Shortening of membrane lipid acyl chains compensates for phosphatidylcholine deficiency in choline‐auxotroph yeast
2021; Springer Nature; Volume: 40; Issue: 20 Linguagem: Inglês
10.15252/embj.2021107966
ISSN1460-2075
AutoresXue Bao, Martijn C. Koorengevel, Marian J.A. Groot Koerkamp, Amir Homavar, A. Weijn, Stefan Crielaard, Mike F. Renne, Joseph H. Lorent, Willie J. C. Geerts, Michał A. Surma, Muriel Mari, Frank C. P. Holstege, Christian Klose, Anton I.P.M. de Kroon,
Tópico(s)Endoplasmic Reticulum Stress and Disease
ResumoArticle14 September 2021Open Access Source DataTransparent process Shortening of membrane lipid acyl chains compensates for phosphatidylcholine deficiency in choline-auxotroph yeast Xue Bao Xue Bao orcid.org/0000-0002-5129-0728 Membrane Biochemistry & Biophysics, Bijvoet Center for Biomolecular Research and Institute of Biomembranes, Utrecht University, Utrecht, The Netherlands Search for more papers by this author Martijn C Koorengevel Martijn C Koorengevel Membrane Biochemistry & Biophysics, Bijvoet Center for Biomolecular Research and Institute of Biomembranes, Utrecht University, Utrecht, The Netherlands Search for more papers by this author Marian J A Groot Koerkamp Marian J A Groot Koerkamp orcid.org/0000-0002-0867-0821 Princess Máxima Center for Pediatric Oncology, Utrecht, The Netherlands Search for more papers by this author Amir Homavar Amir Homavar Membrane Biochemistry & Biophysics, Bijvoet Center for Biomolecular Research and Institute of Biomembranes, Utrecht University, Utrecht, The Netherlands Search for more papers by this author Amrah Weijn Amrah Weijn Membrane Biochemistry & Biophysics, Bijvoet Center for Biomolecular Research and Institute of Biomembranes, Utrecht University, Utrecht, The Netherlands Search for more papers by this author Stefan Crielaard Stefan Crielaard Membrane Biochemistry & Biophysics, Bijvoet Center for Biomolecular Research and Institute of Biomembranes, Utrecht University, Utrecht, The Netherlands Search for more papers by this author Mike F Renne Mike F Renne orcid.org/0000-0003-0508-2298 Membrane Biochemistry & Biophysics, Bijvoet Center for Biomolecular Research and Institute of Biomembranes, Utrecht University, Utrecht, The Netherlands Search for more papers by this author Joseph H Lorent Joseph H Lorent orcid.org/0000-0002-7537-8521 Membrane Biochemistry & Biophysics, Bijvoet Center for Biomolecular Research and Institute of Biomembranes, Utrecht University, Utrecht, The Netherlands Search for more papers by this author Willie JC Geerts Willie JC Geerts Cryo-Electron Microscopy, Bijvoet Center for Biomolecular Research, Utrecht University, Utrecht, The Netherlands Search for more papers by this author Michal A Surma Michal A Surma orcid.org/0000-0001-7833-2214 Lipotype GmbH, Dresden, Germany Search for more papers by this author Muriel Mari Muriel Mari Department of Biomedical Sciences of Cells & Systems, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands Search for more papers by this author Frank C P Holstege Frank C P Holstege orcid.org/0000-0002-8090-5146 Princess Máxima Center for Pediatric Oncology, Utrecht, The Netherlands Search for more papers by this author Christian Klose Christian Klose Lipotype GmbH, Dresden, Germany Search for more papers by this author Anton I P M de Kroon Corresponding Author Anton I P M de Kroon [email protected] orcid.org/0000-0003-1209-756X Membrane Biochemistry & Biophysics, Bijvoet Center for Biomolecular Research and Institute of Biomembranes, Utrecht University, Utrecht, The Netherlands Search for more papers by this author Xue Bao Xue Bao orcid.org/0000-0002-5129-0728 Membrane Biochemistry & Biophysics, Bijvoet Center for Biomolecular Research and Institute of Biomembranes, Utrecht University, Utrecht, The Netherlands Search for more papers by this author Martijn C Koorengevel Martijn C Koorengevel Membrane Biochemistry & Biophysics, Bijvoet Center for Biomolecular Research and Institute of Biomembranes, Utrecht University, Utrecht, The Netherlands Search for more papers by this author Marian J A Groot Koerkamp Marian J A Groot Koerkamp orcid.org/0000-0002-0867-0821 Princess Máxima Center for Pediatric Oncology, Utrecht, The Netherlands Search for more papers by this author Amir Homavar Amir Homavar Membrane Biochemistry & Biophysics, Bijvoet Center for Biomolecular Research and Institute of Biomembranes, Utrecht University, Utrecht, The Netherlands Search for more papers by this author Amrah Weijn Amrah Weijn Membrane Biochemistry & Biophysics, Bijvoet Center for Biomolecular Research and Institute of Biomembranes, Utrecht University, Utrecht, The Netherlands Search for more papers by this author Stefan Crielaard Stefan Crielaard Membrane Biochemistry & Biophysics, Bijvoet Center for Biomolecular Research and Institute of Biomembranes, Utrecht University, Utrecht, The Netherlands Search for more papers by this author Mike F Renne Mike F Renne orcid.org/0000-0003-0508-2298 Membrane Biochemistry & Biophysics, Bijvoet Center for Biomolecular Research and Institute of Biomembranes, Utrecht University, Utrecht, The Netherlands Search for more papers by this author Joseph H Lorent Joseph H Lorent orcid.org/0000-0002-7537-8521 Membrane Biochemistry & Biophysics, Bijvoet Center for Biomolecular Research and Institute of Biomembranes, Utrecht University, Utrecht, The Netherlands Search for more papers by this author Willie JC Geerts Willie JC Geerts Cryo-Electron Microscopy, Bijvoet Center for Biomolecular Research, Utrecht University, Utrecht, The Netherlands Search for more papers by this author Michal A Surma Michal A Surma orcid.org/0000-0001-7833-2214 Lipotype GmbH, Dresden, Germany Search for more papers by this author Muriel Mari Muriel Mari Department of Biomedical Sciences of Cells & Systems, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands Search for more papers by this author Frank C P Holstege Frank C P Holstege orcid.org/0000-0002-8090-5146 Princess Máxima Center for Pediatric Oncology, Utrecht, The Netherlands Search for more papers by this author Christian Klose Christian Klose Lipotype GmbH, Dresden, Germany Search for more papers by this author Anton I P M de Kroon Corresponding Author Anton I P M de Kroon [email protected] orcid.org/0000-0003-1209-756X Membrane Biochemistry & Biophysics, Bijvoet Center for Biomolecular Research and Institute of Biomembranes, Utrecht University, Utrecht, The Netherlands Search for more papers by this author Author Information Xue Bao1, Martijn C Koorengevel1, Marian J A Groot Koerkamp2, Amir Homavar1, Amrah Weijn1, Stefan Crielaard1, Mike F Renne1, Joseph H Lorent1, Willie JC Geerts3, Michal A Surma4, Muriel Mari5, Frank C P Holstege2, Christian Klose4 and Anton I P M de Kroon *,1 1Membrane Biochemistry & Biophysics, Bijvoet Center for Biomolecular Research and Institute of Biomembranes, Utrecht University, Utrecht, The Netherlands 2Princess Máxima Center for Pediatric Oncology, Utrecht, The Netherlands 3Cryo-Electron Microscopy, Bijvoet Center for Biomolecular Research, Utrecht University, Utrecht, The Netherlands 4Lipotype GmbH, Dresden, Germany 5Department of Biomedical Sciences of Cells & Systems, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands *Corresponding author (lead contact). Tel: +31 30 253 3424; E-mail: [email protected] The EMBO Journal (2021)40:e107966https://doi.org/10.15252/embj.2021107966 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Phosphatidylcholine (PC) is an abundant membrane lipid component in most eukaryotes, including yeast, and has been assigned multiple functions in addition to acting as building block of the lipid bilayer. Here, by isolating S. cerevisiae suppressor mutants that exhibit robust growth in the absence of PC, we show that PC essentiality is subject to cellular evolvability in yeast. The requirement for PC is suppressed by monosomy of chromosome XV or by a point mutation in the ACC1 gene encoding acetyl-CoA carboxylase. Although these two genetic adaptations rewire lipid biosynthesis in different ways, both decrease Acc1 activity, thereby reducing average acyl chain length. Consistently, soraphen A, a specific inhibitor of Acc1, rescues a yeast mutant with deficient PC synthesis. In the aneuploid suppressor, feedback inhibition of Acc1 through acyl-CoA produced by fatty acid synthase (FAS) results from upregulation of lipid synthesis. The results show that budding yeast regulates acyl chain length by fine-tuning the activities of Acc1 and FAS and indicate that PC evolved by benefitting the maintenance of membrane fluidity. SYNOPSIS Saccharomyces cerevisiae suppressor mutants overcoming choline auxotrophy allow growth in the absence of the major membrane lipid phosphatidylcholine (PC). This study reveals that the interplay between acetyl-CoA carboxylase (Acc1) and fatty acid synthase (FAS), which regulates lipid acyl chain length, renders PC redundant in such mutant. Most PC-free choline auxotrophy suppressors are 2n-1 aneuploids lacking one copy of chromosome XV. Rare diploid suppressor strains contain a homozygous point mutation in the ACC1 gene encoding acetyl-CoA carboxylase. Aneuploid suppressors rely on feed-back inhibition of Acc1 by FAS-derived acyl-CoA, resulting from up-regulation of lipid synthesis. Inhibition of Acc1 activity is crucial for suppression by reducing average lipid acyl chain length to maintain membrane fluidity. Introduction The glycerophospholipid phosphatidylcholine (PC) is an essential membrane lipid accounting for at least 50% of total phospholipids in most eukaryotes (van Meer et al, 2008). The exception is presented by several species of green algae that often contain the phosphorus-free betaine lipid diacylglyceryl-N, N, N-trimethylhomoserine (DGTS) instead of PC (Sato & Furuya, 1985; Giroud et al, 1988). DGTS and PC both carry a quaternary amine-containing zwitterionic head group and share similar biophysical properties (Sato & Murata, 1991). PC is also present in more than 10% of Bacteria, however, bacterial PC has not been assigned any essential function (Geiger et al, 2013). Besides its role as a building block of lipid bilayers, PC has regulatory functions in signal transduction and metabolic regulation in eukaryotes. For example, specific molecular species of PC serve as endogenous ligands for peroxisome proliferator-activated receptor-α (PPARα) and liver receptor homologue 1 (LRH1), respectively (Chakravarthy et al, 2009; Lee et al, 2011). Loss-of-function mutations of key PC biosynthetic enzymes cause a wide spectrum of human pathologies (van der Veen et al, 2017). Furthermore, alterations in PC metabolism have been implicated in cancer (Ridgway, 2013). By their sheer abundance, PC and its metabolic precursor phosphatidylethanolamine (PE) are important players in determining physical membrane properties such as membrane fluidity and intrinsic curvature that impact the function of membranes and membrane proteins (de Kroon et al, 2013; Covino et al, 2018). Whereas PC has an overall cylindrical molecular shape that makes it ideally suited to build the membrane bilayer matrix, PE is a lipid with non-bilayer propensity that can adopt a conical shape depending on its acyl chain composition (Renne & de Kroon, 2018). The increased PC/PE ratio induced by obesity in mouse liver was found to inhibit Ca2+ transport by SERCA, causing ER stress (Fu et al, 2011). A decrease in PC/PE ratio in mouse liver induces steatohepatitis and ultimately causes liver failure due to loss of membrane integrity (Li et al, 2006). The tolerance of the model eukaryote Saccharomyces cerevisiae towards variation in membrane lipid composition, makes it ideally suited for addressing the functions of lipid classes in membrane lipid homeostasis (de Kroon et al, 2013). The yeast double deletion mutant cho2opi3 lacking the methyltransferases converting PE to PC, relies on supplementation with choline for the synthesis of PC by the CDP-choline route (Fig 1A) (Summers et al, 1988; Kodaki & Yamashita, 1989), and has been used to manipulate cellular PC content. Both DGTS and phosphatidyldimethylethanolamine (PDME), a lipid containing two instead of three N-methyl groups with physical properties similar to PC, can substitute for PC in cho2opi3 (McGraw & Henry, 1989; Boumann et al, 2006; Riekhof et al, 2014), demonstrating that PC is dispensable for yeast growth. Figure 1. Phenotype and karyotype of evolved cho2opi3 suppressors A. Cartoon depicting the biosynthetic pathways producing PC in yeast. B. 10-fold serial dilutions of 1 OD600 unit/ml of the indicated strains were spotted on SD plates containing 0 (C−) or 1 mM choline (C+) and 0 (I−) or 75 µM inositol (I+) and incubated at 30°C for 3 days. A representative experiment is shown (from n = 5). C. 2D-TLC analysis of total lipid extracts of coS#2 cells cultured in SD C+I+ and C−I+; ori, origin; NL, neutral lipids. D. Read-depth analysis indicating monosomy of chromosome XV in coS#3, S#4, and S#5, but not in coS#2. Each data point represents the median chromosome copy number per 5-kb bin plotted over the genome, with alternating colours for each successive chromosome and the mitochondrial DNA. E. Representative DNA content profiles of haploid and diploid cho2opi3 controls (cultured in C+) and the indicated cho2opi3 suppressor strains. Download figure Download PowerPoint Here, we report the isolation and characterization of cho2opi3 suppressor mutants that exhibit sustained growth in the absence of choline. As the suppressors do not contain PC or a PC substitute, elucidation of the mechanism of suppression provides an unbiased route to address PC function. The choline auxotrophy of cho2opi3 is suppressed by 2n-1 monosomy of chromosome XV or by a point mutation in the ACC1 gene encoding acetyl-CoA carboxylase. The genetic changes in both suppressors shorten average acyl chain length due to reduced activity of Acc1. Inhibition of Acc1 is sufficient for suppressing choline auxotrophy as evidenced by the rescue of cho2opi3 by soraphen A, a specific inhibitor of Acc1. The results indicate that the suppression by chromosome XV monosomy relies on inhibition of Acc1 by accumulating acyl-CoA, providing novel clues about the regulation of acyl chain length by the interplay between Acc1 and the fatty acid synthase complex (FAS). Based on the compensatory changes in the PC-free lipidomes, we propose that the acquisition of PC during evolution provided selective advantage in maintaining membrane physical properties, membrane fluidity in particular. Results Phenotype and genotype of evolved PC-free yeast cho2opi3 suppressors After incubating the cho2opi3 mutant on choline-free agar plates at 30°C for 14 days, cho2opi3 suppressor clones were obtained. Most of the clones exhibit sustained growth in the absence of choline, and can be stored as and revived from −80°C glycerol stocks in choline-free SD medium (SD C−). A subset of four cho2opi3 (co) suppressor clones, coS#2-S#5, was characterized in detail (Fig 1). In contrast to their choline auxotroph cho2opi3 parent, coS#2-#5 grow robustly in the absence of choline, albeit slower than the corresponding WT, irrespective of supplementation with inositol (Fig 1B). The effect of inositol was examined because of its key role in the phosphatidic acid (PA)-mediated transcriptional regulation of phospholipid biosynthesis genes containing UASINO (Henry et al, 2012). Remarkably, in the absence of inositol, coS#3-#5 grow slightly better without than with choline present (Fig 1B), suggesting a choline-sensitive requirement for inositol. The doubling times observed in the corresponding liquid media (Fig EV1A) are consistent with the growth phenotypes on agar plates. Click here to expand this figure. Figure EV1. Characterization of evolved cho2opi3 suppressors A. Doubling times of WT (BY4742), cho2opi3, and co S#3, S#4 and S#2, cultured in SD medium supplemented with 1 mM choline and/or 75 µM inositol as indicated. Data are presented as the mean of 2 (wild type) or 3 (other strains) biological replicates, with the individual values indicated. B. 9 out of 10 independent cho2opi3 suppressor strains exhibit monosomy of chr XV. Copy numbers of chr I, IV, VI, IX and XV derived from qPCR and FACS analysis of cellular DNA content (lower panel and Fig 1E) are shown for the haploid co parent and 10 suppressor strains compared to wild type. C. Serial dilution experiment (100–10−5) comparing haploid co MATa and co MATα to co diploid on SD C+/− after incubation for 14 days at 30°C, and absolute copy numbers of chr I, IV, VI, IX and XV in a co diploid and 4 derived suppressor strains as determined by qPCR and FACS. D. The cho2opi3 suppressor strains retain the α-mating type. Suppressor and control strains were mated with SH85 (MATa) and SH80 (MATα) as indicated and then streaked on SD C− plates without amino acids (AA−). The amino acid containing plate (AA+) serves as control. The reduced mating efficiency of coS#4 may be due to haploinsufficiency originating from the loss of one copy of the MAT locus on chr III. The ability of BY4743 (MATa/MATα) to mate with SH85 is attributed to loss of heterozygosity by homologous recombination (Harari et al, 2018). E. Characterization of 3 cho2opi3 suppressor clones generated on SD C−I− plates supplemented with 1 mM propanolamine (Prn). Growth phenotype on SD with 1 mM Prn or without supplement after 5 days at 30°C, DNA content by FACS, and absolute copy numbers of chr I, IV, VI, IX and XV in co SPrn#1, #2, #3 and haploid co parent compared to wild type. Data information: Chromosome copy numbers in panels B, C and E are presented as mean values from 2 assays using primers complementary to non-coding regions on the left and right arm of each chr, respectively, with the individual values indicated. Download figure Download PowerPoint Analysis by thin layer chromatography (TLC) of total lipid extracts of the suppressors cultured in SD C- indicated that the suppressors are devoid of PC, leaving PE as the predominant membrane lipid (Fig 1C, Appendix Fig S1A). MS analysis corroborated this result. PC could not be detected in negative ion mode as acetate-adduct, nor in positive ion mode as H+-adduct. Fragmentation in the positive ion mode did not reveal the phosphocholine head group. To elucidate the nature of the adaptation, coS#2-#5 were subjected to whole genome sequencing (WGS). WGS did not reveal single nucleotide polymorphisms (SNPs), insertions or deletions shared by the four suppressors (Table EV1). However, analysis of chromosome copy number by WGS and fluorescence-activated cell sorting (FACS) revealed changes in ploidy (Fig 1D and E). Suppressors coS#3, #4 and #5 exhibit 2n-1 aneuploidy, by losing a copy of chromosome XV (chr XV) after genome duplication. In addition, coS#4 lost part of the right arm of one copy of chr III, whereas coS#5 gained an extra copy of chr IX and lost its mitochondrial DNA. Ploidy changes, including aneuploidy with gain or loss of chromosomes, are common in adaptive evolution of yeast mutants lacking (non-)essential genes (Storchova, 2014; Szamecz et al, 2014; Liu et al, 2015). Partial karyotype analysis by FACS analysis and a quantitative polymerase chain reaction (qPCR)-based assay (Pavelka et al, 2010) addressing chr XV with chr I, IV, VI and IX as controls, was applied to an extended set of suppressor clones. Like coS#3-#5, suppressors coS#6-#11 exhibit chr XV monosomy, and similar to coS#5, coS#8 and S#9 gained extra copies of chr IX (Fig EV1B). Generation of (2n-1) suppressors from a diploid co strain proceeds more readily than from its haploid counterparts (Fig EV1C), suggesting that genome duplication is limiting. The odd one out is coS#2 that turned diploid and retained both copies of chr XV (Fig 1D and E). WGS of coS#2 revealed a homozygous point mutation in the ACC1 gene encoding acetyl-CoA carboxylase, catalysing the rate limiting step of FA synthesis (Tehlivets et al, 2007). Adenosine at position 657039 of both copies of chr XIV is replaced by cytosine, resulting in the substitution of asparagine at position 1446 of Acc1 by histidine (N1446H; Table EV1). Suppressors coS#2-S#5 retained the MATα mating type as shown by their ability to mate with a threonine-auxotrophic MATa strain (Fig EV1D), indicating that genome duplication happened through endoreduplication rather than mating preceded by mating type switch (Harari et al, 2018). Previous research showed that propanolamine (Prn) could substitute for choline in supporting growth of cho2opi3 (Choi et al, 2004). This finding was unexpected, since the physical properties of phosphatidylpropanolamine (PPrn) resemble those of PE rather than PC (Storey et al, 2001). In our hands, Prn does not support growth of cho2opi3 cells. However, suppressors generated on choline-free agar plates supplemented with 1 mM Prn also grow without supplements and exhibit chr XV monosomy (Fig EV1E). In retrospect, our data suggest that Choi et al, (2004) may have studied cho2opi3 suppressors. We conclude that PC biosynthesis is essential in yeast. However, the requirement for PC can be overcome by adaptive evolution. Ultrastructure of PC-free yeast Electron microscopy for morphological examination of PC-free cells revealed that compared to WT and the cho2opi3 parent cultured with choline (Fig 2A and B), PC-free coS#3, S#4 and, to a lesser extent, coS#2 (Fig 2E, C and J) show accumulation of lipid droplets (LD). Quantitation of the area occupied by LD in 2D projection images shows a nearly 3-fold increase in coS#3 and S#4 compared to WT and parent (Appendix Fig S1B). Other salient features of PC-free cells include the "spikes" of ER often surrounding LD (Fig 2D), in agreement with LD being formed at and staying connected to the ER (Jacquier et al, 2011). In PC-free coS#3 and S#2, proliferation of the ER is apparent from protrusions in the nuclear envelope, adopting a "brass-knuckles" shape that occasionally pushes into the vacuole (Fig 2F, I and J). These structures are reminiscent of the nuclear envelope morphology of a temperature-sensitive acc1 mutant at the restrictive temperature (Schneiter et al, 1996). In coS#2 unidentified vacuolar structures accumulate at the limiting membrane of the star-shaped vacuole (Fig 2K). Some mitochondria have aberrant morphology with sheet-like cristae membranes, often detached from the inner boundary membrane (Fig 2G). Given the defects in mitochondrial structure, it is not surprising that cho2opi3 suppressors do not grow on the non-fermentable carbon source glycerol without choline (Appendix Fig S1C). Upon supplementing choline, the PC-free cells return to wild-type morphology after 3 doublings (Fig 2H and L), with smaller LD (Appendix Fig S1B) often found anchored to the vacuole, suggesting that removal of LD involves lipophagy (van Zutphen et al, 2014). Figure 2. Ultrastructure of PC-free yeast cells A–L. Wild type cultured in C− (A), cho2opi3 cultured in C+ (B), coS#4 (C, D), coS#3 (E-G) and coS#2 (I-K) cultured in C− were analysed by electron microscopy. In addition, coS#3 (H) and coS#2 (L) are shown after culture in C+ for 3 generations. The arrow heads (F, I, J) point to protrusions in the nuclear envelope. CW, cell wall; ER, endoplasmic reticulum; PM, plasma membrane; M, mitochondria; N, nucleus; V, vacuole; *, lipid droplet. Scale bars correspond to 200 nm (A, B, D, F, G) or 500 nm (C, E, H, I, J, K, L). Download figure Download PowerPoint Monosomy of chromosome XV or a point mutation in ACC1 is sufficient to suppress choline auxotrophy in a cho2opi3 mutant To investigate whether chr XV monosomy is sufficient to suppress choline auxotrophy, a 2n-1 cho2opi3 strain was constructed by counter selection against a conditionally stable copy of chr XV as described (Reid et al, 2008). Insertion of the GAL1 promoter and a URA3 marker adjacent to the centromere (CEN15) enabled CEN destabilization on galactose-containing medium, and counter selection against URA3 with 5-fluoroorotic acid (5-FOA), respectively. 5-FOA-induced loss of the destabilized copy of chr XV conferred uracil-auxotrophy while suppressing choline auxotrophy (Fig 3A), unequivocally demonstrating that chr XV monosomy rescues the choline auxotrophy of cho2opi3. In the absence of FOA, suppressors of choline auxotrophy appear more frequently with than without uracil present (Fig 3A), as expected based on probability theory. Engineered co S(2n-1) and evolved coS#3 and S#4 exhibit similar growth phenotypes in the presence or absence of choline and/or inositol (Fig 3B). Figure 3. Choline auxotrophy of cho2opi3 is suppressed by monosomy of chromosome XV or a point mutation in ACC1 A. Induction of chr XV loss in three independent diploid co/co clones containing a conditionally stable chr XV centromere (left panel), suppresses choline auxotrophy. After culture on solid YPGal, cell patches were replica-plated on SD plates with or without choline, uracil and 5-FOA, as indicated (right panel) and incubated at 30°C for 4 days. B. Growth of 10-fold serial diluted engineered co S(2n-1) and evolved co S#3 and S#4 on C+/− I+/− SD at 30°C for 4 days. C. Choline supplementation induces endoduplication of chr XV in aneuploid cho2opi3 suppressors. Growth phenotype on SD C− and C+ (4 days at 30°C) and absolute copy number of chr I, IV, VI, IX and XV as determined by qPCR and FACS after culturing co S#4 in SD C+ for the number of days indicated with daily passage to fresh medium at OD600 0.05. Representative data are shown with chromosome copy number presented as mean value from 2 assays using primers complementary to non-coding regions on the left and right arm of each chr, respectively, with the individual values indicated. D. Serial dilutions of the strains indicated were spotted on SD C+/− I+/− and incubated at 30°C for 4 days. Download figure Download PowerPoint Upon culture in SD C+, 2n-1 suppressors gradually lose the ability to grow in SD C− (and improve growth in SD C+) over a period of 6–10 days (Fig 3C, Appendix Fig S1D). This is accompanied by gain of a second copy of chr XV by endoduplication, in agreement with restoration of euploidy after removal of selection pressure (Reid et al, 2008; Chen et al, 2012). The N1446H mutation was introduced in Acc1 in the co background by CRISPR-Cas9. The engineered haploid co acc1N/H mutant recapitulates the growth phenotype of coS#2 (Fig 3D), proving that a single point mutation in ACC1 renders PC redundant. By crossing co acc1N/H to co, a co ACC1/acc1N/H heterozygous diploid was generated that shows intermediate growth in SD C−. PC-free yeast cells accumulate triacylglycerol and exhibit shortening of average acyl chain length Since Acc1 activity is directly linked to changes in lipid metabolism, we subjected the engineered suppressor strains, parent and WT to mass spectrometry-based shotgun lipidomics (Fig 4, Dataset EV1). After culture in choline-free medium, co acc1N/H and co S(2n-1) show an almost twofold increase in membrane lipid content compared to WT and parent strain, accompanied by 3- and 10-fold increases in triacylglycerol (TAG) in co acc1N/H and co S(2n-1), respectively, reflecting increased FA and glycerolipid synthesis in PC-free suppressors (Fig 4A). Supplementation of choline reduces the level of membrane lipids and TAG in co acc1N/H to and below WT level, respectively. In co S(2n-1), lipid content is also reduced by choline but stays above WT/parent levels (Fig 4A). Compared to TAG, the ergosterolester content shows a modest increase in the suppressors that in co S(2n-1) is not affected by choline (Fig 4A). Figure 4. The lipidome of PC-free cho2opi3 suppressors shows increased lipid content and shortening of average acyl chain length A. Membrane lipid and TAG content, and ergosterolester content (EE, inset) per OD600 unit of the yeast strains indicated after culture to mid-log phase in SD with or without 1 mM choline (C); *P < 0.05, **P < 0.01, unpaired two-tailed t-test of the indicated bar compared to the C+ condition. B. Membrane lipid class composition of classes contributing at least 1% of total membrane lipids of the indicated strains cultured to mid-log phase in SD C+/−, the inset shows CDP-DAG and the separate lyso(L)-phospholipids (lyso-PL). C. Fatty acyl chain profiles of the total lipid, the membrane glycerolipid (ML) and the TAG fraction of the indicated strains cultured to mid-log phase in SD C+/−, showing acyl chains that contribute at least 1% of total, with the C10-C14 acyl chains in the insets. D. PE molecular species profile (sum of carbon atoms in the acyl chains: sum of double bonds in the acyl chains) of the indicated strains cultured to mid-log phase in SD C+/−, showing species that contribute at least 2% of total PE. E, F. Percentage of molecular species containing more than 32 carbon atoms in both acyl chains (C34+C36) (E) and of saturated acyl chains (SFA) (F) in the membrane glycerolipids (ML) and the major membrane lipids, of the indicated strains cultured to mid-log phase in SD C+/−. Data information: All data were obtained by mass spectrometry and are presented as mean ± SD (n = 3 biological replicates); *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, unpaired two-tailed t-test of the indicated bar compared to the cho2opi3 parent unless indicated otherwise. Download figure Download PowerPoint Lipidomics analysis of the membrane lipid class distribution in co acc1N/H and co S(2n-1) cultured in SD C− showed that PE and PI take over as major membrane lipids in the absence of PC (Fig 4B). CDP-DAG and PS, the metabolic precursors of membrane lipids and PE, respectively, are depleted, reflecting upregulated lipid synthesis, in agreement with cho2opi3 mutants derepressing UASINO genes in the absence of choline (McGraw & Henry, 1989; Boumann et al, 2006). Derepression of the INO1 gene, the UASINO gene with t
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