Synthesis and elongation of fatty acids

Transcription

1 Synthesis and elongation of fatty acids A molecular caliper mechanism for determining very long-chain fatty acid length Vladimir Denic and Jonathan S. Weissman (2007) Cell 130, February 28, 2008 Toon de Kroon Membrane Enzymology

2 Reasons for choosing this topic/paper: Acyl chains: membrane building blocks Synthesis of fatty acids Specific functions of very long chain fatty acids Yeast as powerful model eukaryote (in lipid research) Functional reconstitution of membrane protein enzymes

3 Outline of introduction Fatty acids Function Review of FA synthesis general Very long chain fatty acids Definition Function Synthesis: elongation Yeast tricks yeast

4 Fatty acids Essential in all organisms except archaea Constituents of membranes Posttranslational protein modification (myristoylation, palmitoylation) Storage of chemical energy (TAG, sterolesters)

5 Eukaryotic membrane lipids

6 Acyl chain composition determines physicochemical membrane parameters: { acyl chain length degree of unsaturation membrane thickness membrane fluidity membrane curvature (PE, DAG) which affect membrane protein function molecular shape PE confers negative curvature stress PC PI PE

7 Synthesis of fatty acids The formation of malonyl-coa is the committed step in fatty acid synthesis acetyl-coa carboxylase (biotin)

8 Intermediates in fatty acid synthesis are attached to ACP

9

10 Fatty acid synthesis: sequential addition of two-carbon units up to C16, by fatty acid synthase (FAS), localized in the cytosol

11 acyl-malonyl condensing enzyme (β-ketoacyl synthase) KS/CE β-ketoacyl reductase KR

12 β-hydroxyacyl dehydratase DH enoyl reductase ER

13 Stoichiometry of the synthesis of C16:0 8 acetyl CoA + 7 ATP + 14 NADPH + 6 H + palmitate (C16:0) + 14 NADP CoA + 6 H 2 O + 7 ADP + 7 P i

14 Animal fatty acid synthase: homodimer of single chains (540 kda) AT, acetyltransferase MT, malonyltransferase KS/CE, condensing enzyme KR, β-ketoacyl reductase DH, dehydratase ER, enoylreductase TE, thioesterase

15 FAS in fungi: α 6 β MDa particle Jenni et al., 2007

16 Domain organization and structure of the subunits of fungal FAS Jenni et al., 2007

17 Reaction chambers of yeast FAS pathway traversed by ACP ACP in modeled positions at each active site side view top view Lomakin et al., 2007

18 Desaturation of fatty acids: by electron transport chain in the ER

19 Phospholipid and acyl chain composition of wild type yeast phospholipid class mol% phosphatidylcholine (PC) 41 phosphatidylethanolamine (PE) 26 phosphatidylinositol (PI) 18 phosphatidylserine (PS) 9 cardiolipin (CL) 5 fatty acid mol% C14 2 C16:0 15 C16:1 48 C18:0 4 C18:1 30 C26:0 1

20 Very long chain fatty acids (VLCFA): > C18 Functions in mammals: - >C30: skin permeability barrier - signaling: e.g. C20:4 (arachidonic acid) - constituent of myelin in plants: - oils and waxes in S. cerevisiae: - stabilization highly curved membranes - required for sphingolipid synthesis - required for GPI-anchors

21 Morphological phenotype of yeast acc1 ts mutant at 37 o C (is not rescued by C16 supplementation) Separation nuclear inner and outer membranes (asterisk) Formation of vesicle-like structures (star) Arrow points to nuclear pore 0.6µm Schneiter & Kohlwein, 1997

22 Models of highly curved membrane domains and their possible stabilization by VLCFA-containing lipids C26-PI Schneiter et al., 2004

23 Sphingolipid metabolism in Saccharomyces cerevisiae long chain bases (LCB)

24 Structure of GPI anchors of yeast: C26:0

25 Synthesis and elongation by different enzyme complexes: similar reactions little sequence homology

26 Fatty acid elongation machinery in S. cerevisiae Sur4p (Elo3p) Fen1p (Elo2p) (Elo1p Ybr159wp: elo2 elo3: inviable elongation up to C26 elongation up to C22 elongation up to C18 elo1 mutants can t use C12-14 as substituents for endogenous fatty acid synthesis) Dehydratase:?? Tsc13p: β-ketoacylreductase: not essential enoyl reductase: essential

27 Issues addressed by Denic and Weissman: Function of the Elops Fen2p and Sur4p in elongation Gene-enzyme relationships in FA elongation Assignment of the β-hydroxyacyldehydratase How is the length of the fatty acid determined? to be presented by: Agon Hyseni Tessa Quax Bastiaan Bijl Myrthe Braam

28 Yeast tricks 1. DAmP (decreased abundance by mrna perturbation) trick to include essential genes in large-scale screens for genetic interactions (e.g. synthetic lethal/sick screens): the 3 -UTR (untranslated region) of the gene is disrupted by insertion of an antibiotic resistance marker, destabilizing the corresponding mrna, yielding proteins under their natural transcriptional regulation but at substantially reduced levels

29 2. The plasmid shuffle Generation of a library of mutant alleles of an essential gene leu2 ura3 trp1 yfg::leu2 leu2 ura3 trp1 yfg::leu2 YFG1 yfg1-x yfg1-x URA3 TRP1 TRP1 Selection for loss of URA3 plasmid on 5-FOA (ura + ) Only cells that have lost the wild type gene on the URA3 plasmid can grow on the 5-FOA agar plate