Date of Award:

5-1-2004

Document Type:

Dissertation

Degree Name:

Doctor of Philosophy (PhD)

Department:

Biology

Committee Chair(s)

Jon Y. Takemoto

Committee

Jon Y. Takemoto

Committee

Michelle M. Grilley

Committee

Daryll B. DeWald

Committee

Gregory J. Podgorski

Committee

Marie K. Walsh

Abstract

Sphingolipids are ubiquitous membrane components of all eukaryotic cells with important regulatory functions. The roles and biochemistry of two structural features of sphingolipids were studied: C4 hydroxylation and the addition of a terminal phosphoinositol. Structure-function studies of the yeast sphingolipid C4 long chain base hydroxylase (Sur2p) revealed that alanine replacement of any of eight histidine residues from three histidine-rich motifs, as well as additional histidine 249, eliminated hydroxylase activity. Seven additional residues were also found to be important elements of the active site. These results show that Sur2p is a member of the family of eight-histidine motif hydroxylases and desaturates. Syringomycin E is an antifungal agent that is speculated to interact with lipid rafts, and Sur2p promotes its action. Therefore, the effect of C4 sphingoid base hydroxylation on lipid raft properties was studied. Biochemical preparations of lipid rafts from a yeast strain lacking C4 hydroxylated sphingoid bases (sur2Δ) had a lower buoyant density than lipid raft preparations from the isogenic wild type. Also, lipid raft preparations from mutant were composed of linear membrane fragments rather than vesicular structures observed for preparations from wild type strain. While the lipid compositions were similar, several proteins found in preparations from wild type were significantly lower in amounts in the preparations from sur2Δ strain. These results show that C4 sphingoid base hydroxylation influences protein composition of lipid rafts and suggest that lipid rafts play a role in the antifungal action of syringomycin E. The dependence of syringomycin E and plant defensin action on the terminal step in sphingolipid biosynthesis was also investigated. Under certain nutritional conditions, an ipt1Δ deletion strain, normally resistant to these peptides, still produced a small amount of mannosyldiphosphoinositolceramide. Under these conditions it was susceptible to both syringomycin E and the defensin. These results suggest that the terminal addition of phosphoinositol is required for the fungicidal action of these peptides. Overall, the research demonstrates that sphingolipid structure influences the properties of yeast membranes and the susceptibility of yeast to fungicidal peptides.

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