Members of the Arabidopsis FAE1-like 3-Ketoacyl-CoA Synthase Gene Family Substitute for the Elop Proteins of Saccharomyces cerevisiae

938

1,002

Although the transcriptome, proteome, and interactome of several eukaryotic model organisms have been described in detail, lipidomes remain relatively uncharacterized. Using Saccharomyces cerevisiae as an example, we demonstrate that automated shotgun lipidomics analysis enabled lipidome-wide absolute quantification of individual molecular lipid species by streamlined processing of a single sample of only 2 million yeast cells. By comparative lipidomics, we achieved the absolute quantification of 250 molecular lipid species covering 21 major lipid classes. This analysis provided Ϸ95% coverage of the yeast lipidome achieved with 125-fold improvement in sensitivity compared with previous approaches. Comparative lipidomics demonstrated that growth temperature and defects in lipid biosynthesis induce ripple effects throughout the molecular composition of the yeast lipidome. This work serves as a resource for molecular characterization of eukaryotic lipidomes, and establishes shotgun lipidomics as a powerful platform for complementing biochemical studies and other systems-level approaches.fatty acid elongation ͉ S. cerevisiae ͉ shotgun lipidomics T he lipidome of eukaryotic cells consists of hundreds to thousands of individual lipid species that constitute membranes, store metabolic energy and function as bioactive molecules (1-3). Despite the extensive characterization of proteins, their association into complexes and activities (4-6), it is still difficult to assess how perturbations within the lipid metabolic network affect the full lipidome of cells. This work shows that lipidome-wide quantification of individual molecular lipid species (molecules with defined chemical structure) by absolute quantification (expressed in mol or mol%) provides a new approach to relate lipidomics and functional genomics studies.The yeast Saccharomyces cerevisiae serves as a prime model organism for studying the molecular organization and regulatory circuitry of eukaryotic lipidomes (7-9). It uses a relatively simple and conserved network of lipid metabolic pathways (Fig. 1) that synthesize a few hundred molecular lipid species constituting its full lipidome (3). The lipidome diversity is primarily determined by the fatty acid synthase (10), the ⌬-9 desaturase (11) and the fatty acid elongation complex (12) that produce only saturated or mono-unsaturated fatty acids having 10 to 26 carbon atoms for the biosynthesis of glycerolipids, glycerophospholipids, and sphingolipids. Importantly, several metabolic conversions interlink sphingolipid, glycerophospholipid, and glycerolipid metabolism such that any perturbation within the metabolic network is prone to induce lipidome-wide ripple effects. Remarkably, numerous genes involved in lipid metabolism and trafficking can be mutated or deleted without apparent physiological consequences (Fig. 1).Despite remarkable methodological advances, lipidomics seldom complements functional genomics efforts owing to three major factors. First, analysis of glycerophospholipids and sphingolipids requires ...

Section: Lipidome-wide Consequences Of Defective Fatty Acid Elongationsupporting

confidence: 80%

Section: Lipidome-wide Consequences Of Defective Fatty Acid Elongationmentioning

confidence: 99%

Global analysis of the yeast lipidome by quantitative shotgun mass spectrometry

Ejsing

Sampaio

Surendranath

et al. 2009

938

1,002

“…The present study also provides supporting evidence that 18:1⌬ 11 is primarily synthesized via the prokaryotic pathway, i.e., through KASII, because 18:1⌬ 11 levels did not increase in response to elevated 16:1⌬ 9 in seeds expressing HPFAB1 as they would be expected to do if 18:1⌬ 11 elongation was mediated by one or more of the Ϸ20 cytoplasmic Arabidopsis ␤-ketoacyl-CoA synthase enzymes acting in the eukaryotic pathway (20)(21)(22).…”

Section: Discussionsupporting

confidence: 63%

Modulating seed β-ketoacyl-acyl carrier protein synthase II level converts the composition of a temperate seed oil to that of a palm-like tropical oil

Pidkowich

Nguyen

Heilmann

et al. 2007

128

110

␤-Ketoacyl-acyl carrier protein (ACP) synthase II (KASII) elongates 16:0-ACP to 18:0-ACP in the plastid, where it competes with three other enzymes at the first major branch point in fatty acid biosynthesis. Despite its key metabolic location, the influence of KASII in determining seed oil composition remains unclear, in part because the biochemical consequences of the fab1-1 mutation were unresolved. Thus, fab1-1, and a newly identified knockout allele, fab1-2, were analyzed in the context of the hypothesis that modulating KASII activity is sufficient to convert the composition of a temperate seed oil into that of a palm-like tropical oil. No homozygous fab1-2 individuals were identified in progeny of self-fertilized heterozygous fab1-2 plants, Ϸ1/4 of which aborted before the torpedo stage, suggesting that fab1-2 represents a complete loss of function and results in lethality when homozygous. Consistent with this hypothesis, homozygous fab1-2 plants were identified when a fab1-1 transgene was introduced, demonstrating that fab1-1 encodes an active KASII. Strong seed-specific hairpin-RNAi reductions in FAB1 expression resulted in abortion of Ϸ1/4 of the embryos in an apparent phenocopy of fab1-2 homozygosity. In less severe FAB1 hairpin-RNAi individuals, embryos developed normally and exhibited a 1:2:1 segregation ratio for palmitate accumulation. Thus, early embryo development appears sensitive to elevated 16:0, whereas at later stages, up to 53% of 16:0, i.e., a 7-fold increase over wild-type levels, is tolerated. These results resolve the role of KASII in seed metabolism and demonstrate that modulation of Arabidopsis KASII levels is sufficient to convert its temperate oilseed composition to that of a palm-like tropical oil.condensing enzyme ͉ fatty acid biosynthesis ͉ metabolic engineering ͉ plant oil T emperate crops such as canola, soybean, and sunflower contain predominantly unsaturated 18-carbon fatty acids in their seed oils, whereas tropical oils such as palm oil contain higher proportions (Ϸ50%) of 16-carbon saturated fatty acids (1). Whereas oils with high contents of long-chain or very-longchain polyunsaturated fatty acids are desirable for many purposes including human nutrition, oils with highly saturated 16-carbon-chain-length fatty acids, including palm oil, can provide the starting materials for many industrial applications. It is well documented that different membrane fatty acid compositions, i.e., the ratio of saturated to unsaturated fatty acids, play key roles in adaptation to ambient temperatures (2); however, we reasoned that this need not be the case for seed oils. Thus, we hypothesized that it should be possible to mimic a palm-like oil composition in a temperate crop by genetic manipulation of oilseed biosynthesis.The two-carbon elongation steps in fatty acid biosynthesis (3) are catalyzed by a small family of ␤-ketoacyl-acyl-carrier protein (ACP) synthases, commonly referred to as condensing enzymes of the KAS family (4). Three distinct plastidial KAS activities with characteristic c...

“…erucic acid). The Arabidopsis genome encodes 21 FAE-like KCSs, and although these enzymes are structurally unrelated to the ELO class of condensing enzymes, it has been demonstrated that several Arabidopsis FAE-KCSs can rescue the otherwise lethal yeast elo2⌬/elo3⌬ double mutant (10,11). The Arabidopsis CER10 protein shows significant homology with yeast enoyl-CoA reductase Tsc13p, because it rescues the temperature-sensitive lethality of the tsc13-1 yeast mutant (12) and is involved in VLCFA synthesis (13).…”

mentioning

confidence: 99%

The very-long-chain hydroxy fatty acyl-CoA dehydratase PASTICCINO2 is essential and limiting for plant development

Bach

Michaelson

Haslam

et al. 2008

Self Cite

204

197

Very-long-chain fatty acids (VLCFAs) are synthesized as acyl-CoAs by the endoplasmic reticulum-localized elongase multiprotein complex. Two Arabidopsis genes are putative homologues of the recently identified yeast 3-hydroxy-acyl-CoA dehydratase (PHS1), the third enzyme of the elongase complex. We showed that Arabidopsis PASTICCINO2 (PAS2) was able to restore phs1 cytokinesis defects and sphingolipid long chain base overaccumulation. Conversely, the expression of PHS1 was able to complement the developmental defects and the accumulation of long chain bases of the pas2-1 mutant. The pas2-1 mutant was characterized by a general reduction of VLCFA pools in seed storage triacylglycerols, cuticular waxes, and complex sphingolipids. Most strikingly, the defective elongation cycle resulted in the accumulation of 3-hydroxy-acyl-CoA intermediates, indicating premature termination of fatty acid elongation and confirming the role of PAS2 in this process. We demonstrated by in vivo bimolecular fluorescence complementation that PAS2 was specifically associated in the endoplasmic reticulum with the enoyl-CoA reductase CER10, the fourth enzyme of the elongase complex. Finally, complete loss of PAS2 function is embryo lethal, and the ectopic expression of PHS1 led to enhanced levels of VLCFAs associated with severe developmental defects. Altogether these results demonstrate that the plant 3-hydroxy-acyl-CoA dehydratase PASTICCINO2 is an essential and limiting enzyme in VLCFA synthesis but also that PAS2-derived VLCFA homeostasis is required for specific developmental processes. cuticular wax ͉ elongase ͉ sphingolipid ͉ triacylglycerol ͉ leaf development