Mitochondrial glycerol-3-P acyltransferase 1 is most active in outer mitochondrial membrane but not in mitochondrial associated vesicles (MAV).
Glycerol 3-phosphate acyltransferase-1 (GPAT1), catalyzes the committed step in phospholipid and triacylglycerol synthesis. Because both GPAT1 and carnitine-palmitoyltransferase 1 are located on the outer mitochondrial membrane (OMM) it has been suggested that their reciprocal regulation controls acyl-CoA metabolism at the OMM. To determine whether GPAT1, like carnitine-palmitoyltransferase 1, is enriched in both mitochondrial contact sites and OMM, and to correlate protein location and enzymatic function, we used Percoll and sucrose gradient fractionation of rat liver to obtain submitochondrial fractions. Most GPAT1 protein was present in a vesicular membrane fraction associated with mitochondria (MAV) but GPAT specific activity in this fraction was low. In contrast, highest GPAT1 specific activity was present in purified mitochondria. Contact sites from crude mitochondria, which contained markers for both endoplasmic reticulum (ER) and mitochondria, also showed high expression of GPAT1 protein but low specific activity, whereas contact sites isolated from purified mitochondria lacked ER markers and expressed highly active GPAT1. To determine how GPAT1 is targeted to mitochondria, recombinant protein was synthesized in vitro and its incorporation into crude and purified mitochondria was assayed. GPAT1 was rapidly incorporated into mitochondria, but not into microsomes. Incorporation was ATP-driven, and lack of GPAT1 removal by alkali and a chaotropic agent showed that GPAT1 had become an integral membrane protein after incorporation. These results demonstrate that two pools of GPAT1 are present in rat liver mitochondria: an active one, located in OMM and a less active one, located in membranes (ER-contact sites and mitochondrial associated vesicles) associated with both mitochondria and ER.
Pellon-Maison
Magalí
M
Instituto de Investigaciones Bioquímicas de La Plata (CONICET-UNLP), 60 and 120, 1900 La Plata, Argentina.
Montanaro
Mauro A
MA
Coleman
Rosalind A
RA
Gonzalez-Baró
María R
MR
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D015415:Biomarkers / Q000378:metabolism; D002458:Cell Fractionation; D045424:Complex Mixtures; D004721:Endoplasmic Reticulum / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism; D005992:Glycerol-3-Phosphate O-Acyltransferase / Q000378:metabolism*; D008297:Male; D008930:Mitochondria, Liver / Q000187:drug effects / Q000201:enzymology* / Q000648:ultrastructure; D051336:Mitochondrial Membranes / Q000187:drug effects / Q000201:enzymology* / Q000648:ultrastructure; D021381:Protein Transport / Q000187:drug effects; D051381:Rats; D017208:Rats, Wistar; D013367:Submitochondrial Particles / Q000187:drug effects / Q000201:enzymology* / Q000648:ultrastructure
D015415:Biomarkers; D045424:Complex Mixtures; D000255:Adenosine Triphosphate; D005992:Glycerol-3-Phosphate O-Acyltransferase