A Specific Structural Requirement for Ergosterol in Long-chain Fatty Acid Synthesis Mutants Important for Maintaining Raft Domains in Yeast

Abstract

Fungal sphingolipids contain ceramide with a very-long-chain fatty acid (C26). To investigate the physiological significance of the C26-substitution on this lipid, we performed a screen for mutants that are synthetically lethal with ELO3. Elo3p is a component of the ER-associated fatty acid elongase and is required for the final elongation cycle to produce C26 from C22/C24 fatty acids. elo3⌬ mutant cells thus contain C22/C24-instead of the natural C26-substituted ceramide. We now report that under these conditions, an otherwise nonessential, but also fungal-specific, structural modification of the major sterol of yeast, ergosterol, becomes essential, because mutations in ELO3 are synthetically lethal with mutations in ERG6. Erg6p catalyzes the methylation of carbon atom 24 in the aliphatic side chain of sterol. The lethality of an elo3⌬ erg6⌬ double mutant is rescued by supplementation with ergosterol but not with cholesterol, indicating a vital structural requirement for the ergosterol-specific methyl group. To characterize this structural requirement in more detail, we generated a strain that is temperature sensitive for the function of Erg6p in an elo3⌬ mutant background. Examination of raft association of the GPI-anchored Gas1p and plasma membrane ATPase, Pma1p, in the conditional elo3⌬ erg6 ts double mutant, revealed a specific defect of the mutant to maintain raft association of preexisting Pma1p. Interestingly, in an elo3⌬ mutant at 37°C, newly synthesized Pma1p failed to enter raft domains early in the biosynthetic pathway, and upon arrival at the plasma membrane was rerouted to the vacuole for degradation. These observations indicate that the C26 fatty acid substitution on lipids is important for establishing raft association of Pma1p and stabilizing the protein at the cell surface. Analysis of raft lipids in the conditional mutant strain revealed a selective enrichment of ergosterol in detergent-resistant membrane domains, indicating that specific structural determinants on both sterols and sphingolipids are required for their association into raft domains.

Lipid-dependent surface transport of the proton pumping ATPase: A model to study plasma membrane biogenesis in yeast

“…We became interested in the role of lipids in surface transport of Pma1 because we observed that Pma1 was rapidly degraded in cells that fail to elongate the ceramide-bound C22 fatty acid to the mature C26 very long-chain fatty acid, as is the case in cells lacking ELO3, a component of the ER localized acyl chain elongase [28,29]. Interestingly, this rapid turnover of Pma1 in the elo3D mutant correlates with a lack of the newly synthesized protein to acquire detergent resistance [29] (Fig.…”

Match-making for posaconazole through systems thinking

“…Evidence in the budding yeast, S. cerevisiae, suggests a genetic interaction between mutants in sterol and sphingolipid biosynthesis [4,5,29,30]. For example, mutants that affect the hydroxylation pattern of sphingolipids display synthetic growth defects with mutations in late-acting ergosterol biosynthetic genes [4].…”

“…For example, mutants that affect the hydroxylation pattern of sphingolipids display synthetic growth defects with mutations in late-acting ergosterol biosynthetic genes [4]. By contrast, mutations that affect the synthesis of the sphingolipidspecific very-long chain C 26 fatty acid display strong synthetic lethality with mutations in ERG6, a methyltransferase that catalyzes the addition of a fungal-specific methyl group at position C 24 in the aliphatic side chain or ergosterol [5]. Figure 1 summarizes experimental evidence on genetic interactions between the sterol and sphingolipid synthetic genes.…”

“…Figure 1 summarizes experimental evidence on genetic interactions between the sterol and sphingolipid synthetic genes. While the bulk of experimental evidence comes from high throughput screens and needs to be treated with caution, there is solid support for synthetic lethality between ELO3 and ERG6 [5], and for synthetic growth defects of sterol synthetic genes with ISC1, SUR2, and SCS7 [4]. Strikingly, synthetic lethality has been demonstrated between CYP51 and SCS7 [31].…”

“…Systems biology aims at revealing interconnections of biological networks and these works serve as useful resources for rational identification of potential interacting partners for chemotherapy. Based on genetic, physical, and functional interactions between sterols and sphingolipids [4] and due to the synthetic lethality of Saccharomyces cerevisiae double mutants of sterol and sphingolipid anabolism [5], our opinion is that inhibitors of sphingolipid biosynthesis are promising combination partners for posaconazole or ravuconazole.…”