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• W2148053098 abstract "Very long-chain fatty acids (VLCFAs) are essential lipids whose functional diversity is enabled by variation in their chain length. The full VLCFA biosynthetic machinery and how this machinery generates structural diversity remain elusive. Proteoliposomes reconstituted here from purified membrane components—an elongase protein (Elop), a novel dehydratase, and two reductases—catalyzed repeated rounds of two-carbon addition that elongated shorter FAs into VLCFAs whose length was dictated by the specific Elop homolog present. Mutational analysis revealed that the Elop active site faces the cytosol, whereas VLCFA length is determined by a lysine near the luminal end of an Elop transmembrane helix. By stepping the lysine residue along one face of the helix toward the cytosol, we engineered novel synthases with correspondingly shorter VLCFA outputs. Thus the distance between the active site and the lysine residue determines chain length. Our results uncover a mutationally adjustable, caliper-like mechanism that generates the repertoire of cellular VLCFAs. Very long-chain fatty acids (VLCFAs) are essential lipids whose functional diversity is enabled by variation in their chain length. The full VLCFA biosynthetic machinery and how this machinery generates structural diversity remain elusive. Proteoliposomes reconstituted here from purified membrane components—an elongase protein (Elop), a novel dehydratase, and two reductases—catalyzed repeated rounds of two-carbon addition that elongated shorter FAs into VLCFAs whose length was dictated by the specific Elop homolog present. Mutational analysis revealed that the Elop active site faces the cytosol, whereas VLCFA length is determined by a lysine near the luminal end of an Elop transmembrane helix. By stepping the lysine residue along one face of the helix toward the cytosol, we engineered novel synthases with correspondingly shorter VLCFA outputs. Thus the distance between the active site and the lysine residue determines chain length. Our results uncover a mutationally adjustable, caliper-like mechanism that generates the repertoire of cellular VLCFAs. Very long-chain fatty acids (VLCFAs) are structurally diverse biological molecules with unusually long hydrocarbon chains, ranging from 20 to 36 carbons (C20–36) or more (Leonard et al., 2004Leonard A.E. Pereira S.L. Sprecher H. Huang Y.S. Elongation of long-chain fatty acids.Prog. Lipid Res. 2004; 43: 36-54Crossref PubMed Scopus (388) Google Scholar). These lipids perform essential roles in a wide range of biological processes that cannot be supported by the more common shorter fatty acids (i.e., C16 and C18). For example, the chain length of certain VLCFAs allows them to simultaneously reside in both leaflets of the lipid bilayer, thereby stabilizing highly curved cellular membranes, such as those surrounding nuclear pore complexes (Schneiter et al., 2004Schneiter R. Brugger B. Amann C.M. Prestwich G.D. Epand R.F. Zellnig G. Wieland F.T. Epand R.M. Identification and biophysical characterization of a very-long-chain-fatty-acid-substituted phosphatidylinositol in yeast subcellular membranes.Biochem. J. 2004; 381: 941-949Crossref PubMed Scopus (68) Google Scholar, Schneiter et al., 1996Schneiter R. Hitomi M. Ivessa A.S. Fasch E.V. Kohlwein S.D. Tartakoff A.M. A yeast acetyl coenzyme A carboxylase mutant links very-long-chain fatty acid synthesis to the structure and function of the nuclear membrane-pore complex.Mol. Cell. Biol. 1996; 16: 7161-7172Crossref PubMed Scopus (144) Google Scholar). Notably, the variation in VLCFA chain length across different species and different tissues has enabled the numerous functional specializations of these lipids. In S. cerevisiae, for example, while C22 is able to support the essential functions of VLCFAs, C26 is specifically required for a variety of membrane-based processes, including the formation of GPI lipid anchors and the trafficking of proteins in the secretory pathway (Dickson et al., 2006Dickson R.C. Sumanasekera C. Lester R.L. Functions and metabolism of sphingolipids in Saccharomyces cerevisiae.Prog. Lipid Res. 2006; 45: 447-465Crossref PubMed Scopus (201) Google Scholar, Toulmay and Schneiter, 2007Toulmay A. Schneiter R. Lipid-dependent surface transport of the proton pumping ATPase: a model to study plasma membrane biogenesis in yeast.Biochimie. 2007; 89: 249-254Crossref PubMed Scopus (30) Google Scholar). Similarly, in mammals, VLCFAs with lengths greater than C30 allow the formation of a permeability barrier that is critical for the normal structure and function of the skin (McMahon et al., 2007McMahon A. Butovich I.A. Mata N.L. Klein M. Ritter 3rd, R. Richardson J. Birch D.G. Edwards A.O. Kedzierski W. Retinal pathology and skin barrier defect in mice carrying a Stargardt disease-3 mutation in elongase of very long chain fatty acids-4.Mol. Vis. 2007; 13: 258-272PubMed Google Scholar, Vasireddy et al., 2007Vasireddy V. Uchida Y. Salem Jr., N. Kim S.Y. Mandal M.N. Reddy G.B. Bodepudi R. Alderson N.L. Brown J.C. Hama H. et al.Loss of functional ELOVL4 depletes very long-chain fatty acids (> or =C28) and the unique omega-O-acylceramides in skin leading to neonatal death.Hum. Mol. Genet. 2007; 16: 471-482Crossref PubMed Scopus (183) Google Scholar). Finally, VLCFAs and their derivatives act as signaling molecules (e.g., arachidonic acid; Leonard et al., 2002Leonard A.E. Kelder B. Bobik E.G. Chuang L.T. Lewis C.J. Kopchick J.J. Mukerji P. Huang Y.S. Identification and expression of mammalian long-chain PUFA elongation enzymes.Lipids. 2002; 37: 733-740Crossref PubMed Scopus (142) Google Scholar) and are dominant lipid constituents of certain tissues in animals (e.g., photoreceptor cells and myelin; Poulos et al., 1992Poulos A. Beckman K. Johnson D.W. Paton B.C. Robinson B.S. Sharp P. Usher S. Singh H. Very long-chain fatty acids in peroxisomal disease.Adv. Exp. Med. Biol. 1992; 318: 331-340Crossref PubMed Scopus (22) Google Scholar) and plants (e.g., oils and waxes; Kunst and Samuels, 2003Kunst L. Samuels A.L. Biosynthesis and secretion of plant cuticular wax.Prog. Lipid Res. 2003; 42: 51-80Crossref PubMed Scopus (642) Google Scholar). Efforts to understand the mechanistic principles enabling their structural diversity have been hampered by the inability to reconstitute VLCFA synthesis from purified components (Cinti et al., 1992Cinti D.L. Cook L. Nagi M.N. Suneja S.K. The fatty acid chain elongation system of mammalian endoplasmic reticulum.Prog. Lipid Res. 1992; 31: 1-51Crossref PubMed Scopus (182) Google Scholar, Jakobsson et al., 2006Jakobsson A. Westerberg R. Jacobsson A. Fatty acid elongases in mammals: their regulation and roles in metabolism.Prog. Lipid Res. 2006; 45: 237-249Crossref PubMed Scopus (570) Google Scholar, Leonard et al., 2004Leonard A.E. Pereira S.L. Sprecher H. Huang Y.S. Elongation of long-chain fatty acids.Prog. Lipid Res. 2004; 43: 36-54Crossref PubMed Scopus (388) Google Scholar). This has been largely due to the insoluble nature of the VLCFA biosynthetic machinery which has been known since the 1960s to consist of detergent-labile complexes embedded within the endoplasmic reticulum (ER) membrane (for a historical overview see Cinti et al., 1992Cinti D.L. Cook L. Nagi M.N. Suneja S.K. The fatty acid chain elongation system of mammalian endoplasmic reticulum.Prog. Lipid Res. 1992; 31: 1-51Crossref PubMed Scopus (182) Google Scholar). These pioneering studies demonstrated that VLCFAs are synthesized by the elongation of shorter fatty acids (C16 and C18) that are produced in the cytosol by the well-characterized, multienzyme complex termed the fatty acid synthase (FAS; Jenni et al., 2007Jenni S. Leibundgut M. Boehringer D. Frick C. Mikolasek B. Ban N. Structure of fungal fatty acid synthase and implications for iterative substrate shuttling.Science. 2007; 316: 254-261Crossref PubMed Scopus (163) Google Scholar, Lomakin et al., 2007Lomakin I.B. Xiong Y. Steitz T.A. The Crystal Structure of Yeast Fatty Acid Synthase, a Cellular Machine with Eight Active Sites Working Together.Cell. 2007; 129: 319-332Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar). VLCFA synthesis was shown to proceed by a four-step biochemical cycle (Figure 1B; Nugteren, 1965Nugteren D.H. The enzymic chain elongation of fatty acids by rat-liver microsomes.Biochim. Biophys. Acta. 1965; 106: 280-290Crossref PubMed Scopus (198) Google Scholar): (1) condensation of malonyl-CoenzymeA (CoA) with an acyl-CoA precursor; (2) reduction of the resulting 3-keto intermediate; (3) dehydration of the 3-hydroxy species; and (4) reduction of the enoyl product to yield a saturated FA chain that is two carbons longer than its precursor. The above conceptual framework facilitated more recent genetic approaches in budding yeast that identified several ER membrane proteins required for VLCFA production. Specifically, Ybr159wp and Tsc13p are strong candidates for the enzymes catalyzing the second and fourth steps of the elongation cycle, respectively, as they are homologous to known reductases and their inactivation leads to the accumulation of the expected keto and enoyl intermediates in the ER membrane (Beaudoin et al., 2002Beaudoin F. Gable K. Sayanova O. Dunn T. Napier J.A. A Saccharomyces cerevisiae gene required for heterologous fatty acid elongase activity encodes a microsomal beta-keto-reductase.J. Biol. Chem. 2002; 277: 11481-11488Crossref PubMed Scopus (72) Google Scholar, Han et al., 2002Han G. Gable K. Kohlwein S.D. Beaudoin F. Napier J.A. Dunn T.M. The Saccharomyces cerevisiae YBR159w gene encodes the 3-ketoreductase of the microsomal fatty acid elongase.J. Biol. Chem. 2002; 277: 35440-35449Crossref PubMed Scopus (82) Google Scholar, Kohlwein et al., 2001Kohlwein S.D. Eder S. Oh C.S. Martin C.E. Gable K. Bacikova D. Dunn T. Tsc13p is required for fatty acid elongation and localizes to a novel structure at the nuclear-vacuolar interface in Saccharomyces cerevisiae.Mol. Cell. Biol. 2001; 21: 109-125Crossref PubMed Scopus (160) Google Scholar). Additionally, Fen1p and Sur4p are two yeast members (Oh et al., 1997Oh C.S. Toke D.A. Mandala S. Martin C.E. ELO2 and ELO3, homologues of the Saccharomyces cerevisiae ELO1 gene, function in fatty acid elongation and are required for sphingolipid formation.J. Biol. Chem. 1997; 272: 17376-17384Crossref PubMed Scopus (379) Google Scholar) of a large family of proteins termed the Elops (Jakobsson et al., 2006Jakobsson A. Westerberg R. Jacobsson A. Fatty acid elongases in mammals: their regulation and roles in metabolism.Prog. Lipid Res. 2006; 45: 237-249Crossref PubMed Scopus (570) Google Scholar) which are required for the first (condensation) step in the elongation cycle (Moon et al., 2001Moon Y.A. Shah N.A. Mohapatra S. Warrington J.A. Horton J.D. Identification of a mammalian long chain fatty acyl elongase regulated by sterol regulatory element-binding proteins.J. Biol. Chem. 2001; 276: 45358-45366Crossref PubMed Scopus (260) Google Scholar, Paul et al., 2006Paul S. Gable K. Beaudoin F. Cahoon E. Jaworski J. Napier J.A. Dunn T.M. Members of the Arabidopsis FAE1-like 3-Ketoacyl-CoA synthase gene family substitute for the Elop proteins of Saccharomyces cerevisiae.J. Biol. Chem. 2006; 281: 9018-9029Crossref PubMed Scopus (93) Google Scholar, Westerberg et al., 2006Westerberg R. Mansson J.E. Golozoubova V. Shabalina I.G. Backlund E.C. Tvrdik P. Retterstol K. Capecchi M.R. Jacobsson A. ELOVL3 is an important component for early onset of lipid recruitment in brown adipose tissue.J. Biol. Chem. 2006; 281: 4958-4968Crossref PubMed Scopus (95) Google Scholar). Moreover, heterologous expression of Elop homologs in yeast demonstrated that these proteins determine the length of cellular VLCFA products. Hence, the proliferation and specialization of Elops has been responsible for the observed VLCFA chain length diversity across different organisms and cell types (Jakobsson et al., 2006Jakobsson A. Westerberg R. Jacobsson A. Fatty acid elongases in mammals: their regulation and roles in metabolism.Prog. Lipid Res. 2006; 45: 237-249Crossref PubMed Scopus (570) Google Scholar, Leonard et al., 2004Leonard A.E. Pereira S.L. Sprecher H. Huang Y.S. Elongation of long-chain fatty acids.Prog. Lipid Res. 2004; 43: 36-54Crossref PubMed Scopus (388) Google Scholar). The exact function of Elops in the elongation cycle is uncertain, however, as these proteins lack homology to known condensing enzymes. Therefore, it remains to be established whether Elops are members of a novel family of condensing enzymes or noncatalytic adapters for recruiting specific substrates to the actual condensing enzyme. Finally, no protein has been identified that is specifically required for the dehydratase reaction. Based on these findings, we can now restate the question of VLCFA chain length determination in more specific mechanistic terms. Namely, how do Elops instruct the components of the elongation cycle to extend shorter FAs in two-carbon addition steps so as to yield VLCFA products of defined lengths? The observation that ER membranes containing a single Elop can convert substrates of different lengths to the same-length end product (Paul et al., 2006Paul S. Gable K. Beaudoin F. Cahoon E. Jaworski J. Napier J.A. Dunn T.M. Members of the Arabidopsis FAE1-like 3-Ketoacyl-CoA synthase gene family substitute for the Elop proteins of Saccharomyces cerevisiae.J. Biol. Chem. 2006; 281: 9018-9029Crossref PubMed Scopus (93) Google Scholar) argues against a length-determining mechanism that “counts” a fixed number of two-carbon additions. Conversely, ER membranes with different Elops allow for the elongation of the same substrate to different-length VLCFA products (Paul et al., 2006Paul S. Gable K. Beaudoin F. Cahoon E. Jaworski J. Napier J.A. Dunn T.M. Members of the Arabidopsis FAE1-like 3-Ketoacyl-CoA synthase gene family substitute for the Elop proteins of Saccharomyces cerevisiae.J. Biol. Chem. 2006; 281: 9018-9029Crossref PubMed Scopus (93) Google Scholar). Thus, Elops are able to specify VLCFA length in absolute terms. Efforts to understand the mechanism by which Elops achieve this and how evolutionary diversification of the Elop family has enabled the synthesis of VLCFAs of novel chain lengths have been hampered by the incomplete inventory of the components required for VLCFA synthesis (Paul et al., 2006Paul S. Gable K. Beaudoin F. Cahoon E. Jaworski J. Napier J.A. Dunn T.M. Members of the Arabidopsis FAE1-like 3-Ketoacyl-CoA synthase gene family substitute for the Elop proteins of Saccharomyces cerevisiae.J. Biol. Chem. 2006; 281: 9018-9029Crossref PubMed Scopus (93) Google Scholar). Moreover, the integral membrane nature of the enzymes involved has thus far obstructed the in vitro reconstitution of any step of the elongation cycle (Cinti et al., 1992Cinti D.L. Cook L. Nagi M.N. Suneja S.K. The fatty acid chain elongation system of mammalian endoplasmic reticulum.Prog. Lipid Res. 1992; 31: 1-51Crossref PubMed Scopus (182) Google Scholar, Jakobsson et al., 2006Jakobsson A. Westerberg R. Jacobsson A. Fatty acid elongases in mammals: their regulation and roles in metabolism.Prog. Lipid Res. 2006; 45: 237-249Crossref PubMed Scopus (570) Google Scholar, Leonard et al., 2004Leonard A.E. Pereira S.L. Sprecher H. Huang Y.S. Elongation of long-chain fatty acids.Prog. Lipid Res. 2004; 43: 36-54Crossref PubMed Scopus (388) Google Scholar). Here we identify Phs1p as the VLCFA dehydratase and show that it is the final missing component of the elongation cycle by reconstituting robust VLCFA synthesis using purified Elops, Phs1p, Ybr159wp, and Tsc13p inserted into proteoliposomes. Moreover, we demonstrate that Elops are a novel family of condensing enzymes that specify VLCFA length by terminating processive rounds of the elongation cycle following the synthesis of defined length products. Finally, we exploit this reconstituted system to elucidate the logic by which Elops measure VLCFA length and use this understanding to rationally design VLCFA synthase variants with dramatically altered product length outputs. In our previous large-scale genetic interactions study of the yeast early secretory pathway, we identified Phs1p as a highly conserved ER protein with six predicted transmembrane segments that plays an essential role in sphingolipid metabolism (Schuldiner et al., 2005Schuldiner M. Collins S.R. Thompson N.J. Denic V. Bhamidipati A. Punna T. Ihmels J. Andrews B. Boone C. Greenblatt J.F. et al.Exploration of the function and organization of the yeast early secretory pathway through an epistatic miniarray profile.Cell. 2005; 123: 507-519Abstract Full Text Full Text PDF PubMed Scopus (663) Google Scholar). Several observations suggested to us that Phs1p might be a core component of the elongation cycle, the biosynthetic source of VLCFA substrates critical for the production of sphingolipids (Figure 1A). First, as expected following a block in VLCFA synthesis (Figure 1A; Beaudoin et al., 2002Beaudoin F. Gable K. Sayanova O. Dunn T. Napier J.A. A Saccharomyces cerevisiae gene required for heterologous fatty acid elongase activity encodes a microsomal beta-keto-reductase.J. Biol. Chem. 2002; 277: 11481-11488Crossref PubMed Scopus (72) Google Scholar, Han et al., 2002Han G. Gable K. Kohlwein S.D. Beaudoin F. Napier J.A. Dunn T.M. The Saccharomyces cerevisiae YBR159w gene encodes the 3-ketoreductase of the microsomal fatty acid elongase.J. Biol. Chem. 2002; 277: 35440-35449Crossref PubMed Scopus (82) Google Scholar, Kohlwein et al., 2001Kohlwein S.D. Eder S. Oh C.S. Martin C.E. Gable K. Bacikova D. Dunn T. Tsc13p is required for fatty acid elongation and localizes to a novel structure at the nuclear-vacuolar interface in Saccharomyces cerevisiae.Mol. Cell. Biol. 2001; 21: 109-125Crossref PubMed Scopus (160) Google Scholar, Oh et al., 1997Oh C.S. Toke D.A. Mandala S. Martin C.E. ELO2 and ELO3, homologues of the Saccharomyces cerevisiae ELO1 gene, function in fatty acid elongation and are required for sphingolipid formation.J. Biol. Chem. 1997; 272: 17376-17384Crossref PubMed Scopus (379) Google Scholar), reduction in Phs1p levels resulted in the accumulation of cellular long-chain bases (LCBs; Figure 2A). Second, cells with reduced Phs1p levels fail to grow in the presence of exogenously added myristic acid (C14) when FAS is chemically inhibited (Figure 2B), a hallmark of VLCFA mutants (Rossler et al., 2003Rossler H. Rieck C. Delong T. Hoja U. Schweizer E. Functional differentiation and selective inactivation of multiple Saccharomyces cerevisiae genes involved in very-long-chain fatty acid synthesis.Mol. Genet. Genomics. 2003; 269: 290-298Crossref PubMed Scopus (42) Google Scholar). Lastly, Phs1p coimmunoprecipitates with Fen1p, Sur4p, Ybr159wp, and Tsc13p (Figure 2C).Figure 2Phs1p Catalyzes the Third Step of the Elongation CycleShow full caption(A) Wild-type cells or cells with Phs1p under the transcriptional control of the tetracycline-repressible promoter (downward arrow) were treated with doxycycline (a tetracycline derivative). Cellular LCBs were fluorescently labeled and analyzed by reverse-phase HPLC. ∗ is the free fluorescent dye remaining after the labeling procedure. Chromatogram peaks correspond to various structurally diverse LCB species (data not shown).(B) Serial dilutions (vertical dimension) of the indicated strains (horizontal dimension) were spotted onto plates containing glucose, −/+ cerulenin (cer), and myristic acid (C14). phs1 refers to a strain with GAL1 promoter-driven expression of PHS1 from its genomic locus. Promoter shutoff in the presence of glucose is incomplete and results in a partial loss-of-function phs1 allele (data not shown).(C) Cells expressing Myc-tagged proteins from their genomic loci and either Phs1p or Phs1-FLAG on a plasmid (covering Δphs1), as indicated, were lysed and solubilized with digitonin. Following ultracentrifugation and immunoprecipitation (IP) with antiFLAG beads, bound material and input fractions were resolved by SDS-PAGE followed by western analysis (W) with the indicated antibodies. Note that only full-length Tsc13p (∗) was efficiently pulled down by Phs1-FLAG.(D) Wild-type or genetically depleted Phs1p membranes (see Supplemental Experimental Procedures) were incubated with radiolabeled malonyl-CoA and unlabeled C18-CoA in the presence of reducing cofactors. Acyl-CoA reaction products were converted into FA methyl esters, resolved by reverse-phase TLC, and detected by phosphorimager analysis. ∗ is the fast-migrating species that accumulated specifically in Phs1p-depleted membranes. Both membrane preparations lead to comparable incorporation of the label into species migrating faster than C20 (one of which comigrates with ∗) that disappeared upon preincubation of membranes with cerulenin (data not shown) and thus most likely represent LCFAs produced by copurifying FAS in our membrane preparations.(E) Membranes, prepared as in (C), were incubated with radiolabeled malonyl-CoA and unlabeled C20-CoA (similar results were seen using C18-CoA as the substrate, data not shown) in the absence or presence of reducing cofactors, NAD(P)H, as indicated. Acyl-CoA reaction products were converted into free FAs, resolved by normal-phase TLC, and detected by phosphorimager analysis.(F) Purified Phs1-FLAG (yeast source), Phs1-HIS (bacterial source), or the corresponding mock elutions were incubated with a 3-hydroxy C18-CoA dehydratase substrate. CoA-containing molecules were fluorescently labeled and analyzed by reverse-phase HPLC. ∗ is a nonspecific derivatization breakdown product of 3-hydroxy C18-CoA (data not shown).View Large Image Figure ViewerDownload Hi-res image Download (PPT) (A) Wild-type cells or cells with Phs1p under the transcriptional control of the tetracycline-repressible promoter (downward arrow) were treated with doxycycline (a tetracycline derivative). Cellular LCBs were fluorescently labeled and analyzed by reverse-phase HPLC. ∗ is the free fluorescent dye remaining after the labeling procedure. Chromatogram peaks correspond to various structurally diverse LCB species (data not shown). (B) Serial dilutions (vertical dimension) of the indicated strains (horizontal dimension) were spotted onto plates containing glucose, −/+ cerulenin (cer), and myristic acid (C14). phs1 refers to a strain with GAL1 promoter-driven expression of PHS1 from its genomic locus. Promoter shutoff in the presence of glucose is incomplete and results in a partial loss-of-function phs1 allele (data not shown). (C) Cells expressing Myc-tagged proteins from their genomic loci and either Phs1p or Phs1-FLAG on a plasmid (covering Δphs1), as indicated, were lysed and solubilized with digitonin. Following ultracentrifugation and immunoprecipitation (IP) with antiFLAG beads, bound material and input fractions were resolved by SDS-PAGE followed by western analysis (W) with the indicated antibodies. Note that only full-length Tsc13p (∗) was efficiently pulled down by Phs1-FLAG. (D) Wild-type or genetically depleted Phs1p membranes (see Supplemental Experimental Procedures) were incubated with radiolabeled malonyl-CoA and unlabeled C18-CoA in the presence of reducing cofactors. Acyl-CoA reaction products were converted into FA methyl esters, resolved by reverse-phase TLC, and detected by phosphorimager analysis. ∗ is the fast-migrating species that accumulated specifically in Phs1p-depleted membranes. Both membrane preparations lead to comparable incorporation of the label into species migrating faster than C20 (one of which comigrates with ∗) that disappeared upon preincubation of membranes with cerulenin (data not shown) and thus most likely represent LCFAs produced by copurifying FAS in our membrane preparations. (E) Membranes, prepared as in (C), were incubated with radiolabeled malonyl-CoA and unlabeled C20-CoA (similar results were seen using C18-CoA as the substrate, data not shown) in the absence or presence of reducing cofactors, NAD(P)H, as indicated. Acyl-CoA reaction products were converted into free FAs, resolved by normal-phase TLC, and detected by phosphorimager analysis. (F) Purified Phs1-FLAG (yeast source), Phs1-HIS (bacterial source), or the corresponding mock elutions were incubated with a 3-hydroxy C18-CoA dehydratase substrate. CoA-containing molecules were fluorescently labeled and analyzed by reverse-phase HPLC. ∗ is a nonspecific derivatization breakdown product of 3-hydroxy C18-CoA (data not shown). To further define the role of Phs1p in fatty acid elongation, we incubated membranes that had been genetically depleted of Phs1p with radiolabeled malonyl-CoA and unlabeled C18-CoA in the presence of reducing cofactors (essential for the second and fourth steps of the elongation cycle). The products of this reaction were then separated on the basis of chain length by reverse-phase thin-layer chromatography. As previously seen (Paul et al., 2006Paul S. Gable K. Beaudoin F. Cahoon E. Jaworski J. Napier J.A. Dunn T.M. Members of the Arabidopsis FAE1-like 3-Ketoacyl-CoA synthase gene family substitute for the Elop proteins of Saccharomyces cerevisiae.J. Biol. Chem. 2006; 281: 9018-9029Crossref PubMed Scopus (93) Google Scholar), wild-type membranes elongated C18-CoA up to C26 (Figure 2D), the most abundant VLCFA in S. cerevisiae (Welch and Burlingame, 1973Welch J.W. Burlingame A.L. Very long-chain fatty acids in yeast.J. Bacteriol. 1973; 115: 464-466Crossref PubMed Google Scholar). In contrast, Phs1p-depleted membranes were strongly compromised for elongation and instead accumulated a fast-migrating (i.e., more hydrophilic) species (Figure 2D). Normal-phase TLC, a method better suited for resolving elongation cycle intermediates, identified this species as the 3-hydroxy intermediate (Figure 2E), which is the substrate for the third step in the cycle (Figure 1B). Notably, the first two steps (i.e., formation of the 3-keto intermediate in the absence of NAD(P)H and its reduction in the presence of NAD(P)H, respectively) were not compromised by Phs1p depletion (Figure 2E). Thus, Phs1p is specifically required for the dehydratase step of the elongation cycle. As Phs1p lacks homology to known dehydratases, we sought to determine if it catalyzes the third step of the elongation cycle by purifying epitope-tagged Phs1p. Strikingly, coincubation of either yeast- (Figure S1A) or bacterially produced Phs1p (Figure S1B) and a 3-hydroxy C18-CoA dehydratase substrate (Figure S2A) resulted in accumulation of the dehydrated enoyl-CoA (Figures 2F, S2B, and S2C). Taken together, these data establish that Phs1p is the founding member of a novel and conserved family of membrane dehydratases that catalyze the third step of the elongation cycle. Previous genetic studies have shown that mutations in close PHS1 homologs cause tumorigenesis in plants (Bellec et al., 2002Bellec Y. Harrar Y. Butaeye C. Darnet S. Bellini C. Faure J.D. Pasticcino2 is a protein tyrosine phosphatase-like involved in cell proliferation and differentiation in Arabidopsis.Plant J. 2002; 32: 713-722Crossref PubMed Scopus (57) Google Scholar, Da Costa et al., 2006Da Costa M. Bach L. Landrieu I. Bellec Y. Catrice O. Brown S. De Veylder L. Lippens G. Inze D. Faure J.D. Arabidopsis PASTICCINO2 is an antiphosphatase involved in regulation of cyclin-dependent kinase A.Plant Cell. 2006; 18: 1426-1437Crossref PubMed Scopus (36) Google Scholar) and centronuclear myopathy in a canine model for this human disease (Pele et al., 2005Pele M. Tiret L. Kessler J.L. Blot S. Panthier J.J. SINE exonic insertion in the PTPLA gene leads to multiple splicing defects and segregates with the autosomal recessive centronuclear myopathy in dogs.Hum. Mol. Genet. 2005; 14: 1417-1427Crossref PubMed Scopus (115) Google Scholar). The elucidation of the molecular role of Phs1p now points to the involvement of VLCFAs in these processes. To explore the role of Elops in the condensation reaction, we overexpressed and purified functional epitope-tagged versions of Fen1p and Sur4p, two yeast Elops (Figure S3A), but failed to detect significant condensation activity in our detergent-solubilized preparations (data not shown). We therefore removed the detergent in the presence of synthetic phospholipids of a defined composition approximating that of the ER membrane (Matsuoka and Schekman, 2000Matsuoka K. Schekman R. The use of liposomes to study COPII- and COPI-coated vesicle formation and membrane protein sorting.Methods. 2000; 20: 417-428Crossref PubMed Scopus (27) Google Scholar). Remarkably, the resulting proteoliposomes, containing either Fen1p or Sur4p (Figure S3B), catalyzed formation of the 3-keto product (Figure 3A). We next explored the mechanism by which Elops catalyze the condensation reaction. These membrane-embedded proteins lack an absolutely conserved cysteine-containing catalytic triad found in the condensing enzymes of FAS and FAS-like systems (Paul et al., 2006Paul S. Gable K. Beaudoin F. Cahoon E. Jaworski J. Napier J.A. Dunn T.M. Members of the Arabidopsis FAE1-like 3-Ketoacyl-CoA synthase gene family substitute for the Elop proteins of Saccharomyces cerevisiae.J. Biol. Chem. 2006; 281: 9018-9029Crossref PubMed Scopus (93) Google Scholar). Moreover, the cysteine-targeted FAS inhibitor cerulenin does not interfere with VLCFA synthesis (Figure 2B). We therefore examined absolutely conserved signature sequence motifs within the Elop family (Jakobsson et al., 2006Jakobsson A. Westerberg R. Jacobsson A. Fatty acid elongases in mammals: their regulation and roles in metabolism.Prog. Lipid Res. 2006; 45: 237-249Crossref PubMed Scopus (570) Google Scholar) for their possible involvement in catalyzing the condensation reaction. Experimental analysis of Elop membrane top" @default.
• W2148053098 created "2016-06-24" @default.
• W2148053098 creator A5010255611 @default.
• W2148053098 creator A5026663934 @default.
• W2148053098 date "2007-08-01" @default.
• W2148053098 modified "2023-10-17" @default.
• W2148053098 title "A Molecular Caliper Mechanism for Determining Very Long-Chain Fatty Acid Length" @default.
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