Abstract
To identify genes necessary for sphingolipid synthesis in Saccharomyces cerevisiae we developed a procedure to enrich for mutants unable to incorporate exogenous long chain base into sphingolipids. We show here that a mutant strain, AG84-3, isolated by using the enrichment procedure, makes sphingolipids from endogenously synthesized but not from exogenously supplied long chain base. A gene termed LCB3 (YJL134W, GenBank designation X87371x21), which complements the long chain base utilization defect of strain AG84-3, was isolated from a genomic DNA library. The gene is predicted to encode a protein with multiple membrane-spanning domains and a COOH-terminal glycosylphosphatidylinositiol cleavage/attachment site. Deletion of the lcb3 gene in a wild type genetic background reduces the rate of exogenous long chain base incorporation into sphingolipids and makes the host strain more resistant to growth inhibition by long chain bases. Only one protein in current data bases, the S. cerevisiae open-reading frame YKR053C, whose function is unknown, shows homology to the Lcb3 protein. The two proteins are not, however, functional homologs because deletion of the YKR053C open reading frame does not impair long chain base utilization or enhance resistance of cells to growth inhibition by long chain bases. Based upon these data we hypothesize that the Lcb3 protein is a plasma membrane transporter capable of transporting sphingoid long chain bases into cells. It is the first candidate for such a transporter and the first member of what appears to be a new class of membrane-bound proteins.
Highlights
Sphingolipids comprise a diverse group of complex lipids found primarily in the plasma membrane of all known eukaryotes
Isolation of Strain AG84-3—Strain AG84-3 was isolated by using the procedure described under “Experimental Procedures” to enrich for strains defective in sphingolipid synthesis
We conclude from these data that the LCB3 gene complements the defect in sphingolipid synthesis in AG84-3 cells and restores the rate of PHS incorporation into sphingolipids to the wild type level seen in 7R6 cells
Summary
The LCB3 gene was isolated from a S. cerevisiae genomic DNA (strain 4R3, 10) library made by ligating Sau3AI-cut DNA fragments into the BamHI site of pRS315 (CEN6, LEU2, 15). About 15,000 transformants were tested by replica plating for colonies able to grow at 37 °C on defined agar plates lacking leucine and sodium succinate but containing 25 mM PHS. These conditions permitted growth of the parental strain 7R6 but restricted growth of the mutant derivative AG84-3. MPRS305 was constructed by cloning a 3.1-kb PstI-BamHI DNA fragment from pJAB15-1, containing the promoter and 59 end of the LCB3 gene, into pRS305 cut with the same restriction endonucleases. Two days later only about 15 colonies/plate were visible, suggesting that only 3% of the dense cells were viable
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