Novel proteins unique to either transverse tubules (TS28) or the sarcolemma (SL50) have been identified and characterized, and their in situ distribution in rabbit skeletal muscle has been determined using monoclonal antibodies. TS28, defined by mAb IXE112, was shown to have an apparent relative molecular mass of 28,000 D. Biochemical studies showed that TS28 is a minor membrane protein in isolated transverse tubular vesicles. Immunofluorescence and immunoelectron microscopical studies showed that TS28 is localized to the transverse tubules and in some subsarcolemmal vesicles possibly corresponding to the subgroup of caveolae connecting the transverse tubules with the sarcolemma. In contrast, TS28 is absent from the lateral portion of the sarcolemma. Immunofluorescence studies also showed that TS28 is more densely distributed in type II (fast) than in type I (slow) myofibers. Although TS28 and the 1,4-dihydropyridine receptor are both localized to transverse tubules and subsarcolemmal vesicles, TS28 is not a wheat germ agglutinin (WGA)-binding glycoprotein and does not appear to copurify with the 1,4-dihydropyridine receptor after detergent solubilization of transverse tubular membranes. SL50, defined by mAb IVD31, was shown to have an apparent relative molecular mass of 50,000 D. Biochemical studies showed that SL50 is not related to the 52,000-D (beta subunit) of the dihydropyridine receptor but does bind to WGA-Sepharose. Immunofluorescence labeling imaged by standard and confocal microscopy showed that SL50 is associated with the sarcolemma but apparently absent from the transverse tubules. Immunofluorescence labeling also showed that the density of SL50 in type II (fast) myofibers is indistinguishable from that of type I (slow) myofibers. The functions of TS28 and SL50 are presently unknown. However, the distinct distribution of TS28 to the transverse tubules and subsarcolemmal vesicles as determined by immunocytochemical labeling suggests that TS28 may be directly involved in excitation-contraction coupling. Our results demonstrate that, although transverse tubules are continuous with the sarcolemma, each of these membranes contain one or more unique proteins, thus supporting the idea that they each have a distinct protein composition.

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