Cellular Morphogenesis Under Stress Is Influenced by the Sphingolipid Pathway Gene ISC1 and DNA Integrity Checkpoint Genes in Saccharomyces cerevisiae

Abstract

In Saccharomyces cerevisiae, replication stress induced by hydroxyurea (HU) and methyl methanesulfonate (MMS) activates DNA integrity checkpoints; in checkpoint-defective yeast strains, HU treatment also induces morphological aberrations. We find that the sphingolipid pathway gene ISC1, the product of which catalyzes the generation of bioactive ceramides from complex sphingolipids, plays a novel role in determining cellular morphology following HU/MMS treatment. HU-treated isc1D cells display morphological aberrations, cell-wall defects, and defects in actin depolymerization. Swe1, a morphogenesis checkpoint regulator, and the cell cycle regulator Cdk1 play key roles in these morphological defects of isc1D cells. A genetic approach reveals that ISC1 interacts with other checkpoint proteins to control cell morphology. That is, yeast carrying deletions of both ISC1 and a replication checkpoint mediator gene including MRC1, TOF1, or CSM3 display basal morphological defects, which increase following HU treatment. Interestingly, strains with deletions of both ISC1 and the DNA damage checkpoint mediator gene RAD9 display reduced morphological aberrations irrespective of HU treatment, suggesting a role for RAD9 in determining the morphology of isc1D cells. Mechanistically, the checkpoint regulator Rad53 partially influences isc1D cell morphology in a dosage-dependent manner.

Morphogenesis checkpoint kinase Swe1 is the executor of lipolysis-dependent cell-cycle progression

“…In addition to its role in establishing actin polarity and functioning as a cell-cycle checkpoint (22,29,32), Swe1 was also identified in a large-scale study to be connected to sphingolipid metabolism by its genetic interaction with LCB1, encoding the catalytic subunit of SPT (44,45). A related sphingolipid-associated checkpoint function of Swe1 was also reported in the context of inositol sphingolipase C (Isc1), which catalyzes the formation of bioactive ceramides from complex sphingolipids (46,47). Based on these observations, Swe1 appeared as a likely candidate for regulating cell-cycle progression in response to altered lipolysis and the subsequent sphingolipid deficiencies.…”

The yeast sphingolipid signaling landscape

“…The strains csg1 Δ, ipt1 Δ and isc1 Δ were clearly shown to have disrupted actin organization. Both csg1 Δ and ipt1 Δ show defective actin depolymerization following salt stress (Balguerie et al, 2002), and isc1 Δ shows defective actin polarization (Tripathi et al, 2011). Several sphingolipid pathway mutants elo2 Δ, elo3 Δ, sur2 Δ, csg1 Δ, and ipt1 Δ were shown to suppress defective actin repolarization in the rvs161 Δ and act1-1 mutants (Balguerie et al, 2002).…”

Identification of C18:1-Phytoceramide as the Candidate Lipid Mediator for Hydroxyurea Resistance in Yeast

“…Isc1 has also been found to be involved in cellular morphogenesis under genotoxic stress (14). Deletion of the ISC1 gene conferred sensitivity to HU, mediated by the activation of a latent G 2 /M checkpoint.…”

“…Briefly, sphingomyelin labeled with 14 C on the choline moiety was used as a substrate, and the hydrolysis of choline-methyl-14 C-sphingomyelin was determined by liquid scintillation counting.…”