7-II) was resistant to both alkaline and saline extractions. 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 usually 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 Nedocromil sodium lack of GPAT1 removal by alkali and a chaotropic agent showed that GPAT1 had become an integral membrane protein after incorporation. These results demonstrate Nedocromil sodium 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. at 4 C for 10 min, and the supernatant (postnuclear homogenate) was centrifuged for 10 min at 10,000at 4 C in a Sorvall refrigerated centrifuge. The resulting pellet was resuspended in 1 ml of buffer H/g of liver and homogenized in a Dounce tissue grinder with glass pestle to obtain the crude mitochondrial fraction. The supernatant was centrifuged for 1 h at 100,000(Beckman LE-80K ultracentrifuge, rotor 70.1 Ti) to obtain microsomes. All actions were performed at 4 C. 2.2. Purification of crude mitochondria Mitochondria were purified by a self-forming Percoll gradient centrifugation [17,18] (Fig. 1). Briefly, 1 ml of the crude mitochondria suspension was loaded in a tube made up of 9 ml 30% (v/v) Percoll in buffer H and centrifuged for 30 min at 95,000in a 70.1 Ti rotor, in a Beckman LE-80K ultracentrifuge. After centrifugation, the tubes contained two distinct bands separated by a clear zone. The lower brownish band was collected and diluted 4-fold in cooled buffer H. To remove the Percoll, the samples were centrifuged for 10 min at 6300and the pellet was washed twice with cold buffer H to obtain pure mitochondria. The whitish-upper layers of the self-forming Percoll gradient were collected and diluted 4-fold in cold buffer H. They were centrifuged at 10,000for 10 min, and the resulting pellet was washed twice with cold buffer H to obtain the mitochondrial associated vesicle (MAV) fraction. The supernatant of this centrifugation was centrifuged at 100,000for 1 h to obtain the MAM fraction. Open in a separate window Fig. 1 Submitochondrial fractionation and purification of rat-liver mitochondria. Rat liver crude mitochondrial fraction (pellet from a 10,000centrifugation, 10 min) was further purified by a 30% self-forming Percoll gradient. Two bands were separated, an upper band, which was separated into mitochondrial associated membranes (MAM) and mitochondrial associated vesicles (MAV), and a lower band corresponding to pure mitochondria. Crude and pure mitochondria were further fractionated in a discontinuous sucrose gradient. Outer mitochondrial membrane (OMM), inner mitochondrial membrane (IMM) and contact sites (ER-CS) were obtained from crude mitochondria. Outer membrane (OMMp), inner membrane (IMMp) and contact sites (CSp) were obtained from pure mitochondria. 2.3. Subfractionation of rat liver mitochondria To obtain submitochondrial fractions from either crude or pure mitochondria, we modified a method described previously [15,19]. The mitochondrial pellet was resuspended in a hypotonic 10 mM potassium phosphate buffer pH 7.4 (50 mg of protein/ml) and incubated on ice Cav2 for 20 min before adding 60% (w/v) sucrose. After a 20 min incubation on ice, the suspension was sonicated for three periods of 30 s, during a period of 3 min with constant cooling on ice in a Branson S-450A sonicator. The resulting suspension was centrifuged at 6300to remove debris, and Nedocromil sodium the supernatant was layered on top of a discontinuous sucrose gradient [19] (1.2 ml each of 51.3%, 37.7%, and 25.2% sucrose in 10 mM potassium phosphate pH 7.4) and centrifuged for 60 min at 4 C at 121,000(Beckman LE-80K ultracentrifuge, SW 60 rotor). The outer membrane fraction (OMM) was recovered at the 25.2%/37.7% interface, and the contact-site fraction (CS) was recovered at the 37.7%/51.2% interface (Fig. 1). These fractions were washed by diluting them 10-fold with 10 mM potassium phosphate buffer pH 7.4 and centrifuging at 180,000(Beckman LE-80K ultracentrifuge, 70.1 Ti rotor). The inner mitochondrial fraction (IMM) was collected from the pellet of the tube. 2.4. Enzyme assays.