Identification and Extraction of Proteins That Compose the Triad Junction of Skeletal Muscle ANTHONY H. CASWELL and J.-P. BRUNSCHWIG Department of Pharmacology, University of Miami School of Medicine, Florida 33101 ABSTRACT Treatment of both transverse tubules and terminal cisternae with a combination of Triton X-100 and hypertonic K cacodylate causes dissolution of nonjunctional proteins and selective retention of membrane fragments which are capable of junction formation. Treatment of vesicles with Triton X-100 and either KCI or K gluconate causes complete dissolution of all components. Therefore K cacodylate exerts a specific preservative action on the junctional material. The membrane fragment from treatment of transverse tubules with Triton X-100 + cacodylate contains a protein of Mr = 80,000 in SDS gel electrophoresis as the predominant protein while lipid composition is enriched in cholesterol. The membrane fragment retains in electron microscopy the trilaminar appearance of the intact vesicles. Freeze fracture of transverse tubule fragments reveals a high density of low-profile, intercalated particles, which frequently form strings or occasional small arrays. The fragments from Triton X-100 plus cacodylate treatment of terminal cisternae include the protein of Mr = 80,000 as well as the spanning protein of the triad, calsequestrin, and some minor proteins. The fragments are almost devoid of lipid and display an amorphous morphology suggesting membrane disruption. The ability of the transverse tubular fragment, which contains predominantly the Mr = 80,000 protein, to form junctions with terminal cisternae fragments suggests that it plays a role in anchoring the membrane to the junctional processes of the triad. The junctional proteins may be solubilized in a combination of nonionic detergent and hypertonic NaCI. Subsequent molecular sieve chromatography gives an enriched preparation of the spanning protein. This protein has subunits of Mr = 300,000, 270,000, and 140,000 and migrates in the gel as a protein of Mr = 1.2 x 106 indicating a polymeric structure. The triad junction or dyad junction of muscle is the only tubules to the terminal cisternae (TC) ~ by forming and then known intracellular membrane junction. The morphology of breaking the triad; hence we proposed that the protein this junction has been extensively studied in intact muscle by spanned the gap between the two organelles. The fact that the electron microscopy and has been demonstrated to display a junction could be disrupted without apparent damage to the unique organization very different from such intercellular associated organelles suggested that the spanning protein re- junctions as the gap junction (1). The triad junction almost sided external to the membrane and was associated with it certainly is also the site of message transmission in excitation- through a noncovalent interaction with a recognition site in contraction coupling (2, 3). Our knowledge of the composi- the membrane. Hence it is likely that the junction is composed tion, molecular conformation, and dynamics of the junction not only of a spanning protein, but also of an anchoring is however extremely limited. For these reasons the resolution protein that is attached or embedded in the membrane and of the structure of the junction offers an intriguing challenge. that recognizes and binds the spanning protein. In an earlier article we presented evidence that a protein This article describes a further characterization and extrac- doublet observed in PAGE with molecular weights of Abbreviations used in this paper: HTC, "heavy" terminal cisternae; -325,000 and 300,000 was a constituent of the junction (4). LTC, "light" terminal cisternae; TC, terminal cisternae; T-tubules, This protein could be transferred in part from the transverse transverse tubules. THE JOURNAL OF CELL BIOLOGY . VOLUME 99 SEPTEMBER 1984 929-939 © The Rockefeller University Press • 0021-9525/84/09/0929/11 $1.00 929 tion of proteins that form the triad junction and suggests a glutaraldehyde, 3% sucrose, 50 mM Na cacodylate, pH 7.2. The subsequent molecular structure of the triad. preparation has been described previously (11). For negative staining, a drop of the preparation (-0.05 mg/ml) was placed on top of a 400-mesh parlodion (0.75% in amyl acetate) coated grid and allowed to stand for 1 rain. The drop MATERIALS AND METHODS was then blotted and replaced by a drop of 1% K phosphotungstate, pH 6.7, The preparation of microsomes, TC/triads, ~heavy" terminal cisternae (HTC), allowed to stand for 1 rain, and then gently blotted until almost dry. Specimens were observed under a Philips EM-300 electron microscope (Philips Electronic and transverse tubules (T-tubules) has been described before (5, 6). A brief Instruments, Inc., Mahwah, NJ) operating at 80 kV. description is as follows: TC/triads are prepared from a microsomal fraction from rabbit sacrospinalis muscle by centrifugation on a continuous sucrose- density gradient. The band with isopycnic point ~40% sucrose contains free TC and intact triad junctions consisting of two TC vesicles apposed on each RESULTS side of a T-tubule vesicle. Broken triads are prepared by passing TC/triads Distribution of Proteins between through a French press at 6,000 psi. HTC and T-tubules are prepared by centrifuging the broken triads in a continous sucrose-density gradient on a Subcellular Organelles Sorvall TV850 vertical rotor (DuPont Instruments, Sorvall Biomedical Div., Newtown, CT) for I t/2 h at 130,000 g. Of the three bands formed the lightest is In previous work we have employed specific ligand or T-tubules (22-28% wt/wt sucrose), the intermediate band (30-35% sucrose) is enzyme markers to delineate T-tubules and sarcoplasmic light terminal cisternae (LTC), and the heaviest is HTC (38-42% sucrose). The reticulum subfractions. Several proteins play a role in the organelles were concentrated by centrifugation and resuspended in a medium triad junction which do not exhibit defined enzyme activities. of 250 mM sucrose, 2 mM histidine, pH 7.0. Protein was estimated by the Bradford assay (7) and, where stated, 2 mg of Fig. 1 shows the gel electrophoretic pattern and the densito- Triton X-100/mg of protein was added followed by the salt. The sample was metric assay of some proteins, which are identified as bands layered on a linear continuous sucrose-density gradient between 12.5 and 60% on SDS PAGE. A preparation of TC/triads was passed sucrose in a Beckman SW 41 Ti rotor (Beckman Instruments, Inc., Fullerton, through a French press to break the triad junction and then CA). It was centrifuged for 3 h at 200,000 g. Samples (1.3 ml) were withdrawn sequentially from the top of the gradient for subsequent analysis. centrifuged to equilibrium on a sucrose-density gradient. The samples from the gradient were assayed as follows: protein was assayed Three distinct vesicle bands are discernible with isopycnic by the Folin method. Total lipid phosphorus was assayed by a modified Ames points at 26, 32, and 42% sucrose which we have previously assay (8). Aliquots (50 tzl) were diluted to l ml with water in a small test tube identified as T-tubule, LTC, and HTC, respectively (6). The and lipid was extracted into the organic phase by twice partitioning with l ml first three panels of densitometric analysis show the distribu- of a 2: l ratio of chloroform/methanol. The solvent was removed by passing a stream of air over the sample. H202 (30%, 100 #l) was added and allowed to tion of proteins that are representative markers for the three incubate in an oven at 80"C overnight. To the dry sample was added 0.8 ml of organelles. The 72,000 Mr protein is distributed specifically 0.5 M HC1 and the phosphorus was assayed subsequently by the method of in the T-tubules in accord with our earlier conclusions that Ames (8). Cholesterol and cholesterol ester were estimated from 100-td samples this is a T-tubule specific protein (6). The distribution of this using a SmithKline Instruments reagent cholesterol kit SV/28 (SmithKline protein matches perfectly to that of the [3H]ouabain entrap- Instruments, Inc., Sunnyvale, CA). This assay employs enzymic hydrolysis of cholesterol ester followed by cholesterol oxidase to produce H202 which was ment assay we have employed before (data not shown). The assayed colorimetrically at 500 nm in a l-ml cell in a Zeiss spectrophotometer long tail of activity in the denser region of the gradient reflects, (Carl Zeiss, Inc., New York). All parts of the reaction are present in a single in part, the difficulties in assaying this protein at low concen- reagent sample. A cholesterol standard (0.052 ~tmol) was run simultaneously. tration because it is not a major component of the triad and, PAGE slab gels were those of Laemmli (9). Most gels used 9% acrylamide, 0.24% N,N'-methylene-bis-acrylamide BIS separating phase. The analysis of in part, a possible low extent of T-tubules which were not the spanning protein employed 5% acrylamide, 0.133% N,N-methylene-bis- broken from the triad by French press treatment. The 53,000 acrylamide. Samples were dissolved in an equal volume of sample buffer and Mr protein that has been identified by Michalak et al. (12) as incubated in a boiling water bath for 1 min, and between 100 and 250 ul was an intrinsic glycoprotein shows a preponderant distribution layered in the wells of the gel. Standards are the high-molecular-weight standards towards the LTC although some activity may also exist in the of Bio-Rad Laboratories (Richmond, CA) which contained myosin, B-galacto- sidase, phosphorylase b, BSA, and ovalbumin, whereas calsequestrin and the HTC. Little activity appears to be associated with T-tubules Ca pump protein served as internal standards. Standards for the spanning and the presence of this protein in the T-tubule region prob- protein gel included cross-linked albumin (Sigma Chemical Co., St.