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• W1982040229 abstract "In this study, we investigated the roles of very long-chain fatty acid (VLCFA) synthesis by fatty acid elongase 3 (ELO3) in the regulation of telomere length and life span in the yeast Saccharomyces cerevisiae. Loss of VLCFA synthesis via deletion of ELO3 reduced telomere length, and reconstitution of the expression of wild type ELO3, and not by its mutant with decreased catalytic activity, rescued telomere attrition. Further experiments revealed that alterations of phytoceramide seem to be dispensable for telomere shortening in response to loss of ELO3. Interestingly, telomere shortening in elo3Δ cells was almost completely prevented by deletion of IPK2 or KCS1, which are involved in the generation of inositol phosphates (IP4, IP5, and inositol pyrophosphates). Deletion of IPK1, which generates IP6, however, did not affect regulation of telomere length. Further data also suggested that elo3Δ cells exhibit accelerated chronologic aging, and reduced replicative life span compared with wild type cells, and deletion of KCS1 helped recover these biological defects. Importantly, to determine downstream mechanisms, epistasis experiments were performed, and data indicated that ELO3 and YKU70/80 share a common pathway for the regulation of telomere length. More specifically, chromatin immunoprecipitation assays revealed that the telomere binding and protective function of YKu80p in vivo was reduced in elo3Δ cells, whereas its non-homologues end-joining function was not altered. Deletion of KCS1 in elo3Δ cells recovered the telomere binding and protective function of Ku, consistent with the role of KCS1 mutation in the rescue of telomere length attrition. Thus, these findings provide initial evidence of a possible link between Elo3-dependent VLCFA synthesis, and IP metabolism by KCS1 and IPK2 in the regulation of telomeres, which play important physiological roles in the control of senescence and aging, via a mechanism involving alterations of the telomere-binding/protection function of Ku. In this study, we investigated the roles of very long-chain fatty acid (VLCFA) synthesis by fatty acid elongase 3 (ELO3) in the regulation of telomere length and life span in the yeast Saccharomyces cerevisiae. Loss of VLCFA synthesis via deletion of ELO3 reduced telomere length, and reconstitution of the expression of wild type ELO3, and not by its mutant with decreased catalytic activity, rescued telomere attrition. Further experiments revealed that alterations of phytoceramide seem to be dispensable for telomere shortening in response to loss of ELO3. Interestingly, telomere shortening in elo3Δ cells was almost completely prevented by deletion of IPK2 or KCS1, which are involved in the generation of inositol phosphates (IP4, IP5, and inositol pyrophosphates). Deletion of IPK1, which generates IP6, however, did not affect regulation of telomere length. Further data also suggested that elo3Δ cells exhibit accelerated chronologic aging, and reduced replicative life span compared with wild type cells, and deletion of KCS1 helped recover these biological defects. Importantly, to determine downstream mechanisms, epistasis experiments were performed, and data indicated that ELO3 and YKU70/80 share a common pathway for the regulation of telomere length. More specifically, chromatin immunoprecipitation assays revealed that the telomere binding and protective function of YKu80p in vivo was reduced in elo3Δ cells, whereas its non-homologues end-joining function was not altered. Deletion of KCS1 in elo3Δ cells recovered the telomere binding and protective function of Ku, consistent with the role of KCS1 mutation in the rescue of telomere length attrition. Thus, these findings provide initial evidence of a possible link between Elo3-dependent VLCFA synthesis, and IP metabolism by KCS1 and IPK2 in the regulation of telomeres, which play important physiological roles in the control of senescence and aging, via a mechanism involving alterations of the telomere-binding/protection function of Ku. Very long-chain fatty acids (VLCFAs), 2The abbreviations used are: VLCFA, very long-chain fatty acid; FA, fatty acid; IPC, inositol phosphorylceramide; IP, inositol phosphate; IP4, inositol tetrakisphosphate; IP5, inositol pentakisphosphate; IP6, inositol hexakisphosphate; PP-IP, diphosphoinositol phosphate; ELO, fatty acid elongase; IPK, inositol phosphate kinase; ChIP, chromatin immunoprecipitation; GC/MS, gas chromatography/mass spectrometry; NHEJ, non-homologue end-joining; HPLC, high pressure liquid chromatography; GFP, green fluorescent protein; WT, wild type. containing mainly 26 carbons in yeast, are synthesized from palmitoyl-CoA by microsomal fatty acid (FA) elongases (Elo1–3p) that catalyze the multistep chain elongation reactions (1Toke D.A. Martin C.E. J. Biol. Chem. 1996; 271: 18413-18422Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar, 2Oh C.S. Toke D.A. Mandala S. Martin C.E. J. Biol. Chem. 1997; 272: 17376-17384Abstract Full Text Full Text PDF PubMed Scopus (397) Google Scholar, 3Kohlwein S.D. Eder S. Oh C.S. Martin C.E. Gable K. Bacikova D. Dunn T. Mol. Cell. Biol. 2001; 21: 109-125Crossref PubMed Scopus (176) Google Scholar, 4Dittrich F. Dittrich F. Zajonc D. Hühne K. Hoja U. Ekici A. Greiner E. Klein H. Hofmann J. Bessoule J.J. Sperling P. Schweizer E. Eur. J. Biochem. 1998; 252: 477-485Crossref PubMed Scopus (50) Google Scholar). As summarized in Fig. 1A, the chain elongation, up to C24-FA, is catalyzed by both Elo2p and Elo3p, whereas elongation from C24- to C26-FA is exclusively catalyzed by Elo3p (2Oh C.S. Toke D.A. Mandala S. Martin C.E. J. Biol. Chem. 1997; 272: 17376-17384Abstract Full Text Full Text PDF PubMed Scopus (397) Google Scholar). Yeast sphingolipids contain predominantly C26-phytoceramide (5Dickson R.C. Sumanasekera C. Lester R.L. Prog. Lipid Res. 2006; 45: 447-465Crossref PubMed Scopus (215) Google Scholar, 6Dickson R.C. Lester R.L. Biochim. Biophys. Acta. 2002; 1583: 13-25Crossref PubMed Scopus (198) Google Scholar) with hexacosanoic acid (C26-FA), which is a precursor for the synthesis of complex sphingolipids, such as inositol phosphorylceramide (IPC), mannosylinositol phosphorylceramide, and mannosyldiinositol phosphorylceramide. Null mutations of ELO3 (SUR4, YLR372W, SRE1, and VBM1), therefore, cause striking changes in the synthesis of sphingolipids and phosphatidylinositol/inositol phosphate metabolism (7Kobayashi S.D. Nagiec M.M. Eukaryot. Cell. 2003; 2: 284-294Crossref PubMed Scopus (54) Google Scholar, 8Eisenkolb M. Zenzmaier C. Leitner E. Schneiter R. Mol. Biol. Cell. 2002; 13: 4414-4428Crossref PubMed Scopus (97) Google Scholar, 9Reggiori F. Conzelmann A. J. Biol. Chem. 1998; 273: 30550-30559Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 10Levine T.P. Wiggins C.A. Munro S. Mol. Biol. Cell. 2000; 11: 2267-2281Crossref PubMed Scopus (133) Google Scholar), which are known to mediate diverse biological and signaling functions, including regulation of longevity, senescence, and telomere length (11Obeid L.M. Hannun Y.A. Sci. Aging Knowledge Environ. 2003; 2003: PE27Crossref PubMed Scopus (62) Google Scholar, 12Jazwinski S.M. Conzelmann A. Int. J. Biochem. Cell Biol. 2002; 34: 1491-1495Crossref PubMed Scopus (37) Google Scholar, 13York J.D. Biochim. Biophys. Acta. 2006; 1761: 552-559Crossref PubMed Scopus (127) Google Scholar). Telomeres are multifunctional genetic elements that cap eukaryotic chromosome ends, and they play essential roles in genomic stability, oncogenic transformation, and cellular senescence/aging (14Zakian V.A. Annu. Rev. Genet. 1996; 30: 141-172Crossref PubMed Scopus (170) Google Scholar, 15Bertuch A.A. Lundblad V. Curr. Opin. Cell Biol. 2006; 18: 247-253Crossref PubMed Scopus (43) Google Scholar). Telomeres are maintained by a specialized RNA-dependent DNA polymerase, telomerase, which adds species-specific, long-tandem nucleotide repeats (TG-repeats in yeast) to the ends of chromosomes (16Blackburn E.H. Greider C.W. Szostak J.W. Nat. Med. 2006; 12: 1133-1138Crossref PubMed Scopus (681) Google Scholar). Telomeres are regulated by factors that either play a role in their protection, such as Est1/2, Tel1, Yku70/80, and Rad50, or in their rapid shortening, such as Cdc13, Rif2, and Rap1 (14Zakian V.A. Annu. Rev. Genet. 1996; 30: 141-172Crossref PubMed Scopus (170) Google Scholar, 15Bertuch A.A. Lundblad V. Curr. Opin. Cell Biol. 2006; 18: 247-253Crossref PubMed Scopus (43) Google Scholar, 16Blackburn E.H. Greider C.W. Szostak J.W. Nat. Med. 2006; 12: 1133-1138Crossref PubMed Scopus (681) Google Scholar, 17Takata H. Tanaka Y. Matsuura A. Mol. Cell. 2005; 17: 573-583Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 18Smogorzewska A. de Lange T. Annu. Rev. Biochem. 2004; 73: 177-208Crossref PubMed Scopus (665) Google Scholar, 19Fisher T.S. Zakian V.A. DNA Repair. 2005; 4: 1215-1226Crossref PubMed Scopus (123) Google Scholar). The balance between these positive and negative factors determines the maintenance of telomeres in cells. Therefore, uncovering factors and signaling pathways involved in the regulation of telomeres is very important for senescence- and aging-related research. Interestingly, in a recent study, in which the global analysis of the roles of all non-essential genes involved in the regulation of telomere length in yeast were examined, the deletion of ELO3 was identified as one of the mutations that results in a significant telomere shortening (20Askree S.H. Yehuda T. Smolikov S. Gurevich R. Hawk J. Coker C. Krauskopf A. Kupiec M. McEachern M.J. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 8658-8663Crossref PubMed Scopus (278) Google Scholar). However, mechanisms by which Elo3p and VLCFA synthesis regulate telomeres are still unknown. Therefore, in this study, roles and mechanisms of telomere length regulation by VLCFA synthesis via Elo3p in yeast were examined. Consistent with the previous report (20Askree S.H. Yehuda T. Smolikov S. Gurevich R. Hawk J. Coker C. Krauskopf A. Kupiec M. McEachern M.J. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 8658-8663Crossref PubMed Scopus (278) Google Scholar), our data also showed that deletion of ELO3, which resulted in the loss of VLCFA and C26-phytoceramide synthesis, mediated a rapid attrition of telomere length. Further experiments suggested a novel link between C26-FA synthesis by Elo3p, and inositol polyphosphate (IP) metabolism by Ipk2p and Kcs1p in the regulation of telomere length. Remarkably, the deletion of IPK2 or KCS1, which are involved in the synthesis of IP4, IP5, and PP-IP4, and not IPK1, which generates IP6, completely protected telomere attrition in elo3Δ cells. In addition, data also suggested that telomere length attrition in elo3Δ cells was concomitant with accelerated chronologic aging and reduced replicative life span, biological defects that were also recovered by the deletion of KCS1. In addition, epistasis experiments revealed that ELO3 and YKU70/80 share a common pathway for the regulation of telomere length. More specifically, deletion of ELO3 resulted in alterations of the binding and protection of telomeres by Ku70/80, whereas the non-homologue end-joining (NHEJ) function of Ku was not affected. Importantly, deletion of KCS1 in the elo3Δ strain also recovered the telomere binding and protective function of YKu70/80, consistent with the protection of telomere length shortening in these cells. Thus, these data provide initial evidence for a possible link between VLCFA synthesis by Elo3p, and IP metabolism by Ipk2p/Kcs1p in the regulation of telomeres, which are involved in the control of senescence and life span, via alterations of the telomere-binding/protective functions of Ku70/80. Strains, Media, and Genetic Methods—Yeast strains used in this study are listed in Table 1. Yeast cells were grown at 30 °C in rich (YPD or YPG) or minimal media containing 2% glucose (D) or galactose (G). The elo3::URA3 strains were generated by the one-step gene replacement method (21Baudin A. Ozier-Kalogeropoulos O. Denouel A. Lacroute F. Cullin C. Nucleic Acids Res. 1993; 21: 3329-3330Crossref PubMed Scopus (1120) Google Scholar). The URA3 gene was amplified using 60- and 61-base oligonucleotides consisting of 40-base 5′ or 3′ flanking sequences of the ELO3 gene followed by 20- or 21-base URA3 sequences (5′-ATT CGG CTT TTT TCC GTT TGT TTA CGA AAC ATA AAC AGT CAG CTT TTC AAT TCA ATT CAT C-3′, and 5′-TTT TCT TTT TCA TTC GCT GTC AAA AAT TCT CGC TTC CTA TGG GTA ATA ACT GAT ATA ATT-3′). Yeast cells were transformed with purified PCR products, and Ura+ cells were selected on minimal media lacking uracil. Desired deletion was confirmed by PCR of genomic DNA. The plasmid (designated as pGal-Elo3[wt] in this report) containing the ELO3 gene under the control of the GAL1 promoter, YCpGALELO3(U), was obtained from Dr. Charles E. Martin (Rutgers University, Piscataway, NJ). The pGal-Elo3(mut) containing a 24-bp deletion (corresponding to an 8-amino acid deletion at position 19–26) was generated using the QuikChange mutagenesis kit (Stratagene).TABLE 1Yeast strainsStrainGenotypeSourceBY4742MATΔ his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0InvitrogenBY4742 elo1ΔBY4742 elo1::kanMX4InvitrogenBY4742 elo2ΔBY4742 elo2::kanMX4InvitrogenBY4742 elo3ΔBY4742 elo3::kanMX4InvitrogenBY4742 elo3Δ/pGal-ELO3 (wt)BY4742 elo3::kanMX4/pGal-ELO3(wt)::URA3This studyBY4742 elo3Δ/pGal-ELO3 (mut)BY4742 elo3::kanMX4/pGal-ELO3(mut)::URA3This studyBY4742 ipk1ΔBY4742 ipk1::kanMX4InvitrogenBY4742 ipk2ΔBY4742 ipk2::kanMX4InvitrogenBY4742 kcs1ΔBY4742 kcs1::kanMX4InvitrogenBY4742 yku70Δelo3ΔBY4742 yku70::kanMX4 elo3::URA3This studyBY4742 yku80Δelo3ΔBY4742 yku80::kanMX4 elo3::URA3This studyBY4742 ipk1Δelo3ΔBY4742 ipk1::kanMX4 elo3::URA3This studyBY4742 ipk2Δelo3ΔBY4742 ipk2::kanMX4 elo3::URA3This studyBY4742/yKU80-GFPBY4742 yKU80-GFP::HIS3This studyBY4742 elo3Δ/yKU80-GFPBY4742 elo3::URA3 yKU80-GFP::HIS3This studyBY4742 kcs1Δelo3Δ/yKU80-GFPBY4742 kcs1::kanMX4 elo3::URA3 yKU80-GFP::HIS3This studyJK9-3d elo3ΔJK9-3d elo3::URA3This studyJK9-3d lag1ΔJK9-3d lag1::kanMX4Guillas et al. (31Sawai H. Okamoto Y. Luberto C. Mao C. Bielawska A. Domae N. Hannun Y.A. J. Biol. Chem. 2000; 275: 39793-39798Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar)JK9-3d isc1Δ elo3ΔJK9-3d isc1::kanMX4 elo3::URA3This studyJK9-3d lag1Δelo3ΔJK9-3d lag1::kanMX4 elo3::URA3This studyW303-1AMATa can1-100 ade2-1 his3-11,15 leu2-3, 112 trp1-1 ura3-1Guillas et al. (31Sawai H. Okamoto Y. Luberto C. Mao C. Bielawska A. Domae N. Hannun Y.A. J. Biol. Chem. 2000; 275: 39793-39798Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar)W303-1A lac1Δlag1ΔW303-1A lac1::LEU2 lag1::TRP1Guillas et al. (31Sawai H. Okamoto Y. Luberto C. Mao C. Bielawska A. Domae N. Hannun Y.A. J. Biol. Chem. 2000; 275: 39793-39798Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar) Open table in a new tab Analysis of Telomere Length—Telomere length was measured as described previously (20Askree S.H. Yehuda T. Smolikov S. Gurevich R. Hawk J. Coker C. Krauskopf A. Kupiec M. McEachern M.J. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 8658-8663Crossref PubMed Scopus (278) Google Scholar, 22Sundararaj K.P. Wood R.E. Ponnusamy S. Salas A.M. Szulc Z. Bielawska A. Obeid L.M. Hannun Y.A. Ogretmen B. J. Biol. Chem. 2004; 279: 6152-6162Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar) by Southern blotting using genomic DNA prepared from 1.5 ml of yeast cultures grown to saturation. After digestion with XhoI, DNA fragments were separated by electrophoresis on a 1% Tris/borate/EDTA/agarose gel, and transferred to a nylon membrane (Roche). Then, the membranes were probed with a digoxigenin-labeled C1–3A/TG1–3 yeast-specific telomere probe. The bands present at the bottom of the membranes represent telomere fragments resulting from digestion of the last Y′ elements at chromosome termini. The average telomere length for each lane was estimated by plotting the peak of signal intensity of the Y′ telomeres at the bottom of the blots against the position of the molecular weight markers. FA Measurements—Total lipids were extracted from the logarithmic phase of yeast cultures, and FA levels were measured using gas chromatography/mass spectrometry (GC/MS) as described previously (23Alderson N.L. Rembiesa B.M. Walla M.D. Bielawska A. Bielawski J. Hama H. J. Biol. Chem. 2004; 279: 48562-48568Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). FA contents were normalized to total lipid phosphate (Pi) levels. Measurement of Phytoceramide Levels—The measurement of the levels of endogenous phytoceramides was performed using high performance liquid chromatography-tandem mass spectrometry, as described previously (24Bielawski J. Szulc Z.M. Hannun Y.A. Bielawska A. Methods. 2006; 39: 82-91Crossref PubMed Scopus (424) Google Scholar). Phytoceramide contents were normalized to total lipid phosphate (Pi) levels. Measurement of Inositol Phosphates—The levels of IP molecules after labeling with [3H]inositol were analyzed using HPLC as described previously (25Saiardi A. Sciambi C. McCaffery J.M. Wendland B. Snyder S.H. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 14206-14211Crossref PubMed Scopus (149) Google Scholar). Determination of Chronological Aging—Chronologic aging of yeast population was determined as previously described (26Fabrizio P. Longo V.D. Methods Mol. Biol. 2007; 371: 89-95Crossref PubMed Google Scholar). Yeast cultures were grown for 4–5 days until they reach a stationary phase in 10 ml of YPD media. Then, the cells were washed twice, and an equal number of cells were re-suspended in 10 ml of sterile distilled water, and their growth was monitored on YPD plates after serial dilutions. Analysis of Replicative Life Span in Yeast—The replicative life span was analyzed as described previously (27Kim S. Benguria A. Lai C.Y. Jazwinski S.M. Mol. Biol. Cell. 1999; 10: 3125-3136Crossref PubMed Scopus (182) Google Scholar). In summary, new buds (virgin cells) from overnight cultures were deposited in a row in isolated spots on an YPD plate. The number of buds removed by micro-dissection prior to cell death is defined as the life span of the cell in generations. The significance of the differences in the life spans of aging cohorts of each of the strains in these experiments was assessed using the Mann-Whitney test. Chromatin Immunoprecipitation (ChIP) Analysis—The association of endogenous YKu80p-GFP, which was constructed by insertion of a 2.1-kb GFP fragment containing a histidine marker into the 3′-end of YKU80 by homologous recombination, with telomeric DNA in WT and elo3Δ strains were assessed by ChIP analysis as previously described (28Wooten-Blanks L.G. Song P. Senkal C.E. Ogretmen B. FASEB J. 2007; 21: 3386-3397Crossref PubMed Scopus (56) Google Scholar). After endogenous proteins were cross-linked to DNA by 1% formaldehyde for 1 h, YKu80p-GFP was immunoprecipitated using anti-GFP microbeads (MACS). Then, the cross-links were reversed with 200 mm NaCl, and precipitated DNA bound to Ku was spotted on nylon membranes. Telomeres were detected using a digoxigenin-labeled telomere probe as described above. Deletion of ELO3, Which Results in the Loss of VLCFA Synthesis, Mediates Telomere Shortening—In addition to their structural roles in biological membranes, lipid molecules are known to transduce a myriad of signaling pathways involved in the regulation of a wide variety of biological processes, such as regulation of cell growth, proliferation, and/or senescence (11Obeid L.M. Hannun Y.A. Sci. Aging Knowledge Environ. 2003; 2003: PE27Crossref PubMed Scopus (62) Google Scholar). Recently, global analysis of the roles of all non-essential genes in the regulation of telomeres in yeast revealed that loss of ELO3 results in a significant reduction in telomere length (20Askree S.H. Yehuda T. Smolikov S. Gurevich R. Hawk J. Coker C. Krauskopf A. Kupiec M. McEachern M.J. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 8658-8663Crossref PubMed Scopus (278) Google Scholar). However, mechanisms by which ELO3 and/or VLCFA synthesis regulate telomere length have not been determined previously. Given our interest in the roles of lipid/sphingolipid metabolism and signaling in the regulation of telomeres, we set out experiments to determine mechanisms involved in the regulation of telomeres by VLCFA synthesis, which is also an integral part of sphingolipid metabolism, via the function of Elo3 in yeast. First, to further confirm the possible roles of Elo3-generated VLCFA containing 26 carbon units (Fig. 1A) in the regulation of telomeres, we examined the FA contents and telomere length in elo3Δ compared with WT, elo1Δ, and elo2Δ strains, using GC/MS and Southern blotting, respectively. Consistent with previous reports (2Oh C.S. Toke D.A. Mandala S. Martin C.E. J. Biol. Chem. 1997; 272: 17376-17384Abstract Full Text Full Text PDF PubMed Scopus (397) Google Scholar, 3Kohlwein S.D. Eder S. Oh C.S. Martin C.E. Gable K. Bacikova D. Dunn T. Mol. Cell. Biol. 2001; 21: 109-125Crossref PubMed Scopus (176) Google Scholar, 7Kobayashi S.D. Nagiec M.M. Eukaryot. Cell. 2003; 2: 284-294Crossref PubMed Scopus (54) Google Scholar, 8Eisenkolb M. Zenzmaier C. Leitner E. Schneiter R. Mol. Biol. Cell. 2002; 13: 4414-4428Crossref PubMed Scopus (97) Google Scholar), our data showed that deletion of ELO3 completely prevented the synthesis of C26-FA, and decreased C24-FA synthesis about 33% as compared with controls, with concomitant accumulation of C16–C22-FA, both hydroxylated (Fig. 1B and Table 2) and unhydroxylated (data not shown) forms. A moderate decrease in the levels of C24- and C26-, and an increase in C16–22-FAs were observed in elo2Δ cells, and no significant changes were detected in VLCFA contents in elo1Δ when compared with the WT strain (Fig. 1B and Table 2).TABLE 22-OH fatty acid levels (nmol/nmol of Pi)2-OH C162-OH C182-OH C202-OH C222-OH C242-OH C26WT32.24 ± 5.603.68 ± 0.290.4 ± 0.071.07 ± 0.188.86 ± 1.9853.81 ± 5.67elo1Δ29.67 ± 4.882.81 ± 0.411 ± 0.151.71 ± 0.215.19 ± 0.7549.6 ± 3.1elo2Δ65.37 ± 8.4111.01 ± 1.8712.3 ± 1.223.10 ± 0.283.6 ± 0.4318.23 ± 2.11elo3Δ90.52 ± 9.018.6 ± 1.5414.7 ± 1.3916.92 ± 1.725.92 ± 0.69UD Open table in a new tab More importantly, the data also showed that deletion of ELO3 (Fig. 1C, lane 4) decreased telomere length about 100 (+12) bp, whereas deletion of ELO1 or ELO2 (Fig. 1C, lanes 2 and 3, respectively) did not cause detectable effects when compared with the WT strain (Fig. 1C, lanes 1 and 5). Similar data were also observed in another strain (JK9–3d), in which the deletion of ELO3 decreased telomere length ∼110 bp compared with the WT strain (shown in Fig. 4C). These data are consistent with the previous study (20Askree S.H. Yehuda T. Smolikov S. Gurevich R. Hawk J. Coker C. Krauskopf A. Kupiec M. McEachern M.J. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 8658-8663Crossref PubMed Scopus (278) Google Scholar), and suggest that alterations of the VLCFA synthesis by the deletion of ELO3 participate in a rapid reduction of telomere length. Overexpression of WT ELO3 Reconstitutes the Synthesis of VLCFA, Leading to the Prevention of Telomere Attrition in the elo3Δ Mutant Strain—Next, to examine whether the shortening of telomeres in elo3Δ cells can be reversed, VLCFA synthesis was reconstituted in these mutants using an expression vector containing WT ELO3 under the control of a galactose (GAL)-inducible promoter (2Oh C.S. Toke D.A. Mandala S. Martin C.E. J. Biol. Chem. 1997; 272: 17376-17384Abstract Full Text Full Text PDF PubMed Scopus (397) Google Scholar). Expression of ELO3 in the presence of GAL reconstituted the synthesis of C26-FA within 24, 48, or 72 h of induction (Fig. 2A and Table 3), and more importantly, induction of Elo3p expression markedly reversed the shortening of telomere length in elo3Δ/pGAL1-ELO3(wt) cells at these time points compared with that of uninduced or WT cells (Fig. 2B, lanes 2, 4, and 6; 3, 5, and 7; and 1, respectively). Collectively, these data suggest that deletion of ELO3 results in the reduction of telomere length, which can be recovered in response to reconstitution of WT ELO3 expression and the synthesis of VLCFA in elo3Δ cells.TABLE 32-OH fatty acid levels (nmol/nmol of Pi)24 h48 h72 hC22C24C26C22C24C26C22C24C26WT1.07 ± 0.28.86 ± 3.353.81 ± 11.21.08 ± 0.249.0 ± 3.4550.1 ± 10.21.0 ± 0.258.9 ± 2.951.2 ± 10.9Uninduced18.10 ± 3.45.76 ± 3.41.11 ± 0.235.3 ± 12.312.12 ± 2.50.99 ± 0.228.2 ± 9.89.18 ± 2.61.21 ± 0.2Induced9.11 ± 2.332.09 ± 7.623.19 ± 5.27.15 ± 2.838.63 ± 8.341.85 ± 6.86.67 ± 1.819.27 ± 0.936.79 ± 13.1 Open table in a new tab Then, to determine the functional role of Elo3p activity in the regulation of telomere length at 72 h, a mutant form of ELO3, which is defective in generation of C26-FA, was expressed in elo3Δ mutants, and its effects on FA synthesis and telomere length were examined. Because the domains that are required for distinct catalytic functions of Elo1–3p are still unclear, we truncated a stretch of amino acids (LNSSSSCF) at positions 19–26 near the N terminus of Elo3p, which is present only in yeast Elo3, but not present in Elo1p or Elo2p. The mutant form of Elo3p was then expressed in elo3Δ cells under control of the GAL promoter, and its effects on FA synthesis and on the recovery of telomere length attrition were determined. Interestingly, the data showed that expression of the mutant Elo3p, lacking amino acids 19–26, was unable to generate C26 FA as efficiently, whose activity was about 70% less than that of the WT Elo3 (Fig. 3A and Table 4). In addition, the results also showed that C22- and C24-FA levels were comparable in response to the expression of WT or mutant Elo3p (Fig. 3A and Table 4). Moreover, whereas the expression of WT-ELO3 recovered loss of telomeres of about 70%, expression of the mutated-Elo3p did not efficiently protect telomeres in elo3Δ cells (Fig. 3, B and C, lanes 3 and 4). Taken together, these data suggest that enzymatic activity of Elo3p for C26-FA synthesis, and not in the alterations of C22- or C24-FA synthesis, plays important roles in the regulation of telomere length in yeast.TABLE 42-OH fatty acid levels (nmol/nmol of Pi)2-OH C162-OH C182-OH C202-OH C222-OH C242-OH C26elo3Δ+pGalElo3(wt)38.75 ± 4.974.11 ± 0.821.8 ± 0.278.13 ± 1.912.1 ± 0.1831.64 ± 4.7elo3Δ+pGalelo3(mut)51.44 ± 6.433.8 ± 0.794.2 ± 0.816.93 ± 1.333.96 ± 0.419.92 ± 1.43 Open table in a new tab Alterations of the Phytoceramide Generation Might Be Dispensable for the Shortening of Telomere Length in elo3Δ Cells—In yeast, hydroxy-C26-phytoceramide, containing the VLCFA chain, constitutes the majority of ceramides, therefore, the synthesis of VLCFA is directly involved in the regulation of phytoceramide generation (Fig. 4A). In fact, the loss of VLCFA synthesis is known to result in significant changes in sphingolipid metabolism (2Oh C.S. Toke D.A. Mandala S. Martin C.E. J. Biol. Chem. 1997; 272: 17376-17384Abstract Full Text Full Text PDF PubMed Scopus (397) Google Scholar, 3Kohlwein S.D. Eder S. Oh C.S. Martin C.E. Gable K. Bacikova D. Dunn T. Mol. Cell. Biol. 2001; 21: 109-125Crossref PubMed Scopus (176) Google Scholar, 7Kobayashi S.D. Nagiec M.M. Eukaryot. Cell. 2003; 2: 284-294Crossref PubMed Scopus (54) Google Scholar, 8Eisenkolb M. Zenzmaier C. Leitner E. Schneiter R. Mol. Biol. Cell. 2002; 13: 4414-4428Crossref PubMed Scopus (97) Google Scholar). Indeed, as shown in Fig. 4B and Table 5, C22-phytoceramide was highly elevated, whereas C26-phytoceramide, both 2-hydroxylated and non-hydroxylated forms (data not shown), was almost completely lost in elo3Δ cells. Therefore, we investigated whether alterations in ceramide generation were involved in telomere shortening in elo3Δ cells. Phytoceramide can be generated via de novo synthesis by ceramide synthase activities of the longevity assurance gene 1, Lag1p, which is associated with Lac1p and Lip1 (12Jazwinski S.M. Conzelmann A. Int. J. Biochem. Cell Biol. 2002; 34: 1491-1495Crossref PubMed Scopus (37) Google Scholar, 29Guillas I. Kirchman P.A. Chuard R. Pfefferli M. Jiang J.C. Jazwinski S.M. Conzelmann A. EMBO J. 2001; 20: 2655-2665Crossref PubMed Scopus (220) Google Scholar, 30Vallee B. Riezman H. EMBO J. 2005; 24: 730-741Crossref PubMed Scopus (116) Google Scholar), or by the hydrolysis of complex sphingolipids by Isc1p (31Sawai H. Okamoto Y. Luberto C. Mao C. Bielawska A. Domae N. Hannun Y.A. J. Biol. Chem. 2000; 275: 39793-39798Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar, 32Kitagaki H. Cowart L.A. Matmati N. Vaena de Avalos S. Novgorodov S.A. Zeidan Y.H. Bielawski J. Obeid L.M. Hannun Y.A. Biochim. Biophys. Acta. 2007; 1768: 2849-2861Crossref PubMed Scopus (67) Google Scholar) in yeast (Fig. 4A).TABLE 52-OH phytoceramide levels (pmol/nmol of Pi)OH PhyC16OH PhyC18OH PhyC20OH PhyC22OH PhyC24OH PhyC26WT1.9 ± 0.181.8 ± 0.164.5 ± 1.121.5 ± 0.051.6 ± 0.097.9 ± 0.94Elo1Δ3.3 ± 1.013.7 ± 0.913.5 ± 0.981.9 ± 1.161.3 ± 0.517.6 ± 0.71Elo2Δ16.4 ± 3.2711.3 ± 1.2617.8 ± 2.592.6 ± 1.228.5 ± 1.463.8 ± 0.23Elo3Δ23.4 ± 3.0513.0 ± 2.0310.8 ± 1.4615.8 ± 2.6311.1 ± 1.9UDaUD, undectable.Elo3Δ+pGalElo3(wt)2.8 ± 0.773.2 ± 0.543.9 ± 0.334.9 ± 1.013.8 ± 1.205.6 ± 1.1Elo3Δ+pGalelo3(mut)3.6 ± 1.213.3 ± 0.885.6 ± 1.004.5 ± 0.817.0 ± 1.111.3 ± 0.09a UD, undectable. Open table in a new tab To determine whether the increase in phytoceramide with shorter FA chain lengths, especially C22-phytoceramide, which was elevated only in elo3Δ cells, is important in telomere shortening, effects of LAG1 or ISC1 gene deletion on telomere length were determined in Jk93d strain (Fig. 4C). The rationale behind thes" @default.
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• W1982040229 title "Regulation of Telomere Length by Fatty Acid Elongase 3 in Yeast" @default.
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