Identification and Localization of Two Triad Junctional Foot Protein Isoforms in Mature Avian Fast Twitch Skeletal Muscle*

We report evidence for two foot protein isoforms in chicken pectoral muscle. (i) Two polypeptides with molecular masses of -500 kDa copurify with [‘Hlry- anodine binding. (ii) Both polypeptides are associated with oligomeric proteins similar in size to the mam- malian skeletal muscle foot protein. (iii) The polypep- tides are shown to be unique by limited proteolysis. (iv) By using isoform-specific antibodies, the polypeptides are shown to be subunits of different [‘Hlryanodine-binding proteins. Using immunolabeling techniques, we have localized these proteins in chicken breast muscle by both light and electron microscopy. (v) From immunofluorescent light

In conclusion, two distinct homo-oligomeric foot proteins coexist in avian fast twitch skeletal muscle.
We have termed these proteins, (Y and 0 foot proteins. The triad junction, between the sarcoplasmic reticulum and transverse tubule membranes in skeletal muscle cells, is the site where surface membrane depolarization is transduced to release calcium from the sarcoplasmic reticulum and cause muscle contraction (1 protein that interacts with the transverse tubule membrane in an as yet undefined manner (2,3). The foot protein is generally believed to participate in transducing the depolarization signal (1) and embodies an ion channel that may provide the pathway for sarcoplasmic reticulum calcium release (4)(5)(6). In mammalian fast twitch skeletal muscle, a single foot protein has been identified and purified based on its ability to bind [3H]ryanodine (4,7,8). This protein is a homotetramer comprised of a 565-kDa subunit. Recently, cDNA encoding the rabbit skeletal muscle protein monomer has been cloned, sequenced, and functionally expressed (9)(10)(11).
The contributions made by the foot protein to coupling electrical excitation to contraction, and to the formation and structural integrity of the triad junctional structure, are unknown. To gain a different perspective on the role of the foot protein in muscle structure and function, we have initiated investigations of the expression of the foot protein and of the identity and nature of the processes involved in the formation of the triad junction during embryonic skeletal muscle development. We have characterized the foot proteins in mature avian fast twitch skeletal muscle to acquire the basic information necessary for these investigations.
In this report, we describe biochemical and immunocytochemical evidence for the existence of two foot protein isoforms in mature chicken fast twitch skeletal muscle that co-localize to triad junctions in the same muscle fibers. These results have been presented in abstract (12 the foot proteins were either used immediately or rapidly frozen in liquid NZ and stored at -90 "C until use.
For the experiments shown in Fig. 9, the a and @ ryanodine-binding protein isoforms were separated from one another by deleting one of the isoforms by immunoprecipitation with an antibody specific for that isoform.
The  Fig. 3A). As shown in Fig. 2B, the differential detergent extraction/sucrose gradient sedimentation procedure resulted in a significant purification of avian skeletal muscle high molecular mass foot proteins. From comparison with solubilized rabbit skeletal muscle foot protein separated in parallel on identical gradients, the chicken muscle proteins appear to have native and denatured subunit molecular masses of -2000 and -500 kDa, respectively.
Overloading a SDS gel with sample revealed minor contaminating polypeptides of 200, -160, -100, and 45 kDa. The 200-and 45-kDa polypeptides are likely to be myosin and actin. The material apparent between 60 and 80 kDa is due to the CHAPS/phospholipid and is observed in the absence of any added muscle protein. Using this procedure, the solubilized [3H]ryanodine binding activity in the sucrose gradient fraction shown in Fig. 2 polypeptides have lower molecular masses than the rabbit muscle protein subunit (Fig. 3A), which has been identified as -565 kDa based on cDNA sequence analysis (9). We have termed the higher and lower molecular mass polypeptides from chicken muscle, LY and 0, respectively. Third, the polypeptides from avian and mammalian skeletal muscle exhibit limited immunologic cross-reactivity. Affinity-purified polyclonal sera against both chicken muscle polypeptides reacts weakly with rabbit muscle protein (Fig. 3B), and polyclonal sera against rat muscle foot protein reacts with only the (Y polypeptide from avian muscle (Fig. 3C). In both cases Western blots had to be developed beyond when the primary antigen was clearly visible to demonstrate cross-reactivity.
Second, the chicken muscle high molecular mass protein The High Molecular Mass Proteins from Mature Avian Skeletal Muscle Have Different Subunit Compositions-DEAE anion exchange chromatography of the high molecular mass chicken muscle proteins, partially purified as described in Fig.  2, yielded fractions containing different amounts of the cy and @ polypeptides (Figs. 4B). This differential enrichment of these polypeptides could be due to either the purification of individual polypeptide subunits or a selective enrichment of large oligomeric proteins differing in subunit composition.
To decide whether individual polypeptides or large oligomeric proteins were present, aliquots from selected DEAE column fractions were centrifuged in individual sucrose gradients. In each case the two polypeptides were found to be associated with large proteins that sedimented to the same gradient fractions as the freshly solubilized ryanodine-binding protein (compare Fig. 4, A and C). These results suggested that large proteins comprised of different ratios of the two polypeptide subunits were present in the DEAE column fractions.
Avian these studies was determined for both denatured and native antigen by Western blotting (Fig. 5) and immunoprecipitation (Fig. 6A), respectively. Western blots of pectoral muscle microsomal membranes probed with anti-a polypeptide and anti-b polypeptide antibodies are shown in Fig. 5, B and C, respectively.
If hetero-oligomeric isoforms are present, then antibodies specific for either polypeptide should precipitate proteins containing both polypeptides.
On the other hand, if two homooligomeric isoforms exist, then the antibodies should precipitate proteins containing only the polypeptide recognized by that antibody. As shown in Fig. 6A, polypeptide-specific antibodies precipitated proteins containing only a single polypeptide, indicating the existence of two homo-oligomeric foot protein isoforms.
Both Avian Muscle High Molecular Mass Proteins Bind ['H]Ryanodine-Two variations of the immunoprecipitation protocol were used to determine whether both high molecular mass proteins bound [3H]ryanodine.
The proteins were solubilized and either labeled with [3H]ryanodine and precipitated with polypeptide-specific antibodies or immunoprecipitated and then allowed to bind [3H]ryanodine.
As described under "Experimental Procedures" and shown in Fig. 6A, the conditions used for immunoprecipitation resulted in precipitation of equivalent amounts of both isoform. The proteins precipitated by antibodies specific for either polypeptide bound [3H] ryanodine in a specific manner (Fig. 6B) to such an extent that the observed binding could not be attributed to contamination of the precipitate by the alternative isoform (Fig. 6A,  lanes 3 and 4). These data indicate the existence of two distinct high molecular mass ryanodine-binding proteins, comprised of either a or /3 polypeptide subunits. The a and p Polypeptides Are Unique-Several pieces of circumstantial evidence argued that the (Y and p polypeptides are unique and that /3 does not result from either in uivo or in vitro proteolysis of (Y. First, the relative abundance of the two polypeptides did not vary between microsomal membrane preparations. Similar ratios of (Y and p proteins were consistently observed when sarcoplasmic reticulum membranes were isolated in the absence or presence of the following combinations of protease inhibitors: leupeptin + PMSF (7) A, CHAPS-solubilized partially purified muscle microso-ma1 proteins were precipitated with 1lOF and llOE, monoclonal antibodies specific for the cy and 6 polypeptide, respectively. Lanes 1 and 6, solubilized proteins used as starting material for the immunoprecipitations; lanes 2 and 5, proteins remaining in the supernatants after immunoprecipitation; lanes 3 and 4, proteins selectively immunoprecipitated. Mab, monoclonal antibody. B, the proteins precipitated by polypeptide-specific antibodies shown in A bind [3H] ryanodine in a specific manner regardless of whether the proteins bind the ligand prior to (left panel) or after immunoprecipitation (right panel). + PMSF + aprotinin + benzamide + iodoacetamide + pepstatin A (8), or diisopropyl fluorophosphate + PMSF (4). Second, antibodies exist that recognize only one of the polypeptides. If the p polypeptide resulted from proteolysis of the (Y polypeptide, then it would be likely that the /3 polypeptide would be recognized by antibodies specific for the (Y polypeptide, particularly in the case of polyclonal sera. However, as shown in Fig. 3, anti-rat muscle ryanodine-binding protein polyclonal sera reacts with only the o( polypeptide, and a number of polypeptide-specific monoclonal antibodies have been identified (cf. Fig. 6A).
Two different experimental protocols utilizing limited proteolysis were employed to demonstrate further that the two polypeptides are unique. (Y or p polypeptides were either immunoprecipitated and then proteolyzed, or solubilized proteins were proteolyzed and the resulting fragments precipitated with polypeptide-specific antibodies. Both approaches yielded different peptide maps for each polypeptide. The results obtained with the first protocol are shown in Fig. 7. The differences observed in the respective maps are clearest in the region of the gels above 100 kDa as can be appreciated from comparison of the densitometric scans of lanes 4 and 5 shown in the lower portion of Fig. 7. Polypeptides at 25, 55, 80 and 105 kDa are immunoglobulin light and heavy chains and were observed in the absence of antigen. In the second approach, the LY and @ polypeptides were proteolyzed both as a mixture (Fig. 9A)  and in the latter with an antibody that recognized an epitope common to both isoforms (Fig. 8). Again, comparison of the resulting immuno-maps shows a lack of similarity between the proteolytic fragments resulting from the trypsinization of the two polypeptides.
Comparable differences were obtained using S. aureus V8 protease and the calcium-activated neutral protease (data not shown).
The (Y and p polypeptides can also be differentiated by the greater post-solubilization lability of the (Y polypeptide (cf. Fig. 7, compare lanes 2 and 3, and Fig. 9A, compare lanes 9-12) and by the greater susceptibility of this polypeptide to proteolysis by trypsin (Fig. 7B, compare lanes I-4).
The Avian Muscle Ryanodine-binding Proteins Are Present in the Same Muscle Cells-The biochemical identification of two ryanodine-binding protein isoforms in avian skeletal muscle raised questions concerning whether they are phenotypespecific isoforms expressed in different muscle fiber types or if both isoforms co-exist in the same fiber.
The muscle fiber distribution of the EY and /3 ryanodinebinding proteins was compared using immunofluorescent detection of isoform-specific monoclonal antibodies on l-pmthick serial cryosections of chick breast muscle. The immunofluorescent localization of the (Y and fi isoforms in longitudinal sections appeared as discrete points equidistant along the length of the fibers in close proximity to the Z-lines (Fig.  10, A and B). This was confirmed by co-labeling muscle sections with an antibody against the Z-line protein, a-actinin (data not shown). The staining pattern of the (Y and /3 isoforms in serial sections appear identical (Fig. 10). In serial crosssections both the (Y (Fig. 1OC) and /3 (Fig. 1OD)  Localize to the Triad Junction-The Z-line localized immunofluorescent staining observed at the light microscope level was consistent with localization of both isoforms to the triad junction. This question was examined further by more precisely localizing the isoforms with immunogold labeled secondary antibodies and (Y and p protein-specific primary antibodies in ultrathin cryosections of chick breast muscle at the electron microscopic level. These studies yielded two major observations. First, both the (Y (Fig. lL4) and /3 (Fig. 11B) isoforms are localized to the terminal cisternae membrane of the sarcoplasmic reticulum at the triad junction. Second, both isoforms were detected in virtually every triad junction observed in serial sections of the same fiber suggesting that both proteins co-exist in the same junctions. A similar intracellular localization has been observed for the mammalian fast twitch skeletal muscle foot protein (33).

DISCUSSION
The results of the present studies suggest that avian and mammalian fast twitch skeletal muscle fibers differ in that the former contain two ryanodine-binding protein isoforms. The large size of these proteins, their ability to bind [3H] ryanodine, and their localization to the triad junction support the conclusion that the avian muscle proteins are foot protein isoforms.
The molecular similarities between these proteins have not yet been determined. It is not known whether the isoforms are the products of separate genes or represent either post-transcriptional or post-translational modifications of a single gene product. The differences in subunit molecular mass and peptide maps and the identification of isoformspecific antibodies suggest that the avian muscle (Y and p foot proteins differ in both their primary and secondary structures.  Fig. 7. The molecular mass standards used in the gels shown in this figure are as described for pattern of expression of single or multiple foot proteins and the functional contributions made by the different protein isotypes. Both birds and mammals arose from reptilian ancestors (34). Therefore, whether the expression of multiple foot protein isoforms is unique to avian fast twitch skeletal muscle or a trait shared with muscles from other nonmammalian vertebrates is relevant to considerations of the functional significance of the multiple foot protein isoforms. In preliminary studies we have observed multiple high molecular polypeptides that are immunologically related to the avian skeletal muscle foot protein subunits in microsomes prepared from both frog and fish muscles. 3 We are currently determining the tissue and cellular distributions of the frog and fish muscle proteins. The identification of multiple foot protein isoforms in fish muscle could provide a biochemical correlate to the morphological evidence for two foot protein types reported by Block et al. (35). In these studies a tetrameric transverse tubule membrane protein was observed to associate with alternating foot proteins suggesting the existence of structurally distinct foot proteins types. The present results suggest that mammalian fast twitch skeletal muscle may be distinguished from similar muscles in other vertebrates by the expression of a single foot protein type. If this is the case, then comparisons of the different foot protein isotypes may provide insights into whether the functional characteristics of these proteins are such that expression of a single foot protein isotype provided a selective advantage during the evolution of mammalian striated muscle.
Whether the presence of single or multiple foot proteins confers advantages for specific types of muscle function can only be determined once the molecular and functional characteristics of the various foot protein isotypes have been established. We are currently investigating the [3H]ryanodine binding abilities and calcium channel characteristics of the avian muscle proteins. In other studies, we have established that the avian muscle foot protein isoforms are expressed at different times during the embryonic development of the pectoral muscle.4 The differential expression of these proteins during muscle development and their continued expression in mature muscle strongly suggests that the two isoforms make different functional contributions to the muscle cell.
In conclusion, we have presented evidence for the existence of two foot protein isoforms in chicken pectoral muscle. Both isoforms are found in the same muscle cell and both are localized to the triad junction. The functional significance of these proteins is currently being investigated. A and B, localization of the 01 (A) and p (B) isoforms in consecutive serial longitudinal sections of muscle showing identical discrete repetitive staining pattern using monoclonal antibodies 1lOF and llOE, respectively. In both panels the staining coincided with the Z-lines, as demonstrated by staining a third serial section with an antibody against the Z-line protein cY-actinin (data not shown). C and D, consecutive serial muscle cross-sections stained with the antin antibody, 1lOF (C), and the anti-0 antibody, 1lOE (D), respectively. Calibration bar = 20 urn. A, the (Y foot protein isoform is localized to the triad junction at the site of association between the terminal cisternae of the sarcoplasmic reticulum and the transverse tubule (arrows). B, the localization of the p isoform is identical to that of cr. Calibration bars = 0.5 pm.