Genome-wide screen for inositol auxotrophy in Saccharomyces cerevisiae implicates lipid metabolism in stress response signaling

Abstract

Inositol auxotrophy (Ino − phenotype) in budding yeast has classically been associated with misregulation of INO1 and other genes involved in lipid metabolism. To identify all non-essential yeast genes that are necessary for growth in the absence of inositol, we carried out a genome-wide phenotypic screening for deletion mutants exhibiting Ino − phenotypes under one or more growth conditions. We report the identification of 419 genes, including 385 genes not previously reported, which exhibit this phenotype when deleted. The identified genes are involved in a wide range of cellular processes, but are particularly enriched in those affecting transcription, protein modification, membrane trafficking, diverse stress responses, and lipid metabolism. Among the Ino − mutants involved in stress response, many exhibited phenotypes that are strengthened at elevated temperature and/or when choline is present in the medium. The role of inositol in regulation of lipid metabolism and stress response signaling is discussed.

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

“…Solid media had the same composition plus 2% (wt/vol) agar (74). Yeast strains carrying expression plasmids were maintained in uracil drop-out medium.…”

Desumoylation of the Endoplasmic Reticulum Membrane VAP Family Protein Scs2 by Ulp1 and SUMO Regulation of the Inositol Synthesis Pathway

“…Both sumoylation and desumoylation activities are required for normal growth under conditions of inositol starvation. It is noteworthy that earlier genome-wide screens for genes required for inositol prototrophy (45) or for transcriptional changes in response to inositol starvation (17) failed to find any link to the SUMO system. The former screen used the yeast gene deletion collection.…”

Activation of Protein Kinase C-Mitogen-activated Protein Kinase Signaling in Response to Inositol Starvation Triggers Sir2p-dependent Telomeric Silencing in Yeast

“…Many of genes that are induced in the absence of inositol are targets of stress response pathways that have been shown to be activated in the absence of exogenous inositol, including the unfolded protein response and the protein kinase C (PKC) pathway (1)(2)(3)(11)(12)(13). Moreover, mutations in these same stress response pathways confer inositol auxotrophy (Ino Ϫ phenotype) indicating that signaling through these pathways is essential for survival of cells experiencing stress associated with inositol deprivation (3,(11)(12)(13)(14). Thus, growth in the absence of inositol is an inherently stress-activating condition, analogous to growth at elevated temperature, high or low osmolarity, and/or exposure to agents such as tunicamycin, caffeine, or calcofluor white (3,8,13).…”

“…Total RNA was isolated using RNeasy mini kit, including DNA digestion with RNase-free DNase set (Qiagen). cDNA was synthesized from 1 g of total RNA using oligo(dT) [12][13][14][15][16][17][18] primer (0.5 g), PCR grade dNTP mix (0.5 M), First strand buffer (1ϫ), DTT (10 mM), and 100 units of SuperScript II reverse transcriptase (Invitrogen). Real time PCR was performed on a StepOnePlus TM Real Time PCR system (Applied Biosystems) using TaqMan universal PCR master mix, No AmpErase UNG (Applied Biosystems), and the following TaqMan probes and primers: BNA2, TaqMan probe, 5Ј-Fam-ATG GGC TGG ATG TCT-Tamra-3Ј; forward primer (5Ј-AAG GTG CGG AGC GTC ATC-3Ј) and reverse primer (5Ј-GCC CAA GAT CGT CTC ATC CA-3Ј); THI4, TaqMan probe, 5Ј-Fam-TTC TGC GCC AAG AGA ATC GTC GAC ATTTamra-3Ј; forward primer (5Ј-CGG TCA TGA TGG TCC ATT TG-3Ј) and reverse primer (5Ј-CGC CCA ATT TTT GGT TTT GA-3Ј).…”