On the biogenesis of lipid bodies in ancient eukaryotes: synthesis of triacylglycerols by a Toxoplasma DGAT1-related enzyme.
In mammalian cells, the main stored neutral lipids are triacylglycerol and cholesteryl esters, which are produced by two related enzymes, acyl-CoA:diacylglycerol acyltransferase (DGAT) and acyl-CoA:cholesterol acyltransferase (ACAT), respectively. Very little is known about the metabolism, intracellular storage and function of neutral lipids in many pathogenic lower eukaryotes. In this paper, we have characterized the activity of an important triacylglycerol synthetic enzyme in the protozoan Toxoplasma gondii. A full-length cDNA and gene encoding a T. gondii DGAT1-related enzyme were identified and designated TgDGAT1. The gene is composed of 15 exons and 14 introns, and encodes a protein with a predicted M(r) 63.5kDa, containing signature motifs characteristic of the DGAT1 family. The native protein migrates at 44kDa under reducing conditions. TgDGAT1 is an integral membrane protein localized to the parasite cortical and perinuclear endoplasmic reticulum, with the C-terminus oriented to the lumen of the organelle. When a Saccharomyces cerevisiae mutant strain lacking neutral lipid production is transformed with TgDGAT1 cDNA, a significant DGAT activity is reconstituted, resulting in triacylglycerol synthesis and biogenesis of cytosolic lipid inclusions, resembling lipid bodies in T. gondii. No production of steryl esters is observed upon TgDGAT1 expression in yeast. In contrast to human DGAT1 lacking fatty acid specificity, TgDGAT1 preferentially incorporates palmitate. Our results indicate that parasitic protozoa are also neutral lipid accumulators and illustrate the first example of the existence of a functional DGAT gene in an ancient eukaryote, demonstrating that diacylglycerol esterification is evolutionarily conserved.
Quittnat
Friederike
F
Department of Internal Medicine, Yale University School of Medicine, 808 LCI, 333 Cedar Street, P.O. Box 208022, New Haven, CT 06520, USA.
Nishikawa
Yoshifumi
Y
Stedman
Timothy T
TT
Voelker
Dennis R
DR
Choi
Jae-Yeon
JY
Zahn
Matthew M
MM
Murphy
Robert C
RC
Barkley
Robert M
RM
Pypaert
Marc
M
Joiner
Keith A
KA
Coppens
Isabelle
I
D000217:Acyltransferases / Q000235:genetics* / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D002460:Cell Line; D006224:Cricetinae; D003412:Cricetulus; D018076:DNA, Complementary; D051048:Diacylglycerol O-Acyltransferase; D004721:Endoplasmic Reticulum / Q000378:metabolism; D019143:Evolution, Molecular; D006801:Humans; D002479:Inclusion Bodies / Q000235:genetics / Q000502:physiology*; D050356:Lipid Metabolism; D008969:Molecular Sequence Data; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D014122:Toxoplasma / Q000201:enzymology* / Q000235:genetics; D014280:Triglycerides / Q000378:metabolism*
D018076:DNA, Complementary; D014280:Triglycerides; D000217:Acyltransferases; D051048:Diacylglycerol O-Acyltransferase