Sedimentation and immunological analyses of GLUT4 and α2‐Na,K‐ATPase subunit‐containing vesicles from rat skeletal muscle: evidence for segregation

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Abstract
In skeletal muscle insulin induces the translocation of both the GLUT4 glucose transporter and the α2 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 that 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 α2 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 α2 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 α2‐subunit. By contrast, the slow sedimenting vesicles were enriched by ∼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 α2 or the β1 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.