Two Pathways of Sphingolipid Biosynthesis Are Separated in the Yeast Pichia pastoris

Philipp Ternes,Tobias Wobbe,Marnie Schwarz,Sandra Albrecht,Kirstin Feussner,Isabelle Riezman,James M Cregg,Ernst Heinz,Howard Riezman,Ivo Feussner,Dirk Warnecke

doi:10.1074/jbc.m110.193094

Abstract

Although the yeast Saccharomyces cerevisiae has only one sphingolipid class with a head group based on phosphoinositol, the yeast Pichia pastoris as well as many other fungi have a second class, glucosylceramide, which has a glucose head group. These two sphingolipid classes are in addition distinguished by a characteristic structure of their ceramide backbones. Here, we investigate the mechanisms controlling substrate entry into the glucosylceramide branch of the pathway. By a combination of enzymatic in vitro studies and lipid analysis of genetically engineered yeast strains, we show that the ceramide synthase Bar1p occupies a key branching point in sphingolipid biosynthesis in P. pastoris. By preferring dihydroxy sphingoid bases and C(16)/C(18) acyl-coenzyme A as substrates, Bar1p produces a structurally well defined group of ceramide species, which is the exclusive precursor for glucosylceramide biosynthesis. Correlating with the absence of glucosylceramide in this yeast, a gene encoding Bar1p is missing in S. cerevisiae. We could not successfully investigate the second ceramide synthase in P. pastoris that is orthologous to S. cerevisiae Lag1p/Lac1p. By analyzing the ceramide and glucosylceramide species in a collection of P. pastoris knock-out strains in which individual genes encoding enzymes involved in glucosylceramide biosynthesis were systematically deleted, we show that the ceramide species produced by Bar1p have to be modified by two additional enzymes, sphingolipid Δ4-desaturase and fatty acid α-hydroxylase, before the final addition of the glucose head group by the glucosylceramide synthase. Together, this set of four enzymes specifically defines the pathway leading to glucosylceramide biosynthesis.

Highlights

By a combination of enzymatic in vitro studies and lipid analysis of genetically engineered yeast strains, we show that the ceramide synthase Bar1p occupies a key branching point in sphingolipid biosynthesis in P. pastoris
By analyzing the ceramide and glucosylceramide species in a collection of P. pastoris knock-out strains in which individual genes encoding enzymes involved in glucosylceramide biosynthesis were systematically deleted, we show that the ceramide species produced by Bar1p have to be modified by two additional enzymes, sphingolipid ⌬4-desaturase and fatty acid ␣-hydroxylase, before the final addition of the glucose head group by the glucosylceramide syn
Generating a Collection of P. pastoris Mutant Strains Impaired in Biosynthesis of GlcCer—To investigate the pathway leading to GlcCer biosynthesis, we determined the molecular species of Cer and GlcCer in P. pastoris wild type (WT) and in mutant strains, each impaired in the activity of one enzyme involved in GlcCer biosynthesis

Summary

Introduction

Important insights into sphingolipid metabolism and functions came from studies on the yeast Saccharomyces cerevisiae [8] This yeast is an exception among eukaryotic organisms because it contains only one class of complex sphingolipids, namely (G)IPCs, whereas most other fungi contain both (G)IPCs and glycosylceramides [4, 9]. GlcCer and (G)IPCs in fungi differ by the nature of their polar head groups, glucose or (glycosyl)phosphoinositol, and by the structure of their Cer backbones [4, 9]. These include characteristic differences found in both the long-chain (sphingoid) base (LCB) and the amidelinked acyl group. Elongation of long-chain to very long-chain fatty acids is performed at the level of acyl-coenzyme A (CoA) by the fatty acid elongase complex

Results

Discussion

Conclusion