SWI/SNF chromatin remodelling complex contributes to clearance of cytoplasmic protein aggregates and regulates unfolded protein response in Saccharomyces cerevisiae.

Chromatin remodelling complexes are multi-subunit assemblies, each containing a catalytic ATPase and translocase that is capable of mobilizing nucleosomes to alter the chromatin structure. SWI/SNF remodelling complexes with higher DNA translocation efficiency evict histones or slide the nucleosomes away from each other making DNA accessible for transcription and repair machinery. Chromatin remodelling at the promoter of stress-responsive genes by SWI/SNF becomes necessary during the heat and proteotoxic stress. While the involvement of SWI/SNF in transcription of stress-responsive genes has been studied extensively, the regulation of proteostasis by SWI/SNF is not well understood. This study demonstrates critical functions of SWI/SNF in response to cadmium-induced proteotoxic stress. Deletion of either ATPase-translocase subunit of SWI/SNF complex (Swi2/Snf2) or a regulatory subunit Swi3 abrogates the clearance of cadmium-induced protein aggregates. Our results suggest that Snf2 and Swi3 regulate the protein folding in endoplasmic reticulum (ER) that reduces the chances of forming unfolded protein aggregates under the proteotoxic stress of cadmium. The Ire1-mediated unfolded protein response (UPR) maintains ER homeostasis by upregulating the expression of chaperones and ER-associated degradation (ERAD) components. We found that Snf2 maintains normal oxidative environment essential for Ire1 activity. Deletion of SNF2 reduced the Ire1 activity and UPR, indicating involvement of Snf2 in Ire1-mediated ER proteostasis. Together, these findings suggest that SWI/SNF complex regulates ER homeostasis and protein folding crucial for tolerating proteotoxic stress.

Reverse genetic analysis of yeast YPR099C/MRPL51 reveals a critical role of both overlapping ORFs in respiratory growth and MRPL51 in mitochondrial DNA maintenance.

The Saccharomyces cerevisiae genome contains 6572 ORFs, of which 680 ORFs are classified as dubious ORFs. A dubious ORF is a small, noncoding, nonconserved ORF that overlaps with another ORF of the complementary strand. Our study characterizes a dubious/nondubious ORF pair, YPR099C/MRPL51, and shows the transcript and protein level expression of YPR099C. Its subcellular localization was observed in the mitochondria. The overlapping ORF, MRPL51, encodes a mitochondrial ribosomal protein of large subunit. Deletion of any ORF from YPR099C/MRPL51 pair induces common phenotypes, i.e. loss of mtDNA, lack of mitochondrial fusion and lack of respiratory growth, due to the double deletion (ypr099cΔ/Δmrpl51Δ/Δ) caused by sequence overlap. Hence, we created the single deletions of each ORF of the YPR099C/MRPL51 pair by an alternative approach to distinguish their phenotypes and identify the specific functions. Both the ORFs were found essential for the functional mitochondria and respiratory growth, but MRPL51 showed its specific requirement in mtDNA stability. The mechanism of mtDNA maintenance by Mrpl51 is probably Mhr1 dependent that physically interacts with Mrpl51 and also regulates mtDNA repair. Overall, our study provides strong evidence for the protein level expression of a dubious ORF YPR099C and the bifunctional role of Mrpl51 in mtDNA maintenance.

The Crg1 N-Terminus Is Essential for Methyltransferase Activity and Cantharidin Resistance in Saccharomyces cerevisiae.

Crg1 is an S-adenosylmethionine (SAM)-dependent methyltransferase required for cantharidin resistance in yeast. Crg1 has a well-characterized methyltransferase domain that inactivates cantharidin by methylation. However, the remaining part of the Crg1 protein is yet to be functionally characterized. In this study, we identified an essential role of the N-terminus of Crg1 in methyltransferase activity and cantharidin resistance. Yeast cells lacking 41 residues of the N-terminus of Crg1 ( crg1ΔN) showed hypersensitivity to cantharidin as same as the null mutant, crg1. The mass spectrometry-based biochemical enzyme assay revealed a loss of methyltransferase activity in Crg1ΔN, which justifies the loss of cantharidin resistance, as well. The subcellular distribution of Crg1ΔN-daGFP showed cytoplasmic aggregates, whereas wild-type Crg1-daGFP was distributed normally in the cytoplasm. Interestingly, the Crg1-methyltransferase domain point mutants (D44A, D67A, and E105A/D108A) also showed the same cytoplasmic aggregates as Crg1ΔN-daGFP. In silico prediction of the tertiary structures of these mutants indicated an altered protein conformation. Altogether, these observations suggest that the N-terminal truncation, as well as the point mutations in the methyltransferase domain, alters the native folding of Crg1 methyltransferase, resulting in a loss of enzyme activity. Furthermore, the crg1ΔN mutant showed the same phenotypes as the crg1 null mutant in the presence of cantharidin, i.e., lethal cell growth, PE auxotrophy, temperature sensitivity, endoplasmic reticulum stress, GPI anchor missorting, and cell wall damage. Overall, this study identifies an essential role of the N-terminus of Crg1 in methyltransferase activity and cantharidin resistance.

The natural anticancer agent cantharidin alters GPI-anchored protein sorting by targeting Cdc1-mediated remodeling in endoplasmic reticulum.

Cantharidin (CTD) is a potent anticancer small molecule produced by several species of blister beetle. It has been a traditional medicine for the management of warts and tumors for many decades. CTD suppresses tumor growth by inducing apoptosis, cell cycle arrest, and DNA damage and inhibits protein phosphatase 2 phosphatase activator (PP2A) and protein phosphatase 1 (PP1). CTD also alters lipid homeostasis, cell wall integrity, endocytosis, adhesion, and invasion in yeast cells. In this study, we identified additional molecular targets of CTD using a Saccharomyces cerevisiae strain that expresses a cantharidin resistance gene (CRG1), encoding a SAM-dependent methyltransferase that methylates and inactivates CTD. We found that CTD specifically affects phosphatidylethanolamine (PE)-associated functions that can be rescued by supplementing the growth media with ethanolamine (ETA). CTD also perturbed endoplasmic reticulum (ER) homeostasis and cell wall integrity by altering the sorting of glycosylphosphatidylinositol (GPI)-anchored proteins. A CTD-dependent genetic interaction profile of CRG1 revealed that the activity of the lipid phosphatase cell division control protein 1 (Cdc1) in GPI-anchor remodeling is the key target of CTD, independently of PP2A and PP1 activities. Moreover, experiments with human cells further suggested that CTD functions through a conserved mechanism in higher eukaryotes. Altogether, we conclude that CTD induces cytotoxicity by targeting Cdc1 activity in GPI-anchor remodeling in the ER.

Zwitterionic BODIPYs with large stokes shift: small molecular biomarkers for live cells.

The two first examples of zwitterionic BODIPYs have been synthesized via a simple SN-Ar methodology. The molecules exhibit excellent optical behavior, such as a large Stokes shift in solution and therefore a very intense emission, and can thus avoid self-quenching. The zwitterionic nature of the molecules was unambiguously elucidated using single crystal XRD studies. The electronic conjugation was investigated by NMR, DFT (NICS (0)) and XRD analysis. Due to their inherent ionic nature, their enhanced solubility in aqueous conditions was exploited for their utility in bio-imaging and cell viability studies. These molecules demonstrate promising localization inside live yeast cells.

Sen1, the homolog of human Senataxin, is critical for cell survival through regulation of redox homeostasis, mitochondrial function, and the TOR pathway in Saccharomyces cerevisiae.

Mutations in the Senataxin gene, SETX are known to cause the neurodegenerative disorders, ataxia with oculomotor apraxia type 2 (AOA2), and amyotrophic lateral sclerosis 4 (ALS4). However, the mechanism underlying disease pathogenesis is still unclear. The Senataxin N-terminal protein-interaction and C-terminal RNA/DNA helicase domains are conserved in the Saccharomyces cerevisiae homolog, Sen1p. Using genome-wide expression analysis, we first show alterations in key cellular pathways such as: redox, unfolded protein response, and TOR in the yeast sen1 ΔN mutant (N-terminal truncation). This mutant exhibited growth defects on nonfermentable carbon sources, was sensitive to oxidative stress, and showed severe loss of mitochondrial DNA. The growth defect could be partially rescued upon supplementation with reducing agents and antioxidants. Furthermore, the mutant showed higher levels of reactive oxygen species, lower UPR activity, and alterations in mitochondrial membrane potential, increase in vacuole acidity, free calcium ions in the cytosol, and resistance to rapamycin treatment. Notably, the sen1 ∆N mutant showed increased cell death and shortened chronological life span. Given the strong similarity of the yeast and human Sen1 proteins, our study thus provides a mechanism for the progressive neurological disorders associated with mutations in human senataxin.