Cloning and functional characterization of a novel mitochondrial N-ethylmaleimide-sensitive glycerol-3-phosphate acyltransferase (GPAT2).
Glycerol-3-phosphate acyltransferase (GPAT) catalyzes the initial and rate-limiting step in glycerolipid synthesis. Several mammalian GPAT activities have been recognized, including N-ethylmaleimide (NEM)-sensitive isoforms in microsomes and mitochondria and an NEM-resistant form in mitochondrial outer membrane (GPAT1). We have now cloned a second mitochondrial isoform, GPAT2 from mouse testis. The open-reading frame encodes a protein of 798 amino acids with a calculated mass of 88.8kDa and 27% amino acid identity to GPAT1. Testis mRNA expression was 50-fold higher than in liver or brown adipose tissue, but the specific activity of NEM-sensitive GPAT in testis mitochondria was similar to that in liver. When Cos-7 cells were transiently transfected with GPAT2, NEM-sensitive GPAT activity increased 30%. Confocal microscopy confirmed a mitochondrial location. Incubation of GPAT2-transfected Cos-7 cells with trace (3 microM; 0.25 microCi) [1-(14)C]oleate for 6h increased incorporation of [(14)C]oleate into TAG 84%. In contrast, incorporation into phospholipid species was lower than in control cells. Although a polyclonal antibody raised against full-length GPAT1 detected an approximately 89-kDa band in liver and testis from GPAT1 null mice and both 89- and 80-kDa bands in BAT from the knockout animals, the GPAT2 protein expressed in Cos-7 cells was only 80 kDa. In vitro translation showed a single product of 89 kDa. Unlike GPAT1, GPAT2 mRNA abundance in liver was not altered by fasting or refeeding. GPAT2 is likely to have a specialized function in testis.
Wang
Shuli
S
Department of Nutrition, University of North Carolina-Chapel Hill, Chapel Hill, NC 27599, USA.
Lee
Douglas P
DP
Gong
Nan
N
Schwerbrock
Nicole M J
NM
Mashek
Douglas G
DG
Gonzalez-Baró
Maria R
MR
Stapleton
Cliona
C
Li
Lei O
LO
Lewin
Tal M
TM
Coleman
Rosalind A
RA
D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular / Q000379:methods; D004789:Enzyme Activation; D005992:Glycerol-3-Phosphate O-Acyltransferase / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism*; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D008928:Mitochondria / Q000201:enzymology*; D009928:Organ Specificity; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013737:Testis / Q000201:enzymology*; D014018:Tissue Distribution
D011994:Recombinant Proteins; D005992:Glycerol-3-Phosphate O-Acyltransferase
J Lipid Res. 1967 Sep;8(5):447-55
4860577
J Biol Chem. 2004 Apr 2;279(14):13488-95
14724270
Cell. 1989 Feb 10;56(3):335-44
2644044
J Biol Chem. 1991 Dec 15;266(35):23834-9
1721057
Endocr Rev. 1994 Jun;15(3):391-407
8076589
J Biol Chem. 1994 Nov 4;269(44):27209-15
7961630
J Biol Chem. 1996 Jul 12;271(28):16644-51
8663220
Biochim Biophys Acta. 1997 Sep 4;1348(1-2):10-6
9370311
J Bacteriol. 1998 Mar;180(6):1425-30
9515909
Biochemistry. 1999 May 4;38(18):5764-71
10231527
Biochim Biophys Acta. 1999 Aug 18;1439(3):415-23
10446428
Can J Biochem Physiol. 1959 Aug;37(8):911-7
13671378
J Biol Chem. 2004 Dec 31;279(53):55866-74
15485873
J Biol Chem. 2005 Jul 8;280(27):25629-36
15878874
Cell Metab. 2005 Jul;2(1):55-65
16054099
Proc Natl Acad Sci U S A. 2006 Dec 26;103(52):19695-700
17170135
Mol Cell Biochem. 2007 Mar;297(1-2):41-51
17013544
J Biol Chem. 2004 Jul 23;279(30):31727-34
15152008
J Biol Chem. 1999 Dec 3;274(49):34728-34
10574940
J Steroid Biochem Mol Biol. 2001 Jan-Mar;76(1-5):23-30
11384860
J Biol Chem. 2001 Jul 6;276(27):24674-9
11319232
J Biol Chem. 2001 Nov 16;276(46):43182-8
11557771
Arch Biochem Biophys. 2001 Dec 1;396(1):119-27
11716470
In Silico Biol. 2002;2(1):S1-15
11808873
Mol Cell Biol. 2002 Dec;22(23):8204-14
12417724
J Biol Chem. 2002 Nov 15;277(46):43918-23
12205087
Prog Lipid Res. 2004 Mar;43(2):134-76
14654091
Biochem J. 1980 Jul 15;190(1):183-9
6255941