Biological membranes are composed of a wide variety of phospholipids with respect to their hydrophilic head moieties and hydrophobic acyl chains. The generation and maintenance of the diversity in phospholipid molecular species are accomplished by the process of synthesis, degradation, and remodeling of each phospholipid. The fatty acyl chains in phospholipids are considered to be remodeled in cells to maintain membrane homeostasis. Despite its potential importance for the structure and function of the biological membranes, the molecular mechanisms underlying the remodeling of acyl chains in phospholipids are poorly understood

Phosphatidylethanolamine (PE) is one of the abundant phospholipids, comprising 20 to 30% of total phospholipids in most eukaryotic cells. Due to its small polar head group, PE tends to form non-bilayer hexagonal-II structure under physiological condition. This property has been considered to affect dynamics of biological membrane, for instance, membrane fission and fusion. In Saccharomyces cerevisiae, PE is biosynthesized through two pathways. In de novo pathway, phosphatidylserine (PS) is decarboxylated to PE by Psdlp or Psd2p. In CDP-ethanolamine branch of the Kennedy pathway, PE is synthesized from exogenous ethanolamine or ethanolamine formed endogenously through a lipid turnover process. In S. cerevisiae, PE is essential for growth and deletion of both PSD1 and PSD2 is lethal without a PE supply through the Kennedy pathway

Kakihara in our laboratory constructed a yeast mutant, TKY12Ga, in which both PSD1 and PSD2 were deleted and the promoter of ECT1 encoding a key enzyme in PE on the Kennedy pathway, was replaced with the galactose-inducible and glucose-repressible GALl promoter. In a medium containing galactose as a carbon source, the expression of ECTl was induced and the mutant grew using PE synthesized via the Kennedy pathway in the presence of ethanolamine. In contrast, the expression of ECTl was repressed and cell growth was arrested in a glucose-containing medium, and PE content was reduced to 1%. However, when didecanoyl PE (diC10PE) was added to the medium containing glucose, TKY12Ga grew despite the inability to synthesize PE. Since a 10-carbon-fatty acyl residue appears to be too short to maintain the structure and function of the biological membrane,diC10PE was assumed to be remodeled to the ones containing acyl chains of normal length to support the growth of the mutant.

This study is aimed at elucidating the mechanism of PE remodeling and the physiological role of PE in S. cerevisiae.

Incorporation and remodelina of PE with short fatty acyl chains in Yeast

It was reported that Lem3p/Ros3p and two P-type ATPase, Dnflp and Dnf2p were involved in the inward-directed trans-bilayer transport of fluorescent-labeled PE and lyso-PE at the plasma membrane in yeast. The involvement of these three proteins in the uptake of PE with short acyl chains was examined. Genes encoding these proteins were deleted in TKY12Ga and the growth of the null mutants in SD medium containing diC10PE was examined. Deletion of DNF2 caused slight growth impairment in the PE-containing medium, while deletion of DNF1 did not. However, deletion of both DNF1 and DNF2 conferred severer growth defect, suggesting their redundant functions in the uptake of diC10PE. In contrast, deletion of LEM3 caused most significant growth impairment. These results indicate that the growth of TKY12Ga in SD medium containing diC10PE requires uptake of PE with short acyl chains or its metabolites from the medium and that Lem3p, Dnflp and Dnf2p are involved in this process.

The metabolism of diC10PE, in which ethanolamine moiety was labeled with deuterium, was analyzed using ESI-MS/MS. Deuterium-labeled diC10PE was synthesized by the base exchange transphosphatidylation by phospholipase D from the didecanoyl PC and ethanol-1,1,2,2-d4-amine. TKY12Ga was pulse-labeled with deuterium-labeled diC10PE for 15 min, and chased with non-labeled diC10PE for 15, 30, 60 and 180 min. Then, the molecular species profile of PE was determined by the neutral loss scan of m/z 145 to detect PEs containing ethanol-1,1,2,2,-d4-amine phosphate headgroup in positive ion mode. After 15 min incubation with deuterium-labeled diC10PE, a portion of the deuterium-labeled diC10PE had been remodeled, as one fatty acyl chain was replaced with a longer monounsaturated C16:1 and C18:1 acyl chains. These remodeling intermediates decreased quickly during chased incubation. In addition, PEs in which two fatty acyl chains were replaced with C16:1 or C18:1 acyl chain (16:1-16:1-PE, 16:1-18:1-PE and 18:1-18:1-PE) were also detected after 15 min incubation with deuterium-labeled diC10PE. These results suggest that PE with short acyl chains was remodeled to PEs containing normal acyl chains. Analysis of structures of the remodeling intermediates in detail using product ion scan by MS/MS showed that the majority of the remodeling intermediates contained C16:1 or C18:1 acyl chain at sn-2 position. These results suggest that diC10PE was first remodeled at sn-2 position and then at sn-1 position. The water-soluble metabolites of diC10PE, which were partitioned in the aqueous phase were also extracted, and their molecular profiles were analyzed. After a 30-min incubation with deuterium-labeled diC10PE, the peak corresponding to monoC10PE was observed, suggesting that lyso-10:0-PE was also an intermediate of remodeling of diC10PE.

Quantification of diC10PE and decanoic acid in the whole culture and the culture supernatant using LC-MS showed that amount of decanoic acid in both of the whole culture and the culture supernatant was increased during the incubation of TKY12Ga with diC10PE, accompanied by the decrease of diC10PE. The amount of released decanoic acid in the culture supernatant was approximately twofold of the consumed diC10PE. These results suggest that most of C10 fatty acid cleaved from diC10PE were excreted into culture medium and it could be concluded that yeast remodeled diC10PE by removing C10 fatty acid and reacylating with normal length acyl chain, but not by elongating the acyl chains.

In vitro remodeling reaction experiment was performed using cell lysate of TKY12Ga with deuterium-labeled diC10PE and palmitoleoyl-CoA. After 15 min incubation, a small portion of deuterium-labeled 10:0-16:1-PE was detected, and its amount was increased with the prolonged incubation. In addition, deuterium-labeled 10:0-18:1-PE, 16:1-16:1-PE and 16:1-18:1-PE were detected after 60 min incubation. These results suggest the PE remodeling reaction can be reconstituted in vitro.

Analysis of genes involved in remodeling of diC10PE

Presence of monoC10PE, 10:0-16:1-PE, and 10:0-18:1-PE as remodeling intermediates of the diC10PE suggests that the remodeling can be dissected into at least two reactions, the cleavage of acyl chains by phospholipase activities and reacylation by acyltransferase activities. Yeast genome contains five genes encoding phospholipase B, PLB1, PLB2, PLB3, SPO1, and NTE1, and a gene, YOR022c, homologous to a gene encoding bovine phospholipase Al. A mutant was constructed by deletion of these six genes in TKY12Ga, and its growth in SD medium containing diC10PE and remodeling of deuterium-labeled diC10PE in vivo and in vitro were examined. No defect of the sextuple mutant in growth and remodeling was observed. In addition, monoC10PE was detected in the cell extract when this mutant was incubated with diC10PE. These data suggested that other enzyme(s) is/are involved in the removal of acyl chains in diC10PE.

Alelp was reported to have an acyltransferase activity against the sn-2 position in various lysophospholipids. Null mutation in ALE1 is synthetically lethal with the deletion of SLC1, which encodes a lyso-PA acyltransferase. Null mutants of ALEI or SLC1 under TKY12Ga background were constructed. Their growth in SD medium containing diC10PE and their remodeling of deuterium-labeled diC10PE in vivo and in vitro were examined. Both mutants were partially impaired in the growth in SD medium containing diC10PE, and also impaired in remodeling of deuterium-labeled diC10PE in vivo and in vitro, suggesting that Alelp and Slclp were involved in the remodeling of diC10PE. To identity genes involved in the remodeling at sn-1 position, same experiments were performed using null mutants of GAT1 and GAT2, which encode glycerol-3-phosphate (Gly-3-P) acyltransferase that were reported to transfer fatty acid from acyl-CoA to the sn-1 position of Gly-3-P.However, no growth and remodeling defect was observed in these two mutants.

Analysis of mutant defective in utilization of exogenous PE

To find enzymes involved in PE remodeling reaction, analysis of mutants defective in utilization of exogenous PE will be a useful way. Kakihara mutagenized TKY12Ga with mutagen ethyl methanesulfonate (EMS) and isolated 33 mutants that have defects in growth in SD medium containing diC10PE. M25 is one of these mutants. Analysis of M25 showed that introduction of a plasmid containing the DCP1 gene to M25 complemented its growth defect in SD medium containing diC10PE. DCP1 gene encodes an mRNA decapping enzyme on the major pathway of mRNA decay in S. cerevisiae. Nucleotide sequence analysis showed a point mutation within the highly conserved region of DCP1 gene in M25, suggesting the possibility that Dcplp is involved in the mRNA degradation process of the transcript(s) that is/are responsible for the utilization of exogenous PE in yeast.

Requirement of de novo synthesis of PE and PC for yeast sporulation

During the sporulation process of S. cerevisiae, meiotic progression is accompanied by de novo formation of the prospore membrane inside the cell. However, it remains to be determined whether de novo synthesis of certain species of lipids is necessary for spore formation. Requirements of PE and another major phospholipid, phosphotidylcholine (PC), in the sporulation of yeast were examined. When TKY12G, diploid of TKY12Ga, was incubated in spore medium (SM) with exogenous ethanolamine, sporulation efficiency was approximately 21%. However, no sporulation was observed in the absence of exogenous ethanolamine, suggesting that de novo PE synthesis was essential to yeast sporulation. On the other hand, when KEY503, a strain that synthesizes PC only via the CDP-choline branch of the Kennedy pathway and is therefore auxotrophic for choline because of the deletion of both PEM1 and PEM2 genes, was incubated in SM in the presence of choline, spore formation was efficient. In contrast, spore formation efficiency of KEY503 was decreased in the absence of choline, but the effect was less drastic than that of repression of PE synthesis. These results suggest that de novo synthesis of PC was also required for efficient spore formation by yeast.

Conclusion

In this study, the incorporation and metabolism of diClOPE in yeast was analyzed. Results suggest that diCl0PE was remodeled to PEs containing normal acyl chains and Alelp and Slclp were involved in this reaction. This system, in which the remodeling process of stable isotope-labeled phospholipids containing short acyl chains in yeast is monitored by ESI-MS/MS, will be applicable to the analysis of each reaction in the acyl chain remodeling of various phospholipid and provide a valuable tool to study the transport and metabolism of PE in yeast.

真核細胞の生体膜の主要構成成分であるリン脂質には、そのアシル鎖の炭素鎖長や不飽和度の点で多様な分子種が存在する。リン脂質のアシル鎖の交換を行うリモデリングは、生体膜の恒常性の維持や情報伝達等に重要な役割を果たしていると考えられているが、その機構や意義は不明である。本論文は、酵母Saccharomyces cerevisiaeにおいて生育に必須な主要リン脂質の1つであるphosphatidylethanolamine (PE)のリモデリングのメカニズムとその生理的意義を明らかにすることを目的とし、PE合成を培地の炭素源で制御できるTKY12G株においてPE合成を抑制した場合にも培地に炭素鎖長10のアシル鎖を持つ短鎖PEを添加することによりその生育が支持されることを利用して、PEのアシル鎖のリモデリングについて解析を行ったものである。第1章ではこれら研究の背景と目的が序論として述べられている。

第2章では、TKY12Ga株において、PE合成を抑制した場合に短鎖PEで生育が支持されるためには、短鎖PEが培地から細胞内に取り込まれる必要があること、この取り込みには、Lem3p、Dnflp、Dnf2pが関与することを示している。更に親水性頭部を安定同位体標識した短鎖PEを酵母に取り込ませてその代謝変換を質量分析装置により解析した結果、短鎖PEはリモデリングを受け、16:1や18:1といった正常な長さのアシル基を持つPEへと変換されていること、そのリモデリングは優先的にsn-2位から始まることを強く示唆している。

第3章では、TKY12Ga株において、ホスホリパーゼをコードするSPO1,PLB1,.PLB2、PLB3、NTE1、YOR022c遺伝子を破壊しても、短鎖PEを正常に脱アシル化できることを示唆している。さらに本章では、リゾリン脂質のsn-2位に対するアシルトランスフェラーゼをコードするALE1とSLC1遺伝子の破壊株は、短鎖PEのsn-2位のリモデリングに欠損をもつこと、それに対して、グリセロール3リン酸とジヒドロキシアセトンリン酸のSn-1位に対するアシルトランスフェラーゼをコードするGAT1とGAT2遺伝子の破壊株では、短鎖PEが正常にリモデリングされることが示唆されている。

第4章では短鎖PEを含むグルコース培地で生育できない変異株、すなわち短鎖PEを取り込み、利用できない変異株の分離とM25株はmRNA decapping enzymeをコードするDCP1に変異を持つことを明らかにしている。

第5章ではTKY12G株においてPEの生合成を抑制した場合に酵母の胞子形成に重篤な欠損が見られたことから、酵母の胞子形成にPE合成が必要であることを占めした。

以上、本研究は安定同位体標識した短鎖PEを用いて酵母における膜リン脂質PEのリモデリングの存在を示し、さらにこの過程におけるアシルトランスフェラーゼの役割を明らかにしたものであり、学術上貢献するところが少なくない。よって審査委員一同は本論文が博士(農学)の学位論文として価値あるものと認めた。