SMALL, MEMBRANE-BOUND, ALTERNATIVELY SPLICED FORMS OF ANKYRIN-1 ASSOCIATED WITH THE SARCOPLASMIC-RETICULUM OF MAMMALIAN SKELETAL-MUSCLE

We have recently found that the erythroid ankyrin gene, Ank1, expresse s isoforms in mouse skeletal muscle, several of which share COOH-termi nal sequence with previously known Ank1 isoforms but have a novel, hig hly hydrophobic 72-amino acid segment at their NH2 termini. Here, thro ugh the use of domain-specific peptide antibodies, we report the prese nce of the small ankyrins in rat and rabbit skeletal muscle and demons trate their selective association with the sarcoplasmic reticulum. In frozen sections of rat skeletal muscle, antibodies to the spectrin-bin ding domain (anti-p65) react only with a 210-kD Ank1 and label the sar colemma and nuclei, while antibodies to the COOH terminus of the small ankyrin (anti-p6) react with peptides of 20 to 26 kD on immunoblots a nd decorate the myoplasm in a reticular pattern. Mice homozygous for t he normoblastosis mutation (gene symbol nb) are deficient in the 210-k D ankyrin but contain normal levels of the small ankyrins in the myopl asm. In nb/nb skeletal muscle, anti-p65 label is absent from the sarco lemma, whereas anti-p6 label shows the same distribution as in control skeletal muscle. In normal skeletal muscle of the rat, anti-p6 decora tes Z lines, as defined by antidesmin distribution, and is also presen t at M lines where it surrounds the thick myosin filaments. Immunoblot s of the proteins isolated with rabbit sarcoplasmic reticulum indicate that the small ankyrins are highly enriched in this fraction. When ex pressed in transfected HEK 293 cells, the small ankyrins are distribut ed in a reticular pattern resembling the ER if the NH2-terminal hydrop hobic domain is present, but they are uniformly distributed in the cyt osol if this domain is absent. These results suggest that the small an kyrins are integral membrane proteins of the sarcoplasmic reticulum. W e propose that, unlike the 210-kD form of Ank1, previously localized t o the sarcolemma and believed to be a part of the supporting cytoskele ton, the small Ank1 isoforms may stabilize the sarcoplasmic reticulum by linking it to the contractile apparatus.