A Systems Biology Approach Reveals the Role of a Novel Methyltransferase in Response to Chemical Stress and Lipid Homeostasis

Using small molecule probes to understand gene function is an attractive approach that allows functional characterization of genes that are dispensable in standard laboratory conditions and provides insight into the mode of action of these compounds. Using chemogenomic assays we previously identified yeast Crg1, an uncharacterized SAM-dependent methyltransferase, as a novel interactor of the protein phosphatase inhibitor cantharidin. In this study we used a combinatorial approach that exploits contemporary high-throughput techniques available in Saccharomyces cerevisiae combined with rigorous biological follow-up to characterize the interaction of Crg1 with cantharidin. Biochemical analysis of this enzyme followed by a systematic analysis of the interactome and lipidome of CRG1 mutants revealed that Crg1, a stress-responsive SAM-dependent methyltransferase, methylates cantharidin in vitro. Chemogenomic assays uncovered that lipid-related processes are essential for cantharidin resistance in cells sensitized by deletion of the CRG1 gene. Lipidome-wide analysis of mutants further showed that cantharidin induces alterations in glycerophospholipid and sphingolipid abundance in a Crg1-dependent manner. We propose that Crg1 is a small molecule methyltransferase important for maintaining lipid homeostasis in response to drug perturbation. This approach demonstrates the value of combining chemical genomics with other systems-based methods for characterizing proteins and elucidating previously unknown mechanisms of action of small molecule inhibitors

Genetic interactions and functional analyses of the fission yeast gsk3 and amk2 single and double mutants defective in TORC1-dependent processes

The Target of Rapamycin (TOR) signalling network plays important roles in aging and disease. The AMP-activated protein kinase (AMPK) and the Gsk3 kinase inhibit TOR during stress. We performed genetic interaction screens using synthetic genetic arrays (SGA) with gsk3 and amk2 as query mutants, the latter encoding the regulatory subunit of AMPK. We identified 69 negative and 82 positive common genetic interactors, with functions related to cellular growth and stress. The 120 gsk3-specific negative interactors included genes functioning in translation and ribosomes. The 215 amk2-specific negative interactors included genes functioning in chromatin silencing and DNA damage repair. Both amk2- and gsk3-specific interactors were enriched in phenotype categories related to abnormal cell size and shape. We also performed SGA screen with the amk2 gsk3 double mutant as a query. Mutants sensitive to 5-fluorouracil, an anticancer drug are under-represented within the 305 positive interactors specific for the amk2 gsk3 query. The triple-mutant SGA screen showed higher number of negative interactions than the double mutant SGA screens and uncovered additional genetic network information. These results reveal common and specialized roles of AMPK and Gsk3 in mediating TOR-dependent processes, indicating that AMPK and Gsk3 act in parallel to inhibit TOR function in fission yeast

Seipin and the membrane-shaping protein Pex30 cooperate in organelle budding from the endoplasmic reticulum

Lipid droplets (LDs) and peroxisomes are ubiquitous organelles with central roles in eukaryotic cells. Although the mechanisms involved in biogenesis of these organelles remain elusive, both seem to require the endoplasmic reticulum (ER). Here we show that in yeast the ER budding of these structurally unrelated organelles has remarkably similar requirements and involves cooperation between Pex30 and the seipin complex. In the absence of these components, budding of both LDs and peroxisomes is inhibited, leading to the ER accumulation of their respective constituent molecules, such as triacylglycerols and peroxisomal membrane proteins, whereas COPII vesicle formation remains unaffected. This phenotype can be reversed by remodeling ER phospholipid composition highlighting a key function of these lipids in organelle biogenesis. We propose that seipin and Pex30 act in concert to organize membrane domains permissive for organelle budding, and that may have a lipid composition distinct from the bulk ER

Systematic exploration of essential yeast gene function with temperature-sensitive mutants

Conditional temperature-sensitive (ts) mutations are valuable reagents for studying essential genes in the yeast Saccharomyces cerevisiae. We constructed 787 ts strains, covering 497 (~45%) of the 1,101 essential yeast genes, with ~30% of the genes represented by multiple alleles. All of the alleles are integrated into their native genomic locus in the S288C common reference strain and are linked to a kanMX selectable marker, allowing further genetic manipulation by synthetic genetic array (SGA)–based, high-throughput methods. We show two such manipulations: barcoding of 440 strains, which enables chemical-genetic suppression analysis, and the construction of arrays of strains carrying different fluorescent markers of subcellular structure, which enables quantitative analysis of phenotypes using high-content screening. Quantitative analysis of a GFP-tubulin marker identified roles for cohesin and condensin genes in spindle disassembly. This mutant collection should facilitate a wide range of systematic studies aimed at understanding the functions of essential genes