Mitochondrially-targeted bacterial phosphatidylethanolamine methyltransferase sustained phosphatidylcholine synthesis of a Saccharomyces cerevisiae Δpem1 Δpem2 double mutant without exogenous choline supply.
In eukaryotic cells, phospholipids are synthesized exclusively in the defined organelles specific for each phospholipid species. To explain the reason for this compartmental specificity in the case of phosphatidylcholine (PC) synthesis, we constructed and characterized a Saccharomyces cerevisiae strain that lacked endogenous phosphatidylethanolamine (PE) methyltransferases but had a recombinant PE methyltransferase from Acetobacter aceti, which was fused with a mitochondrial targeting signal from yeast Pet100p and a 3×HA epitope tag. This fusion protein, which we named as mitopmt, was determined to be localized to the mitochondria by fluorescence microscopy and subcellular fractionation. The expression of mitopmt suppressed the choline auxotrophy of a double deletion mutant of PEM1 and PEM2 (pem1Δpem2Δ) and enabled it to synthesize PC in the absence of choline. This growth suppression was observed even if the Kennedy pathway was inactivated by the repression of PCT1 encoding CTP:phosphocholine cytidylyltransferase, suggesting that PC synthesized in the mitochondria is distributed to other organelles without going through the salvage pathway. The pem1Δpem2Δ strain deleted for PSD1 encoding the mitochondrial phosphatidylserine decarboxylase was able to grow because of the expression of mitopmt in the presence of ethanolamine, implying that PE from other organelles, probably from the ER, was converted to PC by mitopmt. These results suggest that PC could move out of the mitochondria, and raise the possibility that its movement is not under strict directional limitations.
Department of Biotechnology, The University of Tokyo, 1-1-1, Yayoi, Bunkyo-ku, Tokyo, 113-8657, Japan.
Shingo
S
Kobayashi
Department of Biotechnology, The University of Tokyo, 1-1-1, Yayoi, Bunkyo-ku, Tokyo, 113-8657, Japan.
Aya
A
Mizuike
Department of Biotechnology, The University of Tokyo, 1-1-1, Yayoi, Bunkyo-ku, Tokyo, 113-8657, Japan.
Hiroyuki
H
Horiuchi
Department of Biotechnology, The University of Tokyo, 1-1-1, Yayoi, Bunkyo-ku, Tokyo, 113-8657, Japan.
Ryouichi
R
Fukuda
Department of Biotechnology, The University of Tokyo, 1-1-1, Yayoi, Bunkyo-ku, Tokyo, 113-8657, Japan. Electronic address: aaohta@isc.chubu.ac.jp.
Akinori
A
Ohta
D000091:Acetobacter / Q000737:chemistry / Q000201:enzymology; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D002262:Carboxy-Lyases / Q000172:deficiency / Q000235:genetics; D002794:Choline; D019916:Choline-Phosphate Cytidylyltransferase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D019856:Ethanolamine / Q000378:metabolism; D015966:Gene Expression Regulation, Fungal; D005816:Genetic Complementation Test; D007527:Isoenzymes / Q000172:deficiency / Q000235:genetics; D008928:Mitochondria / Q000201:enzymology / Q000235:genetics*; D024101:Mitochondrial Proteins / Q000172:deficiency / Q000235:genetics; D010713:Phosphatidylcholines / Q000096:biosynthesis*; D050918:Phosphatidylethanolamine N-Methyltransferase / Q000172:deficiency / Q000235:genetics*; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics*; D015398:Signal Transduction; D019076:Transgenes
D001426:Bacterial Proteins; D007527:Isoenzymes; D024101:Mitochondrial Proteins; D010713:Phosphatidylcholines; D034741:RNA, Small Interfering; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D019856:Ethanolamine; C494486:CHO2 protein, S cerevisiae; D050918:Phosphatidylethanolamine N-Methyltransferase; D019916:Choline-Phosphate Cytidylyltransferase; D002262:Carboxy-Lyases; C577156:Psd1 protein, S cerevisiae; D002794:Choline
Saccharomyces cerevisiae; mitochondria; phosphatidylcholine; phosphatidylethanolamine; transport