p94, a muscle-specific member of calpain family, is unique in that it undergoes rapid and exhaustive autolysis with a half-life of less than 1 h resulting in its disappearance from muscle. Recently, p94 was shown to be responsible for limb girdle muscular dystrophy type 2A. To elucidate the muscular proteolytic system mediated by p94 and to solve the mystery of its unusually rapid autolysis, we searched for p94-binding proteins by the two-hybrid system. Although calpain small subunit plays a crucial role for regulation of ubiquitous calpains, it did not associate with p94. After a screening of skeletal muscle library, connectin (or titin), a gigantic filamentous protein spanning the M-to Z-lines of muscle sarcomere, was found to bind to p94 through a p94-specific region, IS2. The connectin-insoluble fraction of washed myofibrils contained full-length intact p94, suggesting that connectin regulates p94 activity.Proteolysis in cytosol is a key reaction to modulate various intracellular protein functions such as signal transduction, protein turnover, and cell structure. Calpain, Ca 2ϩ -dependent cysteine proteinase (EC 3.4.22.17), is one of the major intracellular proteinases known as interacting various protein kinases, transcription factors, and cytoskeletal proteins (1-5). Ubiquitous -and m-calpains, dimers of a large catalytic (CL 1 (6) and mCL (7), respectively) and small regulatory (30K (8)) subunit. Recently, we found that the ubiquitous calpain large subunit monomer can express full proteolytic activity, and that 30K dissociates from the large subunit upon activation by Ca 2ϩ (9, 10). In other words, 30K together with calpastatin, a specific proteinaceous inhibitor for calpain, play pivotal roles in regulation of calpain activity.p94 is a muscle-specific member of the calpain large subunit family, and distinct not only from the other members but also from other proteases in that it autolyzes very rapidly and extensively leading to almost complete disappearance right after translation even in the presence of EGTA and leupeptin as observed in vitro (11,12). Ubiquitous calpains as well as many other proteases also undergo autolysis at the NH 2 terminus, but only to a limited extent. Quite recently, p94 was identified as responsible for limb girdle muscular dystrophy type 2A (LGMD2A), the first demonstration of the involvement of an enzyme in muscle dystrophy (13). To elucidate physiological meaning of this exhaustive autolysis and the molecular mechanism connecting LGMD2A and p94 function, it is important to clarify the substrates of p94 and the manner to regulate the proteolytic activity of p94.30K is the first candidate to bind to and regulate p94, since the calmodulin-like Ca 2ϩ -binding domains of CL and mCL (see Fig. 2), which are the binding sites for 30K, are highly homologous to p94 (5). Analysis of p94 at the protein level, however, is very difficult because of the extremely rapid autolysis, and, thus, we examined using the yeast two-hybrid system (14). As a result, 30K was revealed not to bind to p94...

When whole muscle fibers or myofibrils of rabbit and chicken skeletal muscles are directly solubilized in hot SDS solution, a very high molecular protein called titin can be isolated by gel filtration (Wang et al. 1979). Connectin, an elastic protein of muscle (Maruyama et al. 1977), can be isolated by a similar method from thoroughly extracted muscle residues. Studies of electrophoretic mobility on 2-3% polyacrylamide gel electrophoresis, amino acid composition, and localization in myofibrils determined by the indirect immunofluorescence technique showed that titin and connectin are identical. Connectin was found to be unstable in SDS solution on storage for a few days at room temperature; the doublet band of connectin on SDS gel electrophoresis became diffuse and eventually disappeared. Connectin was concentrated around the A-I junction region of a myofibril, although it was present in an entire sarcomere except for the Z lines. On removal of myosin, the A-I junction was still fluorescent, when treated with fluorescent antibody against connectin. In the KI-extracted myofibril, materials accumulated on both sides of the Z lines were strongly stained, and there were fluorescent filaments between the neighboring Z lines, but the Z lines were not stained at all.

The amino acid sequences deduced from cDNA analyses revealed that human leucocyte L-plastin phosphorylated in response to interleukin 1, 2 closely resembles a chicken intestinal microvilli protein, fimbrin, that bundles actin filaments [de Arruda et al. (1990) J. Cell Biol. 111, 1069-1079]. In the present work, it was observed that unphosphorylated L-plastin isolated from human T cells bundled F-actin just as fimbrin does. L-Plastin acted on T cell beta-actin, but hardly acted on muscle alpha-actin or chicken gizzard gamma-actin, whereas fimbrin bundled muscle alpha-actin. Unlike fimbrin, L-plastin's actin-bundling action was strictly calcium-dependent: the bundles were formed at pCa 7, but not at pCa 6. Under suitable conditions, approximately one molecule of L-plastin bound to 8 molecules of actin monomer in the actin filament.

The structure and function of the giant elastic protein connectin/titin are described on the basis of recent investigations. The 3000 kDa protein links the Z line to the myosin filament in striated muscle sarcomeres. The NH2-terminal region of connectin filament is involved in the Z line binding, and the COOH-terminal region is bound onto the myosin filament with an overlap between the counter-connectin filaments at the M line. The PEVK region in the I band is shown to be mainly responsible for passive tension generation. The longitudinal continuity of myosin-, actin-free sarcomeres is explained by the linkage of freed connectin filaments extending from both sides of the Z lines in a sarcomere. The role of connectin in myofibrillar differentiation and the biodiversity of connectin-related proteins in the animal kingdom are briefly reviewed.

No abstract

The elastic protein isolated from myofibrils of chicken skeletal muscle was compared with extracellular non-collagenous reticulin prepared from chicken liver and skeletal muscle. The amino acid compositions of these proteins were similar except that their contents of Phe, Leu, Cys/2, and Hyp were different. The impregnations of the elastic protein and reticulin with silver were also different. The reticulin was not at all elastic. It also differed from reticulin in solubility and antigenicity. It is proposed to call the intracellular elastic protein connectin.

Two kinds of monoclonal antibodies (3B9 and SM1) against connectin, muscle elastic protein, reacted with both alpha- and beta-connectins. Immunofluorescence studies revealed that 3B9 stained both edges of the A band of chicken breast muscle myofibrils and remained as such upon stretching to a sarcomere length of 3.5 microns. On the other hand, SM1 stained the I band very close to the edges of the A band and the SM1-stained stripes moved considerably upon stretching to a sarcomere length of 3.5 microns. Immunoelectron microscopic observations with frog semitendinosus muscle revealed that three distinct stripes bound with 3B9 in the edges of the A band did not move on stretching up to 3.5 microns. On the other hand, the two stripes stained with SM1 in the I band clearly moved to the same extent as the stretching. However, when a sarcomere was stretched to 4.0 microns, all the stripes with 3B9 or SM1 disappeared and diffused deposits of the antibodies were observed. Thus it is concluded that connectin filaments in the I band region are more extensible than those at both edges of the A band.

Abstract. To clarify the full picture of the connectin (titin) filament network in situ, we selectively removed actin and myosin filaments from cardiac muscle fibers by gelsolin and potassium acetate treatment, respectively, and observed the residual elastic filament network by deep-etch replica electron microscopy. In the A bands, elastic filaments of uniform diameter (6-7 nm) projecting from the M line ran parallel, and extended into the I bands. At the junction line in the I bands, which may correspond to the N2 line in skeletal muscle, individual elastic filaments branched into two or more thinner strands, which repeatedly joined and branched to reach the Z line. Considering that cardiac muscle lacks nebulin, it is very likely that these elastic filaments were composed predominantly of connectin molecules; indeed, anti-connectin monoclonal antibody specifically stained these elastic illaments. Further, striations of •4 nm, characteristic of isolated connectin molecules, were also observed in the elastic filaments. Taking recent analyses of the structure of isolated connectin molecules into consideration, we concluded that individual connectin molecules stretched between the M and Z lines and that each elastic filament consisted of laterallyassociated connectin molecules. Close comparison of these images with the replica images of intact and S1-decorated sarcomeres led us to conclude that, in intact sarcomeres, the elastic filaments were laterally associated with myosin and actin filaments in the A and I bands, respectively. Interestingly, it was shown that the elastic property of connectin filaments was not restricted by their lateral association with actin filaments in intact sarcomeres. Finally, we have proposed a new structural model of the cardiac muscle sarcomere that includes connectin filaments.