Sphingolipid long-chain-base auxotrophs of Saccharomyces cerevisiae: genetics, physiology, and a method for their selection

Abstract

A selection method for sphingolipid long-chain-base auxotrophs of Saccharomyces cerevisiae was devised after observing that strains that require a long-chain base for growth become denser when starved for this substance. Genetic analysis of over 60 such strains indicated only two complementation classes, kcbl and kb2. Mutant strains from each class grew equally well with 3-ketodihydrosphingosine, erythrodihydrosphingosine or threodihydrosphingosine, or phytosphingosine. Since these metabolites represent the first, second, and last components, respectively, of the long-chain-base biosynthetic pathway, it is likely that the LCBJ and LCB2 genes are involved in the first step of long-chain-base synthesis. The results of long-chain-base starvation in the Lcb-strains suggest that one or more sphingolipids have a vital role in S. cerevisiae. Immediate sequelae of long-chain-base starvation were loss of viability, exacerbated in the presence of ao-cyclodextrin, and loss of phosphoinositol sphingolipid synthesis but not phosphatidylinositol synthesis. Loss of viability with long-chainbase starvation could be prevented by also blocking either protein or nucleic acid synthesis. Without a long-chain-base, cell division, dry mass accumulation, and protein synthesis continued at a diminished rate and were further inhibited by the detergent Tergitol. The cell density increase induced by long-chain-base starvation is thus explained as a differential loss of cell division and mass accumulation. Long-chain-base starvation in Lcb-S. cerevisiae and inositol starvation of Inos-S. cerevisiae share common features: an increase in cell density and a loss of cell viability overcome by blocking macromolecular synthesis.

Very Long-chain Fatty Acid-containing Lipids rather than Sphingolipids per se Are Required for Raft Association and Stable Surface Transport of Newly Synthesized Plasma Membrane ATPase in Yeast

“…Strains were cultivated at 24, 30, or 37°C in YPD-rich medium (1% Bacto yeast extract, 2% Bacto peptone (USBiological, Swampscott, MA), 2% glucose) or in minimal medium. Cells lacking sphingolipids were cultivated in complete synthetic medium or complete complex medium as described (17). Phytosphingosine (PHS) was supplemented in 0.5% Tergitol at a concentration of 25 M. The presence of the kanMX marker was selected for by growing the cells on medium containing 200 g/ml G418 (Invitrogen).…”

Biochemistry, cell biology and molecular biology of lipids ofSaccharomyces cerevisiae

“…87,180,288 The growth defect produced by mutation of either LCB1 or LCB2 can be circumvented by adding any one of several long-chain bases to the culture medium. 203,289 In the absence of a long-chain base, the cells stop making sphingolipids and die within a few hours, even though protein, DNA and other lipid synthesis continues. 203,289 It is not known how cells take up the relatively hydrophobic long-chain bases from the culture medium.…”

“…203,289 In the absence of a long-chain base, the cells stop making sphingolipids and die within a few hours, even though protein, DNA and other lipid synthesis continues. 203,289 It is not known how cells take up the relatively hydrophobic long-chain bases from the culture medium. Other sphingolipid metabolites, such as ceramides or long-chain base phosphates and the sphingolipids containing inositol, are not taken up by cells and this property has prevented many potentially interesting experiments from being performed.…”

Sphingolipid synthesis is necessary for kinetoplast segregation and cytokinesis in Trypanosoma brucei

“…Mutations in the SPT2 subunit in Saccharomyces cerevisiae and in Chinese hamster ovary cells that render the cells deficient in synthesis of sphingoid bases lead to severe growth retardation and eventual cell death unless the cells are supplemented with exogenous sphingoid bases (Dickson and Lester, 1999;Hanada, 2003;Pinto et al, 1992). Deletion of SPT2 in Drosophila leads to embryonic lethality (Dickson and Lester, 1999;Obeid et al, 2002).…”