Abstract
Ceramide plays a crucial role as a basic building block of sphingolipids, but also as a signalling molecule mediating the fate of the cell. Although Lac1p and Lag1p have been shown recently to be involved in acyl-CoA-dependent ceramide synthesis, ceramide synthase is still poorly characterized. In this study, we expressed tagged versions of Lac1p and Lag1p and purified them to near homogeneity. They copurified with ceramide synthase activity, giving unequivocal evidence that they are subunits of the enzyme. In purified form, the acyl-CoA dependence, fatty acyl-CoA chain length specificity, and Fumonisin B1/Australifungin sensitivity of the ceramide synthase were the same as in cells, showing that these are properties of the enzyme and do not depend upon the membrane environment or other factors. SDS-PAGE analysis of purified ceramide synthase revealed the presence of a novel subunit of the enzyme, Lip1p. Lip1p is a single-span ER membrane protein that is required for ceramide synthesis in vivo and in vitro. The Lip1p regions required for ceramide synthesis are localized within the ER membrane or lumen.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Amino Acid Sequence
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Cytoplasm / metabolism
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Endoplasmic Reticulum / metabolism
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Membrane Proteins / chemistry
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Membrane Proteins / genetics
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Membrane Proteins / isolation & purification
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Membrane Proteins / metabolism*
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Molecular Sequence Data
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Mutation / genetics
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Oxidoreductases / chemistry*
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Oxidoreductases / isolation & purification
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Oxidoreductases / metabolism*
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Protein Binding
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Protein Subunits / chemistry
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Protein Subunits / genetics
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Protein Subunits / isolation & purification
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Protein Subunits / metabolism
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Saccharomyces cerevisiae / cytology
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Saccharomyces cerevisiae / enzymology*
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Saccharomyces cerevisiae / genetics
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Saccharomyces cerevisiae / growth & development
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Saccharomyces cerevisiae Proteins / chemistry
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Saccharomyces cerevisiae Proteins / genetics
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Saccharomyces cerevisiae Proteins / isolation & purification
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Saccharomyces cerevisiae Proteins / metabolism*
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Sequence Alignment
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Sphingolipids / biosynthesis
Substances
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LAG1 protein, S cerevisiae
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Lip1 protein, S cerevisiae
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Membrane Proteins
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Protein Subunits
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Saccharomyces cerevisiae Proteins
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Sphingolipids
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Oxidoreductases
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dihydroceramide desaturase