Abstract
The Saccharomyces cerevisiae gene SYR2, necessary for growth inhibition by the cyclic lipodepsipeptide syringomycin E, is shown to be required for 4-hydroxylation of long chain bases in sphingolipid biosynthesis. Four lines of support for this conclusion are presented: (a) the predicted Syr2p shows sequence similarity to diiron-binding membrane enzymes involved in oxygen-dependent modifications of hydrocarbon substrates, (b) yeast strains carrying a disrupted SYR2 allele produced sphingoid long chain bases lacking the 4-hydroxyl group present in wild type strains, (c) 4-hydroxylase activity was increased in microsomes prepared from a SYR2 overexpression strain, and (d) the syringomycin E resistance phenotype of a syr2 mutant strain was suppressed when grown under conditions in which exogenous 4-hydroxysphingoid long chain bases were incorporated into sphingolipids. The syr2 strain produced wild type levels of sphingolipids, substantial levels of hydroxylated very long chain fatty acids, and the full complement of normal yeast sphingolipid head groups. These results show that the SYR2 gene is required for the 4-hydroxylation reaction of sphingolipid long chain bases, that this hydroxylation is not essential for growth, and that the 4-hydroxyl group of sphingolipids is necessary for syringomycin E action on yeast.
Highlights
Syringomycin E is a member of a family of cyclic lipodepsipeptides produced by strains of the plant bacterium Pseudomonas syringae pv. syringae [1]
In this report we present evidence that S. cerevisiae SYR2 is required for 4-hydroxylation of sphingoid bases and that this activity is necessary for syringomycin E action
We show that strains mutant in SYR2 produce sphingolipids missing the hydroxyl group at the C-4 position of the long chain base moiety, that supplying such cells with C-4 hydroxylated long chain base suppresses the syringomycin E-resistant phenotype of syr2 strains, and that strains that overexpress Syr2p are enriched in 4-hydroxylase activity
Summary
To examine the nature of the long chain bases in the sphingolipid fractions, a portion of the [4,5-3H]DHS-labeled AG4 eluates were dried and hydrolyzed in 1 N HCl in methanol/water (82:18) at 80 °C for 18 h. Incubation was at 25 °C for 90 min, followed by methanol-HCl hydrolysis, 4-biphenylcarbonyl chloride derivatization, and reverse phase HPLC analysis of long chain bases as described above. Syringomycin E Treatment—W⌬LCB1⌬SYR2 cells were grown overnight in modified YPD containing either 50 M DHS or 50 M PHS. Cells were washed once with sterile water and transferred to modified YPD minus Tergitol and long chain base and with the indicated amounts of syringomycin E, prepared as described previously [33]. Syringomycin E was found to be ineffective if added directly to medium containing working concentrations of Tergitol and long chain base. 16 h after syringomycin E addition, aliquots were removed and diluted 10-fold to measure growth by turbidity at A600
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