Abstract
Previous studies have demonstrated roles for de novo production of sphingolipids in Saccharomyces cerevisiae in the regulation of the transient cell cycle arrest and nutrient permease degradation associated with the heat stress response, suggesting multiple functions for yeast sphingolipids in this response. We, therefore, sought to determine the generalized involvement of sphingolipids in the heat stress response by using microarray hybridization of RNA isolated from heat-stressed cultures of the mutant strain lcb1-100, which is unable to produce sphingolipids in response to heat. Approximately 70 genes showed differential regulation during the first 15 min of heat stress in the lcb1-100 strain compared with the wild type strain, indicating a requirement for de novo sphingolipid biosynthesis for proper regulation of these genes during heat stress. Grouping these genes into functional categories revealed several pathways, including some in which sphingolipids were previously suspected to play a role, such as stress response pathways and cell cycle regulation. Hierarchical clustering analysis revealed sphingolipid involvement in regulation of tRNA synthesis and metabolic genes and transporters. Additionally, the microarray results demonstrated novel sphingolipid involvement in transcriptional regulation of pathways of translation and cell wall organization and biogenesis. Our results demonstrate a broad-reaching effect of sphingolipids in the yeast heat stress response and suggest that the mechanism of sphingolipid involvement in several cellular pathways occurs via sphingolipid-mediated regulation of message levels.
Highlights
With the advent of genomic technology, yeast stress responses were among the first experimental targets [4, 5]
Mutants deficient in sphingolipid biosynthesis are deficient in several aspects of the heat stress response, including cell cycle arrest [14] and ubiquitinmediated protein degradation [15]
Specific roles for sphingolipids in regulation of specific responses of the heat stress response (HSR) of S. cerevisiae have been recently studied [8, 14, 15, 18], and data obtained in these studies have demonstrated that sphingolipids are critical for the overall response
Summary
Laboratory strain RH406 (MATa leu trp ura bar1) and the lcb 100 mutant (MATa leu trp ura bar lcb temperature-sensitive) were a generous gift from Dr Howard Riezman. Cultures were seeded at low density and grown at 30 °C to mid-log phase (A600 ϭ 0.5– 0.7). Cultures were transferred to 39 °C for the appropriate times, cells were centrifuged at 500 ϫ g at 4 °C, and pellets were rinsed one time in ice-cold sterile water. Total RNA was isolated using the Qiagen RNeasy kit (Valencia, CA) and the enzymatic lysis protocol according to instructions from the manufacturer
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