeted to PQC sites to influence gene expression changes and facilitate stress recovery.

Systematic identification of genes involved in metabolic acid stress resistance in yeast and their potential as cancer targets.

A hallmark of all primary and metastatic tumours is their high rate of glucose uptake and glycolysis. A consequence of the glycolytic phenotype is the accumulation of metabolic acid; hence, tumour cells experience considerable intracellular acid stress. To compensate, tumour cells upregulate acid pumps, which expel the metabolic acid into the surrounding tumour environment, resulting in alkalization of intracellular pH and acidification of the tumour microenvironment. Nevertheless, we have only a limited understanding of the consequences of altered intracellular pH on cell physiology, or of the genes and pathways that respond to metabolic acid stress. We have used yeast as a genetic model for metabolic acid stress with the rationale that the metabolic changes that occur in cancer that lead to intracellular acid stress are likely fundamental. Using a quantitative systems biology approach we identified 129 genes required for optimal growth under conditions of metabolic acid stress. We identified six highly conserved protein complexes with functions related to oxidative phosphorylation (mitochondrial respiratory chain complex III and IV), mitochondrial tRNA biosynthesis [glutamyl-tRNA(Gln) amidotransferase complex], histone methylation (Set1C-COMPASS), lysosome biogenesis (AP-3 adapter complex), and mRNA processing and P-body formation (PAN complex). We tested roles for two of these, AP-3 adapter complex and PAN deadenylase complex, in resistance to acid stress using a myeloid leukaemia-derived human cell line that we determined to be acid stress resistant. Loss of either complex inhibited growth of Hap1 cells at neutral pH and caused sensitivity to acid stress, indicating that AP-3 and PAN complexes are promising new targets in the treatment of cancer. Additionally, our data suggests that tumours may be genetically sensitized to acid stress and hence susceptible to acid stress-directed therapies, as many tumours accumulate mutations in mitochondrial respiratory chain complexes required for their proliferation.

A conserved endoplasmic reticulum membrane protein complex (EMC) facilitates phospholipid transfer from the ER to mitochondria.

Mitochondrial membrane biogenesis and lipid metabolism require phospholipid transfer from the endoplasmic reticulum (ER) to mitochondria. Transfer is thought to occur at regions of close contact of these organelles and to be nonvesicular, but the mechanism is not known. Here we used a novel genetic screen in S. cerevisiae to identify mutants with defects in lipid exchange between the ER and mitochondria. We show that a strain missing multiple components of the conserved ER membrane protein complex (EMC) has decreased phosphatidylserine (PS) transfer from the ER to mitochondria. Mitochondria from this strain have significantly reduced levels of PS and its derivative phosphatidylethanolamine (PE). Cells lacking EMC proteins and the ER-mitochondria tethering complex called ERMES (the ER-mitochondria encounter structure) are inviable, suggesting that the EMC also functions as a tether. These defects are corrected by expression of an engineered ER-mitochondrial tethering protein that artificially tethers the ER to mitochondria. EMC mutants have a significant reduction in the amount of ER tethered to mitochondria even though ERMES remained intact in these mutants, suggesting that the EMC performs an additional tethering function to ERMES. We find that all Emc proteins interact with the mitochondrial translocase of the outer membrane (TOM) complex protein Tom5 and this interaction is important for PS transfer and cell growth, suggesting that the EMC forms a tether by associating with the TOM complex. Together, our findings support that the EMC tethers ER to mitochondria, which is required for phospholipid synthesis and cell growth.

Polarization of the endoplasmic reticulum by ER-septin tethering.

Polarization of the plasma membrane (PM) into domains is an important mechanism to compartmentalize cellular activities and to establish cell polarity. Polarization requires formation of diffusion barriers that prevent mixing of proteins between domains. Recent studies have uncovered that the endoplasmic reticulum (ER) of budding yeast and neurons is polarized by diffusion barriers, which in neurons controls glutamate signaling in dendritic spines. The molecular identity of these barriers is currently unknown. Here, we show that a direct interaction between the ER protein Scs2 and the septin Shs1 creates the ER diffusion barrier in yeast. Barrier formation requires Epo1, a novel ER-associated subunit of the polarisome that interacts with Scs2 and Shs1. ER-septin tethering polarizes the ER into separate mother and bud domains, one function of which is to position the spindle in the mother until M phase by confining the spindle capture protein Num1 to the mother ER.

Barriers to uniformity within the endoplasmic reticulum.

Differentiating the endoplasmic reticulum (ER) into different physical domains may help the ER spatially regulate its many functions. For example, ER sheets are highly decorated with ribosomes for protein synthesis, whereas tubules usually correspond to smooth ER. Hence, ER morphology may play direct roles in functional diversification within the ER. The ER also makes direct physical contacts with other organelles, called ER junctions, enabling further functional diversification through input from external sources. In yeast, an ER diffusion barrier has now been discovered at the bud neck that compartmentalizes the ER into bud and mother diffusion domains by restricting the lateral diffusion of ER membrane proteins. Therefore, diffusion barriers also likely contribute to functional diversification within the ER by creating suites of molecular factors within ER diffusion domains.

An assembly of proteins and lipid domains regulates transport of phosphatidylserine to phosphatidylserine decarboxylase 2 in Saccharomyces cerevisiae.

Saccharomyces cerevisiae uses multiple biosynthetic pathways for the synthesis of phosphatidylethanolamine. One route involves the synthesis of phosphatidylserine (PtdSer) in the endoplasmic reticulum (ER), the transport of this lipid to endosomes, and decarboxylation by PtdSer decarboxylase 2 (Psd2p) to produce phosphatidylethanolamine. Several proteins and protein motifs are known to be required for PtdSer transport to occur, namely the Sec14p homolog PstB2p/Pdr17p; a PtdIns 4-kinase, Stt4p; and a C2 domain of Psd2p. The focus of this work is on defining the protein-protein and protein-lipid interactions of these components. PstB2p interacts with a protein encoded by the uncharacterized gene YPL272C, which we name Pbi1p (PstB2p-interacting 1). PstB2p, Psd2, and Pbi1p were shown to be lipid-binding proteins specific for phosphatidic acid. Pbi1p also interacts with the ER-localized Scs2p, a binding determinant for several peripheral ER proteins. A complex between Psd2p and PstB2p was also detected, and this interaction was facilitated by a cryptic C2 domain at the extreme N terminus of Psd2p (C2-1) as well the previously characterized C2 domain of Psd2p (C2-2). The predicted N-terminal helical region of PstB2p was necessary and sufficient for promoting the interaction with both Psd2p and Pbi1p. Taken together, these results support a model for PtdSer transport involving the docking of a PtdSer donor membrane with an acceptor via specific protein-protein and protein-lipid interactions. Specifically, our model predicts that this process involves an acceptor membrane complex containing the C2 domains of Psd2p, PstB2p, and Pbi1p that ligate to Scs2p and phosphatidic acid present in the donor membrane, forming a zone of apposition that facilitates PtdSer transfer.

Balony: a software package for analysis of data generated by synthetic genetic array experiments.

BACKGROUND: Synthetic Genetic Array (SGA) analysis is a procedure which has been developed to allow the systematic examination of large numbers of double mutants in the yeast Saccharomyces cerevisiae. The aim of these experiments is to identify genetic interactions between pairs of genes. These experiments generate a number of images of ordered arrays of yeast colonies which must be analyzed in order to quantify the extent of the genetic interactions. We have designed software that is able to analyze virtually any image of regularly arrayed colonies and allows the user significant flexibility over the analysis procedure. RESULTS: "Balony" is freely available software which enables the extraction of quantitative data from array-based genetic screens. The program follows a multi-step process, beginning with the optional preparation of plate images from single or composite images. Next, the colonies are identified on a plate and the pixel area of each is measured. This is followed by a scoring module which normalizes data and pairs control and experimental data files. The final step is analysis of the scored data, where the strength and reproducibility of genetic interactions can be visualized and cross-referenced with information on each gene to provide biological insights into the results of the screen. CONCLUSIONS: Analysis of SGA screens with Balony can be either automated or highly interactive, enabling the user to customize the process to their specific needs. Quantitative data can be extracted at each stage for external analysis if required. Beyond SGA, this software can be used for analyzing many types of plate-based high-throughput screens.

Plasma membrane--endoplasmic reticulum contact sites regulate phosphatidylcholine synthesis.

Synthesis of phospholipids, sterols and sphingolipids is thought to occur at contact sites between the endoplasmic reticulum (ER) and other organelles because many lipid-synthesizing enzymes are enriched in these contacts. In only a few cases have the enzymes been localized to contacts in vivo and in no instances have the contacts been demonstrated to be required for enzyme function. Here, we show that plasma membrane (PM)--ER contact sites in yeast are required for phosphatidylcholine synthesis and regulate the activity of the phosphatidylethanolamine N-methyltransferase enzyme, Opi3. Opi3 activity requires Osh3, which localizes to PM-ER contacts where it might facilitate in trans catalysis by Opi3. Thus, membrane contact sites provide a structural mechanism to regulate lipid synthesis.

Lipids as conductors in the orchestra of life.

The lipid phosphatidic acid is an important metabolic intermediate in the biosynthesis of lipids in all eukaryotic cells, but it is even more than that. Phosphatidic acid is emerging as a lipid that is both composer and conductor, where in addition to its role as biosynthetic precursor (composer) it is also a potent signaling molecule (conductor) that integrates membrane biogenesis with nutrient sensing and cell growth. This article discusses recent advances in yeast that give praise for phosphatidic acid as one of life's conductors.

Putting the pH into phosphatidic acid signaling.

The lipid phosphatidic acid (PA) has important roles in cell signaling and metabolic regulation in all organisms. New evidence indicates that PA also has an unprecedented role as a pH biosensor, coupling changes in pH to intracellular signaling pathways. pH sensing is a property of the phosphomonoester headgroup of PA. A number of other potent signaling lipids also contain headgroups with phosphomonoesters, implying that pH sensing by lipids may be widespread in biology.