Submitochondrial localization and targeting of mitochondrial glycerol-3-phosphate acyltransferase 1

MAGALÍ PELLON-MAISON; MAURO A. MONTANARO; ROSALIND A. COLEMAN; MARÍA R. GONZÁLEZ-BARÓ

Aspen, Colorado, EEUU,

Congreso; Kern Aspen Lipid Conference: Developments in the Pathogenesis of Obesity and the Metabolic Syndrome; 2006

Given Institute

Submitochondrial localization and targeting of mitochondrial glycerol-3-phosphate acyltransferase 1. Magalí Pellon-Maison1, Mauro A. Montanaro1, Rosalind A. Coleman2 and María R. Gonzalez-Baró1. 1Instituto de Investigaciones Bioquímicas de La Plata (CONICET-UNLP), 60 y 120, 1900- La Plata, Argentina and 2Departments of Nutrition and Pediatrics, University of North Carolina, CB 7461, Chapel Hill NC 27599     ABSTRACT             The onset of insulin resistance in obese and elderly patients has been associated with mitochondrial dysfunction and the consequent accumulation of lipids and fatty acids in the cytosol, especially in muscle and liver. In the liver, mitochondria play an important role in lipid synthesis. The first and committed step in phospholipid and triacylglycerol synthesis  is catalyzed by glycerol 3-phosphate acyltransferase (GPAT). Of the three known GPAT isoforms, two are reported to be located in the mitochondria (mtGPAT1 and mtGPAT2). mtGPAT1 is the unique isoform in rat liver mitochondria and is an outer-membrane protein. Recent reports have linked acyl-CoA metabolism in the mitochondria as a balance between the glycerolipid synthesis and fatty acid beta-oxidation, via the reciprocal regulation of mtGPAT1 and carnitine-palmitoyl transferase 1 (CPT1). CPT1 is located in mitochondrial contact sites (CS), dynamic structures reported to be involved in the incorporation of proteins and phospholipids into the mitochondria. We decided to determine whether mtGPAT1 was also located in mitochondrial contact sites, and to correlate protein localization and enzymatic function. With Percoll-purified rat liver mitochondria, we found that the localization of mtGPAT1 protein was not consistent with the enzymatic activity. The protein was detected by western blot mainly in a membrane fraction associated with mitochondria (P10), whereas higher specific activity was found in pure mitochondria. P10 is a membrane fraction suggested to be enriched in mitochondrial contact sites. Thus, we hypothesized that  mtGPAT1 would be localized in this CS subfraction. CS from crude mitochondria had both ER and mitochondrial components demonstrated by ER- and mitochondria-marker-enzyme activities. CS isolated from pure mitochondria expressed only mitochondrial marker enzyme activities and very little mtGPAT1 protein. The presence of mtGPAT1 in CS associated with the ER is consistent with studies of two other enzymes reported to localize in CS: CPT1 and long-chain acyl-CoA synthetase-1. These three enzymes have in common one of their substrates, the long-chain acyl-CoAs. In order to determine whether mtGPAT1 is first targeted to ER or to mitochondria, or whether both subcellular fractions are required to target the enzyme, recombinant protein was synthesized in vitro and its incorporation into crude and pure mitochondria was assayed. mtGPAT1 was rapidly incorporated into both pure and crude mitochondrial fractions, whereas it was not incorporated at all into the microsomal fraction. The incorporation process was ATP-driven. Lack of removal by a chaotropic agent showed that mtGPAT1 entered the mitochondrial membrane during this process. These results indicate that mtGPAT1 is first targeted to mitochondria and then localizes to CS that may have physical interactions with both ER and the mitochondrial outer membrane.