Abstract
Sphingolipids are abundant components of eucaryotic membranes, where they perform essential functions. To uncover new roles for sphingolipids, we studied Saccharomyces cerevisiae lcb1-100 cells, which have a temperature-sensitive block in the first step in sphingolipid synthesis. We find that the level of all five species of the sphingoid long chain base intermediates is reduced 2-7-fold in cells grown at a permissive temperature, and the level of complex sphingolipids is reduced 50%. In addition, lcb1-100 cells make no detectable phosphorylated sphingoid bases. After transfer to a restrictive temperature (a heat shock), the level of the major sphingoid bases drops rather than transiently rising, as in wild type cells. These changes affect lcb1-100 cells in multiple ways. Basal uracil transport by Fur4p is reduced 25%, and when cells are heat-shocked, uracil transport activity falls rapidly and is not restored as it is in wild type cells. Restoration requires a functional secretory pathway and synthesis of complex sphingolipids, leading us to hypothesize that Fur4p associates with lipid rafts. The finding that Fur4p is insoluble in TritonX-100 at 4 degrees C and behaves like a raft-associated protein on a density gradient supports this hypothesis. Raft association may be essential for regulating breakdown of Fur4p in response to stresses and other factors that govern uracil transport activity. Our results show that long chain bases do not contribute to the inactivation of Fur4p transport activity after heat stress, but they are essential for some later, but unknown, process that leads to degradation of the protein. Further studies using lcb1-100 cells should reveal new roles of sphingolipids in nutrient uptake and other membrane-dependent processes.
Highlights
Sphingolipids are essential components of eucaryotic cells and are implicated in a growing number of cellular functions including signal transduction and association with sterols in membranes to form lipid rafts
These data differ from previous data that indicated that lcb1–100 cells grown at 25 °C contain a normal level of long chain bases (LCBs) and that the level did not change during heat shock [19]
Our results demonstrate that sphingolipid synthesis is essential for delivery of Fur4p to the plasma membrane (Fig. 5) and that the protein is present in lipid rafts (Fig. 7)
Summary
Sphingolipids are essential components of eucaryotic cells and are implicated in a growing number of cellular functions including signal transduction and association with sterols in membranes to form lipid rafts. To uncover new roles for sphingolipids we have studied Saccharomyces cerevisiae cells carrying a mutation that inhibits sphingolipid synthesis at a restrictive temperature These studies demonstrate that delivery of the uracil transporter Fur4p to the plasma membrane requires synthesis of complex sphingolipids, which most likely reflects the need for de novo formation of lipid rafts, since we find that Fur4p is present in rafts. Other studies on Pma1p, a proton transporter and the most abundant protein in the plasma membrane of S. cerevisiae cells [14], argue that raft association occurs in the Golgi apparatus [15], where IPC, MIPC, and M(IP)2C would be expected to be the sphingolipid component of rafts, or alternatively, association occurs in the endoplasmic reticulum [16].
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