SEDIMENTATION AND IMMUNOLOGICAL ANALYSES OF GLUT4 AND ALPHA-2-NA,K-ATPASE SUBUNIT-CONTAINING VESICLES FROM RAT SKELETAL-MUSCLE - EVIDENCE FOR SEGREGATION

In skeletal muscle insulin induces the translocation of both the GLUT4 glucose transporter and the alpha 2 subunit of the Na,K-ATPase from a n intracellular membrane (IM) compartment to the plasma membrane (PM). Fractionation studies of rat skeletal muscle using a discontinuous su crose gradient have indicated that the insulin-induced loss of both pr oteins occurs from a fraction containing intracellular membranes (LM) 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 th is study we report that membrane vesicles from the insulin-responsive IM fraction can in fact be separated on the basis of differences in th eir sedimentation velocities; immunoblot analyses of fractions collect ed from a sucrose velocity gradient revealed the presence of two separ ate peaks for GLUT4 and the alpha 2 subunit of the Na,K-ATPase. One of these peaks representing a fast sedimenting population of vesicles (w ith a sedimentation coefficient of 2697 +/- 57 S) reacted against anti bodies 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 similar to 32-fold in GLUT4 relative to the starting IM fr action,when the fractional protein content was taken into account. Imm unoprecipitation of GLUT4-containing vesicles from the insulin-sensiti ve IM fraction revealed that no immunoreactivity towards either the al pha 2 or the beta 1 subunits of the Na,K-ATPase could be observed, sig nifying that the insulin-responsive subunits of the Na,K-ATPase and GL UT4 were present in different membrane vesicles and that it was unlike ly, therefore, that the insulin-induced redistribution of these protei ns to the PM occurs from a common intracellular pool.