title: Sedimentation and immunological analyses of GLUT4 and alpha 2-Na,K-ATPase subunit-containing vesicles from rat skeletal muscle: evidence for segregation.
abstract: In skeletal muscle insulin induces the translocation of both the GLUT4 glucose transporter and the alpha 2 subunit of the Na,K-ATPase from an intracellular membrane (IM) compartment to the plasma membrane (PM). Fractionation studies of rat skeletal muscle using a discontinuous sucrose gradient have indicated that the insulin-induced loss of both proteins occurs from a fraction containing intracellular membranes (IM) of common density. This raises the possibility that both proteins may be colocalized in a single intracellular compartment or are present in separate membrane vesicles that are of similar buoyant density. In this study we report the membrane vesicles from the insulin-responsive IM fraction can in fact be separated on the basis of differences in their sedimentation velocities; immunoblot analyses of fractions collected from a sucrose velocity gradient revealed the presence of two separate peaks for GLUT4 and the alpha 2 subunit of the Na,K-ATPase. One of these peaks representing a fast sedimenting population of vesicles (with a sedimentation coefficient of 2697 +/- 57 S) reacted against antibodies to the alpha 2 subunit of the Na,K-ATPase, whereas, the second peak contained a population of much slower sedimenting vesicles (with a sedimentation coefficient of 209 +/- 4 S) were practically devoid of the alpha 2-subunit. By contrast, the slow sedimenting vesicles were enriched by approximately 32-fold in GLUT4 relative to the starting IM fraction when the fractional protein content was taken into account. Immunoprecipitation of GLUT4-containing vesicles from the insulin-sensitive IM fraction revealed that no immunoreactivity towards either the alpha 2 or the beta 1 subunits of the Na,K-ATPase could be observed, signifying that the insulin-responsive subunits of the Na,K-ATPase and GLUT4 were present in different membrane vesicles and that it was unlikely, therefore, that the insulin-induced redistribution of these proteins to the PM occurs from a common intracellular pool.
mesh_terms: D000818:Animals; D002451:Cell Compartmentation; D002462:Cell Membrane / Q000378:metabolism; D051275:Glucose Transporter Type 4; D007425:Intracellular Membranes / Q000378:metabolism; D008297:Male; D009004:Monosaccharide Transport Proteins / Q000378:metabolism*; D009124:Muscle Proteins; D009132:Muscles / Q000378:metabolism*; D011233:Precipitin Tests; D051381:Rats; D017207:Rats, Sprague-Dawley; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism*; D014461:Ultracentrifugation
chemical_list: D051275:Glucose Transporter Type 4; D009004:Monosaccharide Transport Proteins; D009124:Muscle Proteins; C495694:Slc2a4 protein, rat; D000254:Sodium-Potassium-Exchanging ATPase