The GLUT4 glucose transporter and the α2 subunit of the Na+,K+‐ATPase do not localize to the same intracellular vesicles in rat skeletal muscle

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Abstract
The GLUT4 glucose transporter and the α 2 subunit of the Na+,K+-ATPase of rat skeletal muscle are two proteins which redistribute from intracellular membranes to plasma membranes following in vivo insulin stimulation. Here we show that although both proteins co-segregate after subcellular fractionation of unstimulated rat hindlimb muscles, they do not share the same intracellular residence inside the muscle fibre. By immunogold single- and double-labeling on ultrathin muscle cryosections with specific antibodies, the GLUT4 glucose transporter and the Na+,K+-ATPase α 2 subunit were observed on different vesicular structures within the cell. GLUT4 was detected on subsarcolemmal and perinuclear membranes, and at the junction between myofibrillar A and I bands where triads are localized. The α 2 subunit of the Na+,K+-ATPase was observed at the plasma membrane and in distinct subsarcolemmal vesicles and intermyofibrillar membranes. Quantitative analysis of double-labeling of GLUT4 and Na+,K+-ATPase α 2 subunit revealed that less than 6% of the two proteins co-localize in the same continuous vesicular structures. The differential intracellular localization of the two proteins was further confirmed by immunopurification of GLUT4-containing membranes from muscle homogenates, in which the α 2 subunit of the Na+,K+-ATPase was found only at the same extent as the α 1 subunit of the enzyme, a protein exclusively present at the plasma membrane.