36

Noda Institute for Scientific Research GRANT
2010 Research Grant

Maintenance of membrane homeostasis by the remodeling ofacyl residues in phospholipids
Ryouichi Fukuda
Department of Biotechnology, The University of Tokyo

Research aims
Biological membranes are composed of a wide variety ofphospholipids diverse in their hydrophilic head moieties
and hydrophobic acyl residues. The remodeling of acyl residues in phospholipids contributes to the generation of thediversity and the variation of phospholipid molecular species. In addition, improper or oxidized acyl residues inphospholipid molecules are supposed to be exchanged withappropriate ones by remodeling reaction to maintain membrane homeostasis. In higher eukaryotic cells, polyunsaturated fatty acids are introduced into phospholipids by remodeling reaction. Despite the importance of the remodeling of phospholipid acyl residues for the structure and function of biological membranes, its molecular mechanisms remain to be established. Recently, several lysophospholipid
acyltransferases belonging to the membrane bound O-acyltransferase (MBOAT) family were reported to be involvedin the remodeling of acyl residues at sn-2 position of various phospholipids.1)
In order to address the mechanism of metabolism ofphospholipids, it is effective to establish a system, in which
an exogenous phospholipid or a phospholipid analog is incorporated into the cells, and to monitor its metabolism inthe cells. We constructed a system to analyze the remodeling of acyl residues of phosphatidylcholine (PC), one of themost abundant and essential phospholipids, in Saccharomyces cerevisiae. In this system, exogenous PC containing
short acyl residues, which are more accessible to theplasma membrane of yeast cells due to their higher solubility in water than phospholipids containing longer acyl residues, is incorporated into yeast mutants defective in PC
synthesis, and the remodeling is evaluated by observing thegrowth of the mutants and more directly by analyzing themetabolism of incorporated phospholipids by mass spectrometry.2), 3) In this study, we constructed a system to evaluate the remodeling of phosphatidylethanolamine (PE), an
essential phospholipid with some unique functions, and analyzed the mechanism of the remodeling at its sn-2 position. In addition, we synthesized new PC analogs and constructed a system to specifically analyze the introduction ofacyl residues at sn-1 position.
MethodsS. cerevisiae strains TKY12Ga (MATa his3 leu2 trp1

ade2 ura3 psd1D::kanMX4 psd2D::LEU2 PGAL1ECT1::HIS3) and PCY12G (MATa his3 leu2 trp1 ade2
ura3 pem1::HIS3 pem2::hph PGAL1-CCT1::nat1) were derived from the strain W3031A (MATa his3 leu2 trp1 ade2ura3).
Phospholipids were extracted by the method of Brighand Dyer. MS/MS spectra were obtained on API3000 triple
quadrupole instrument (Applied Biosystem). Phospholipids
were dissolved in acetonitrile/methanol/water (4 : 4 : 1)
containing 0.1% ammonium formate and infused into ESI
source with a Harvard syringe pump at the flow rate of
10 ml/min. The ion spray voltage was set at 5.0 kV for positive scans and −3.8 kV for negative scans. Experiments
were conducted with nitrogen as a collision gas and collision energy of 25~60 Vdc. For each spectrum, 100~200
scans were summed.
Results
Remodeling of PE containing short acyl residues
We constructed a yeast strain TKY12Ga, the endogenous
PE synthesis of which was controllable, by deletion of both
PSD1 and PSD2 encoding phosphatidylserine (PS) decarboxylases and replacement of the promoter of chromosomal ECT1 encoding CTP:phosphoethanolamine cytidylyltransferase with the GAL1 promoter.4) In a glucose-containing medium, PE synthesis in TKY12G was repressed
and its growth was arrested. However, addition of PE containing decanoyl residues (diC10PE) supported the growthof this strain on glucose.
We synthesized diC10PE, in which an ethanolamine
moiety was labeled with deuterium, to analyze its metabolism. TKY12Ga was incubated with deuterium-labeled
diC10PE. Lipids were extracted and subjected to electrospray ionization tandem mass spectrometry (ESI-MS/MS)
analysis.5) Deuterium-labeled diC10PE was rapidly converted to PE species containing C16 or C18 acyl residues.
PE containing decanoic acid and C16 or C18 fatty acids asacyl residues, which are probably remodeling intermediates, were also detected. During growth in the presence of
diC10PE, a substantial amount of decanoic acid was released into the culture medium. These results suggest that
diC10PE was remodeled to PE species containing longer
acyl residues and used as membrane components (Fig. 1).
We further investigated the mechanism of the remodelingof diC10PE. We constructed deletion mutants of genes en-		37Fig. 1. Remodeling of PE containing short acyl residues.
PE containing decanoyl residues is remodeled to those with acyl residuesof physiological length and used in S. cerevisiae.

coding various acyltransferases in TKY12Ga background,and analyzed the remodeling of diC10PE in them. The deletion mutants for ALE1 encoding yeast MBOAT family acyltransferase and SLC1 encoding lysophosphatidic acid acyltransferase exhibited the growth defects in the medium
containing diC10PE. The analysis of the remodeling of
deuterium-labeled diC10PE revealed that the amount of theremodeling products and the remodeling intermediates, particularly the remodeling intermediates containing a longer
acyl residue at sn-2 position were decreased in these mutants. When deuterium-labeled diC10PE was incubatedwith the extract of the wild-type cells, the remodeling intermediates, i.e., the 10:0–16:1-PE and 10:0–18:1-PE weredetected. In contrast, the amount of these remodeling intermediates was decreased in the reaction with the extract ofthe deletion mutant of ALE1 or SLC1. These results imply
the involvement of Ale1p and Slc1p in the remodeling ofacyl residues at sn-2 position of diC10PE.

1-myristoyl-2-hexadecyl-PC (MHPC), and 1-hydroxyl2-hexadecyl-PC (2-alkyl-lysoPC, ALPC), in which a hexadecyl residue is placed at sn-2 position through an ether
linkage to prevent intramolecular transacylation.
We constructed a yeast strain PCY12G, the endogenous
PC synthesis of which can be controlled, by deletion ofboth PEM1 and PEM2 encoding PE methyltransferases andthe replacement of the promoter of chromosomal CCT1 encoding CTP:phosphocholine cytidylyltransferase with theGAL1 promoter. In a glucose-containing medium, the PC
synthesis of PCY12G was repressed and its growth was arrested. However, addition of ALPC to the medium supported its growth on glucose. Analysis of the metabolism ofALPC using ESI-MS/MS suggested that ALPC was converted to the PC species containing C16 or C18 acyl residue at its sn-1 position. These results imply that incorporated ALPC was acylated at sn-1 position and used insteadof endogenous PC.
Conclusion
Our results suggest the presence of a mechanism tomaintain the physiological length of acyl residues of PE inyeast. In addition, it was suggested that Ale1p and Slc1p
are involved in the remodeling of acyl residues at sn-2 position of PE. We also successfully synthesized PC analogs
and constructed a system to specifically evaluate the remodeling at sn-1 position of PC. This system will be applicable to the identification and characterization of enzymes
involved in the remodeling at sn-1 position of PC.

Remodeling of acyl residue at sn-1 position of PC

References

2-Acyl-lysophospholipids are less stable than 1-acyl-lysophospholipids, and acyl residues at sn-2 positions of
2-acyl-lysophospholipids produced by the reaction catalyzed by phospholipase A1 can be easily transacylated tosn-1 positions. This interferes with the accurate assessmentof deacylation and reacylation at sn-1 position of phospholipids. To analyze the mechanism of the remodeling at sn-1
position of PC, we synthesized three PC analogs as substrates, namely, 1-palmitoyl-2-hexadecyl-PC (PHPC),

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