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69 Journal of Cell Science 112, 69-79 (1999) Printed in Great Britain © The Company of Biologists Limited 1998 JCS0137 Identification of the A-band localization domain of myosin binding proteins C and H (MyBP-C, MyBP-H) in skeletal muscle Rénald Gilbert*, Julie A. Cohen, Sherly Pardo, Amartya Basu and Donald A. Fischman‡ Department of Cell Biology, Cornell University Medical College, 1300 York Avenue, New York, NY 10021, USA *Present address: Montreal Neurological Institute, 3801 University Street, Montreal, QC, Canada H3A 2B4 ‡Author for correspondence (e-mail: [email protected]) Accepted 26 October; published on WWW 8 December 1998 SUMMARY Although major constituents of the thick filaments of vertebrate striated muscles, the myosin binding proteins (MyBP-C and MyBP-H) are still of uncertain function. Distributed in the cross-bridge bearing zone of the A-bands of myofibrils, in a series of transverse 43 nm stripes, the proteins are constructed of a tandem series of small globular domains, each composed of ~90-100 amino acids, which have sequence similarities to either the C2-set of immunoglobulins (IgC2) and the fibronectin type III (FnIII) motifs. MyBP-C is composed of ten globular domains (~130 kDa) whereas MyBP-H is smaller (~58 kDa) and consists of a unique N-terminal segment followed by four globular domains, the order of which is identical to that of MyBP-C (FnIII-IgC2-FnIII-IgC2). To improve our understanding of this protein family we have characterized the domains in each of these two proteins which are required for targeting the proteins to their native site(s) in the sarcomere during myogenesis. Cultures of skeletal muscle myoblasts were transfected with expression plasmids encoding mutant constructs of the MyBPs bearing an N-terminal myc epitope, and their localization to the A-band examined by immunofluorescence microscopy. Based on the clarity and intensity of the myc A-band signals we concluded that constructs encoding the four C-terminal motifs of MyBP-C and MyBP-H (~360 amino acids) were all that was necessary to efficiently localize each of these peptides to the A-band. Truncation mutants lacking one of these 4 domains were less efficiently targeted to the C-zone of the sarcomere. Deletion of the last C-terminal motif of MyBP-H, its myosin binding domain, abolished all localization to the A-band. A chimeric construct, HU-3C10, in which the C-terminal motif of MyBP-H was replaced by the myosin binding domain of MyBP-C, efficiently localized to the A-band. Taken together, these observations indicate that MyBP-C and MyBP-H are localized to the A-band by the same Cterminal domain, composed of two IgC2 and two FnIII motifs. A model has been proposed for the interaction and positioning of the MyBPs in the thick filament through a ternary complex of the four C-terminal motifs with the myosin rods and titin. INTRODUCTION isoforms have been reported (Takano-Ohmuro et al., 1989). In contrast, only one isoform of MyBP-H has been isolated to date (Starr and Offer, 1983; Bahler et al., 1985a; Vaughan et al., 1993a,b). In skeletal muscle, MyBP-H is principally associated with fibers of fast twitch muscles (Bahler et al., 1985b; Bennett et al., 1986; Vaughan et al., 1993a). In cardiac tissue it is restricted to the myofibrils of Purkinje fibers (Alyonycheva et al., 1997a). The MyBPs are predicted to be composed of a series of globular motifs, each 90-100 amino acids in length, which bear resemblance to the C2-set of the immunoglobulin superfamily (IgC2) and the fibronectin type III motif (FnIII). Depending on the isoform, MyBP-C is composed of ten or eleven motifs (skeletal muscle isoforms have 10 motifs, cardiac muscle isoforms 11) whereas MyBP-H is smaller and contains only four motifs (Fig. 1). Except for its unique N-terminal sequence of 131 amino acids, MyBP-H is quite homologous to Two closely related members of the family of myosin binding proteins (MyBPs), myosin binding protein C (MyBP-C) and myosin protein H (MyBP-H), also known as C-protein and Hprotein, respectively, are significant myofibrillar constituents of vertebrate skeletal and cardiac muscles. Located in the A-band of the myofibrils in close association with the thick filaments, they are restricted to a series of transverse stripes, 43 nm apart, in the central cross-bridge bearing region (Craig and Offer, 1976; Dennis et al., 1984; Bahler et al., 1985b; Bennett et al., 1986). Three isoforms of MyBP-C (skeletal fast, skeletal slow and cardiac) have been characterized in adult muscles (Einheber and Fischman, 1990; Fürst et al., 1992; Weber et al., 1993; Okagaki et al., 1993; Kasahara et al., 1994; Gautel et al., 1995; Yasuda et al., 1995) and additional embryonic Key words: C-protein, H-protein, A-band, Thick filament, Sarcomere, Muscle protein, Development, Familial hypertrophic cardiomyopathy, Titin 70 R. Gilbert and others MyBP-C and the arrangement of its four globular motifs is identical to the last four C-terminal motifs of MyBP-C (Fig. 1). The unique N-terminal region of MyBP-H, composed of short repetitive sequences enriched in prolines and alanines, is responsible for the abnormally slow mobility of MyBP-H upon SDS-polyacrylamide gel electrophoresis (SDS-PAGE) (Vaughan et al., 1993a). One of the best characterized properties of the MyBPs is their relatively strong affinity for myosin (Offer et al., 1973; Moos et al., 1975; Yamamoto, 1984). Using an in vitro binding assay it has been shown that the principle myosin binding domain of MyBP-C resides within the C-terminal IgC2 motif, a region that is highly conserved in all MyBPs (Okagaki et al., 1993). MyBP-C also binds to titin and weakly to actin (Moos et al., 1978; Yamamoto, 1986; Fürst et al., 1992; Koretz et al., 1993). Its titin binding domain has been mapped to the last three or four C-terminal motifs (Freiburg and Gautel, 1996). It has been reported that mammalian MyBP-H does not bind to titin (Soteriou et al., 1993) but avian MyBP-H appears to do so (D. A. Fischman and M. Gautel, unpublished observations). The precise functions of the MyBPs are uncertain. In the case of MyBP-C, evidence exists for a role in myofibril assembly and the regulation of contraction. When purified myosin is polymerized in vitro at physiological stoichiometries with MyBP-C, the resulting thick filaments are slightly longer and more regular than in the absence of MyBP-C (Koretz, 1979; Davis, 1988). The presence of MyBP-C reduces the critical concentration required for myosin polymerization suggesting that it may regulate thick filament assembly (Davis, 1988). This is consistent with the observation that coexpression of the MyBPs with myosin heavy chain in COS cells promotes the formation of long cable-like co-polymers of both proteins (Seiler et al., 1996). Deletion of the C-terminal IgC2 motif from MyBP-H or MyBP-C prevents such cable formation. In addition, we have observed that the expression of truncated forms of MyBP-C lacking the myosin binding domain in muscle cultures inhibit myofibrillogenesis (Gilbert et al., 1996). It has recently been shown that mutations of the MyBP-C cause familial hypertrophic cardiomyopathy (Watkins et al., 1995; Bonné et al., 1995). Most but not all of these mutations generate truncated forms of MyBP-C that alter or lack the myosin or the myosin and titin binding domains (Watkins et al., 1995; Carrier et al., 1997; Yu et al., 1998). It is not clear how expression of these truncated proteins cause this disease, but our work in muscle cultures suggests that they may interfere with the assembly of the myofibrils. The observation that MyBP-C inhibits actin-activated skeletal muscle myosin ATPase but stimulates actin-activated cardiac muscle myosin ATPase (Offer et al., 1973; Watkins, 1998; Yamamoto and Moos, 1983; Hartzell, 1985) suggests a potential role in the regulation of contraction. The cardiac isoform of MyBP-C is phosphorylated by two sets of kinases: one that is calcium-calmodulin regulated (Gautel et al., 1995) the other by a cAMP dependent kinase (PKA); and this phosphorylation correlates with the rate of twitch relaxation (Hartzell and Titus, 1982; Hartzell, 1984). The reactive serine residues have been identified and are located at the N terminus within the linker between motifs I and II of the cardiac isoform of MyBP-C (Gautel et al., 1995). Recent studies indicate that phosphorylation of cardiac MyBP-C by PKA increases the orientation of crossbridges and thereby affects crossbridge cycling (Weisberg and Winegrad, 1996, 1998). Partial extraction of MyBP-C from myofibrils enhances tension generation at submaximal concentrations of Ca2+ and accelerates the contractile velocity at low levels of Ca2+ activation, again supporting a role in the regulation of contraction (Hofmann et al., 1991a,b). MyBP-H may also be involved in the regulation of contraction since it inhibits actinactivated skeletal muscle myosin ATPase in vitro (Yamamoto, 1984). We have previously demonstrated that the C terminus of MyBP-C encodes the information that specifies its targeting to the A-bands of myofibrils (Gilbert et al., 1996). However, in that study it was not certain if this information was encoded by the last four or by the last three C-terminal motifs. In addition, the motifs of MyBP-H that interact with the in vivo thick filament, and those essential for its accurate localization to the A-band had not been identified. To address these questions, we have analyzed the expression and distribution of a series of truncation mutants of the MyBPs in muscle cultures. We show that both MyBP-C and MyBP-H are targeted to the A-band by the same domain composed of two IgC2 and two FnIII motifs at their C-termini. We also demonstrate that the C-terminal IgC2 motif of MyBP-C (its myosin binding domain) can replace the C-terminal IgC2 motif of MyBP-H without affecting its localization to the A-band, indicating that these motifs share similar targeting properties. The data suggest a topological model in which the MyBPs are associated with the thick filament through the same four globular motifs at their C-termini. MATERIALS AND METHODS Vector constructs Recombinant DNA procedures followed standard methods (Sambrook et al., 1989). The polymerase chain reaction (PCR) products and the junctions of the spliced regions of the various constructs were all confirmed by DNA sequencing, which was accomplished by the DNA Services facility of Cornell University (Ithaca, NY). Unless otherwise specified, the linkers and enzymes were purchased from New England Biolabs. For PCR, the primers were synthesized by the DNA Services facility of Cornell University (Ithaca, NY) and the PCR reactions were accomplished using the GeneAmp PCR Reagent Kit with AmpliTaq DNA Polymerase (Perkin Elmer) according to the manufacturer’s recommendations. Mutant C7-10 was generated by PCR using pMC (Gilbert et al., 1996) as the template and the following two primers: 5′ATTCTGCAGTCCGACGAGCGAACCGACCCACGTG-3′ and 5′GACTCTAGAACCCAAAACGCCGCTC-3′. The first primer generated a PstI site in front of proline 728 in the protein sequence. The amplified DNA fragment was digested with PstI and NotI and cloned into the PstI and NotI sites of pBSmyc (Gilbert et al., 1996). This reaction added the linker encoding the myc tag at the 5′ end of C7-10. The insert was then excised by digestion with HindIII and NotI and cloned into the HindIII and NotI sites of pMC. Mutant C8-10, was generated by PCR using pMC as the template and the following two primers: 5′-ATTATCTGCAGTCCGCGGCACCTCCGCCAG-3′ and 5′-GACTCTAGAACCCAAAACGCCGCTC-3′. The first primer generated a PstI site in front of proline 835 in the protein sequence. The amplified DNA fragment was digested with PstI and NotI and cloned into the PstI and NotI sites of pBSmyc. This reaction added the linker encoding the myc tag at the 5′ end of C8-10. The insert was then excised by digestion with HindIII and NotI and cloned into the Myosin binding proteins HindIII and NotI sites of pMC. The plasmid expressing MyBP-H of chicken skeletal muscle with a myc tag at the N terminus (pMH) was generated according to the following protocol: a plasmid encoding the cDNA of MyBP-H with a NdeI site engineered at its initiation codon (Vaughan et al., 1993a), was digested with NdeI and a double stranded linker (5′-TATGTGAATTCACA-3′) encoding two NdeI and one EcoRI sites was ligated at this site. MyBP-H cDNA was then removed by digestion with EcoRI and cloned into the EcoRI site of pBSmyc. Myc-tagged MyBP-H cDNA was then removed by digestion with HindIII and BalI, the ends were blunted with Klenow, it was then cloned into the SmaI site of pJDp (Mikawa et al., 1992). To construct HU, a PCR reaction was done using pMH as the template and the following two primers: 5′-AGTCTACCATATGCCCAAAGAAGAGCCGCCCAG-3′ and 5′-CTGTGTTGCGGATGTTGACC-3′. The first primer generated an NdeI site in front of proline 130 in the protein sequence. The PCR product was digested with NdeI and BstEII and cloned into the NdeI and BstEII sites of pMH. To construct HU-1, pMH was digested with BamHI and the fragment encoding motifs II, III and IV was isolated, blunted with Klenow and cloned into the SmaI site of pBSmyc. The insert was removed by digestion with NcoI and XbaI and cloned into the NcoI and XbaI sites of pMH. Mutant H4 was generated by PCR using pMH as the template and the following two primers: 5′-GCTGTGAACCTGCTGATCC-3′ and 5′CTACGTCTAGAGGGATCTTCTGTGGCTGG-3′. The second primer generated an XbaI site after proline 438 in the protein sequence. The PCR product was digested with BstEII and XbaI and cloned into the BstEII and XbaI sites of pMH. The resulting plasmid was called pMH4X. A termination codon was generated after proline 438 by inserting into the XbaI site of pMH4X a NheI linker (5′CTAGCTAGCTAG-3′) that encodes a termination codon in any reading frame. To generate the chimera HU-3C10, the C-terminal Ig C2 motif of MyBP-C was amplified by PCR using pMC as the template and the following two primers: 5′ATCTATCTAGAACGCGACCTCCGCGCCGCCCCAC-3′ and 5′CGTCGAAGCTTACCCCAAAACGCCGCTCTG-3′. The first primer provided an XbaI site before glutamic acid 1032 in the protein sequence and the second primer generated an HindIII site after the termination codon. The PCR product was digested with XbaI and HindIII and cloned into the XbaI and HindIII sites of pMH4X. Western blots Canine fibroblasts (D17) and primary cultures of 11-day-old embryonic chicken pectoralis myoblasts were cultured, transfected with a mixture of DNA and lyposomes, and processed for western blotting as described previously (Gilbert et al., 1996). One day after the beginning of transfection of chicken myoblasts, the fetal bovine serum of the culture medium was replaced with 10% heat inactivated horse serum (Hyclone). The fibroblasts and the myoblasts were analyzed one day, or three and four days after the beginning of transfection, respectively. To improve the transfer of positively charged proteins, such as HU-1, the proteins were transferred to nitrocellulose membranes in 25 mM 3-N-cyclohexylamino-2 hydroxypropanesulfonic acid (CAPSO, Sigma), pH 10, 20% methanol (Szewczyk and Kozloff, 1985). The blots were incubated with monoclonal antibodies (mAbs) specific for the fast isoform of chicken skeletal MyBP-C (MF1) (Reinach et al., 1982) or for the myc epitope (9E10) (Evan et al., 1985), followed by horseradish peroxidaselabeled affinity purified goat anti-mouse antibodies (Sigma). Blots were also incubated with an affinity purified anti-H antibody (see below), followed by horseradish peroxidase-labeled affinity purified goat anti-rabbit antibodies (Sigma). The antibody complex was visualized by chemiluminescence (Dupont). Myoblast cultures and immunofluorescence microscopy Myoblasts were isolated, grown and transfected as described above. The cells were processed for indirect immunofluorescence three to four days after transfection as described previously (Gilbert et al., 71 1996). To study the distribution of the recombinant proteins and myosin, the cells were incubated with mAb 9E10, followed by Texas Red-conjugated affinity purified rabbit anti-mouse IgG1 antibodies (American Qualex). They were then incubated with F59, a mAb specific for the S1 fragment of chicken skeletal myosin heavy chain, isotype IgG1 (Miller et al., 1989) which was conjugated to biotin (Harlow and Lane, 1988), followed by FITCconjugated avidin (Cappel). To study the distribution of the endogenous MyBP-H and myosin, the cells were incubated with the affinity purified anti-H antibody (see below), followed by FITCconjugated affinity purified goat anti-rabbit antibodies (Cappel). They were then incubated with F59, followed by Texas Redconjugated affinity purified goat anti-mouse IgG antibodies (Jackson). Samples were examined with a Nikon Microphot SA upright epifluorescence microscope and photographs taken with Kodak TMAX 400 black and white film. Production and purification of antibodies against MyBP-H Native MyBP-H from chicken pectoralis muscle was purified according to the method of Okagaki et al. (1993). The purified protein was resolved in 7% polyacrylamide gel, the 86 kDa band was excised, the protein eluted electrophoretically from the gel, and used as an immunogen. Two female New Zealand white rabbits were injected subcutaneously with 100 µg of MyBP-H mixed with an equal volume of complete Freud’s adjuvant. The rabbits were boosted four times with 50 µg of MyBP-H mixed with an equal volume of incomplete Freud’s adjuvant. The antibody against MyBP-H (anti-H antibody) was affinity purified using a column made with recombinant MyBPH produced in Escherichia coli. Briefly, the cDNA of MyBP-H was excised from pMH by digestion with EcoRI and SalI, cloned in frame into the EcoRI and XhoI sites of the GST-fusion vector pGEX-4T-3 (Pharmacia), and transformed into BL21(DE3)pLys cells. The transformed cells were grown to an OD600 of 0.3 in Luriat Bertani (LB) medium supplemented with 50 µg/ml carbenicillin and 34 µg/ml chloramphenicol. The bacteria were then induced with 0.1 mM isopropyl-1-thio-β-D-galactopyranoside (Sigma) for 4 hours at 37°C. The induced GST-MyBP-H was purified from soluble bacterial extract by affinity chromatography using a glutathione-Sepharose 4B column (Pharmacia) according to the manufacturer’s recommendations. The purified GST-MyBP-H was dialyzed extensively against PBS and was coupled to CNBr-activated Sepharose 4B (Sigma) as described by the instructions of the manufacturer. The immunoglobulins of the anti-H serum were fractionated and concentrated by ammonium sulfate precipitation (Harlow and Lane, 1988). They were resuspended in PBS, dialyzed against PBS and passed twice through the MyBP-Hconjugated Sepharose column. The bound antibodies were eluted with 0.1 M glycine-HCl, pH 3.0, and neutralized with 3 M Tris-HCl, pH 8.0. Fractions containing purified immunoglobulins were pooled and dialyzed against PBS. RESULTS Description of the mutants of MyBP-C In a previous study (Gilbert et al., 1996), we showed that fulllength recombinant MyBP-C was correctly and efficiently incorporated into the A-bands of myofibrils following expression of its cDNA plasmids in cultured myotubes. We also demonstrated that the information specifying localization of MyBP-C to the A-band is encoded by its C terminus. However, it remained unclear if this information was encoded by the last four or the last three C-terminal motifs of MyBPC. To clarify this point, mutants C7-10 and C8-10 were constructed and their distribution in cultured myotubes investigated by immunofluorescence microscopy. C7-10 and 72 R. Gilbert and others Fig. 2. Evidence for the expression of C7-10 and C8-10 in cultured muscle. Myoblasts transfected with plasmids without insert (-), or coding for C7-10, or C8-10 were analyzed by SDS-PAGE after differentiation. The proteins were then transferred to a nitrocellulose membrane and subsequently reacted with mAbs specific for the myc epitope. Numbers at the left of each gel indicate the relative mobilities of molecular mass markers (kDa). Fig. 1. Description of the mutants used in the present study. (A) Mutants of MyBP-C (white). (B) Mutants of MyBP-H (black). The numbers above the proteins correspond to the first and last amino acids, starting from the N terminus (left). The roman numerals below indicate the position of each IgC2 and FnIII motif. The position of the myc epitope at the N terminus is indicated. The distribution of each protein was investigated in well differentiated myotubes and their presence in the A-band is noted. Mutant HU3C10 was constructed by fusing the first 438 amino acids of MyBPH to amino acids 1032 to 1132 of MyBP-C. C8-10 encode motifs VII to X and VIII to X, respectively (Fig. 1). A short linker encoding a myc epitope of 12 amino acids was inserted at the 5′ end of all cDNAs and a mAb specific for this epitope was used to study recombinant protein expression in the muscle cells. We have shown previously that the presence of the myc epitope does not interfere with the localization of MyBP-C to the A-band (Gilbert et al., 1996). Pectoralis myoblasts were isolated from day 11 chicken embryos and transfected with plasmids encoding C7-10 and C8-10 one day after plating in monolayer. Three to four days later, the time required for the myoblasts to fuse and differentiate into myotubes with robust cross-striated myofibrils, the expression of these two constructs was analyzed by western blots and by immunofluorescence microscopy. Bands of 55 kDa and 40 kDa were observed in the lanes containing lysates of myotubes expressing C7-10 and C8-10, respectively (Fig. 2). The relative mobilities of these bands correspond to the expected products of these two constructs. An extra band with a relative mobility ~45 kDa was also present in both lysates. This band was also observed when untransfected myotubes or myotubes transfected with plasmids lacking an insert were analyzed (Fig. 2). Since the predicted molecular mass of the chicken c-myc is 45 kDa (Shih et al., 1984), this extra band most likely corresponds to endogenous c-myc protein expressed in the fibroblasts and undifferentiated myoblasts present in the culture. The products of C7-10 and C8-10 were not recognized by mAb MF1 (Reinach et al., 1982), an antibody specific for the fast isoform of MyBP-C (data not shown). This is in agreement with a previous study indicating that the epitope recognized by MF1 is located within motifs III to VI of MyBP-C (Gilbert et al., 1996). Motifs VII to X encode the A-band localization domain of MyBP-C The peptide encoded by mutant C7-10 was efficiently localized to the A-bands of cross-striated myofibrils. Following expression of its cDNA in cultured muscle, it co-localized with myosin heavy chain (Fig. 3A,B). In the A-band, C7-10 was distributed as a doublet, with one bright stripe on each side of the M-line. At the level of the light microscope, the distribution of this truncation mutant was identical to that of endogenous MyBP-C or full-length recombinant MyBP-C. In addition, its signal intensity in the A-band and the sharpness of the doublet were comparable to that of recombinant full-length MyBP-C. Mutant C8-10 also co-localized with myosin heavy chain in the A-band (Fig. 3C,D). However, its signal intensity in the Abands was weak, sometimes blurred and most of the product of C8-10 remained diffuse in the cytoplasm. At higher magnification, a weak doublet with one faint band on each side of the M-line was apparent. We have previously demonstrated that larger deletions of the N terminus of MyBP-C, or deletion of one or more of the C-terminal motifs, completely abolish localization to the A-band (Gilbert et al., 1996). Taken together, these data indicate that the A-band targeting domain of MyBP-C is encoded by motifs VII to X. This contains the minimal sequence allowing its efficient localization to the Aband, because removal of one more motif at the N- or C terminus reduces or abolishes localization to this site. Description of the mutants of MyBP-H MyBP-H, which shares extensive homologies with MyBP-C Myosin binding proteins (Vaughan et al., 1993a), is another significant constituent of the thick filaments of vertebrate striated muscle (see Introduction). MyBP-H is smaller than MyBP-C and consists of a unique N-terminal sequence of 131 amino acids followed by two IgC2 and two FnIII motifs (Fig. 1B). In chicken muscle, this protein is found in a series of 9 transverse stripes 43 nm apart in the C-zone of each half A-band (Bahler et al., 1985b). To define which motifs specify the precise localization of MyBP-H to the A-band, a series of mutants was constructed and their expression was investigated in muscle cultures by western blot and by immunofluorescence microscopy. The structures of the MyBP-H mutants used in the present study are shown in Fig. 1B. Mutant HU lacks the first 129 amino acids of MyBP-H encoding its unique N-terminal sequence. Mutant HU-1 lacks the first 237 amino acids of MyBP-H containing the unique sequence and the first FnIII motif. The last 101 amino acids are deleted in mutant H4. Therefore, this truncation mutant lacks the C-terminal IgC2 motif (motif IV) of MyBP-H containing its myosin binding domain (Alyonycheva et al., 1997b). Mutant HU-3C10 was generated by replacing motif IV of MyBP-H with the C-terminal IgC2 motif of MyBP-C, motif X, containing its myosin binding domain. To distinguish the recombinant proteins from endogenous MyBP-H normally expressed in cultured muscle, a linker encoding a myc epitope 73 of 12 amino acids was inserted at the 5′ end of all cDNAs. The full-length MyBP-H encoding the myc epitope is termed MyBP-Hmyc. To compare the distribution of MyBP-Hmyc with endogenous MyBP-H, a polyclonal antibody was raised against MyBP-H. The antigen consisted of a pure preparation of MyBP-H isolated from the pectoralis muscle of adult White Leghorn chickens. The antibody was affinity purified against full-length recombinant MyBP-H synthesized in E. coli. The specificity of the anti-H antibody was investigated by western blot using lysates of un-transfected myotubes and lysates of fibroblasts transfected with plasmids encoding MyBP-Hmyc (Fig. 4A). A single product of ~85-86 kDa was detected in the lane containing lysate of myotubes. This product migrated with the predicted mobility of the chicken MyBP-H (Bahler et al., 1985a; Vaughan et al., 1993a). When fibroblasts expressing MyBP-Hmyc were analyzed, a single band was observed that migrated with the same relative mobility as endogenous MyBP-H. This band was absent in the lane containing fibroblasts transfected with plasmids lacking an insert, thus confirming the specificity of the anti-H antibody (Fig. 4A). The mobility of MyBP-H in SDS-PAGE is aberrantly slow; its cDNA encodes a protein with a predicted molecular mass of 54 kDa (Vaughan et al., 1993a). The unique N-terminal sequence of MyBP-H is responsible for this aberrant mobility. Fig. 3. Distribution of C7-10 and C8-10. Myoblasts transfected with plasmids coding for C7-10 (A,B), or C8-10 (C,D) were processed for immunofluorescence after differentiation. The cells were double immunostained with mAbs specific for the myc epitope and sarcomeric myosin. The left row shows the distribution of the recombinant proteins (Texas Red channel), the right row indicates the distribution of myosin heavy chain in the same cells (FITC channel). C7-10 and C8-10 are localized to the A-bands of cross-striated myofibrils. Note that the signal of C8-10 in the A-band is not as sharp as that of C7-10 and that a relatively large fraction of the protein remains diffuse in the cytoplasm. Insets: higher magnifications illustrating that C7-10 and C8-10 are distributed as A-band doublets (arrows, A,C). Bars, 20 µm. 74 R. Gilbert and others When this sequence is removed, the resulting protein migrates with a mobility that corresponds to the value deduced from its amino acid sequence (Vaughan et al., 1993b). Before studying the distribution of MyBP-Hmyc and its mutants in cultured skeletal myoblasts, we examined the size and antigenicity of the expressed proteins in these cells. Chicken myoblasts were transfected one day after plating with plasmids encoding MyBP-Hmyc, HU, HU-1, H4 and HU-3C10. Three to four days later, the cells were lysed and analyzed by western blots using a mAb against the myc epitope. Lysates of myotubes transfected with MyBP-Hmyc and the four mutants contained products that were recognized by the anti-myc antibody and that migrated with the expected mobility (Fig. 4B). A band of 45 kDa was detected in all the lanes containing myotube lysates (Fig. 4B). As mentioned above, the size of this band and the fact that it is recognized by the anti-myc antibody suggest it corresponds to endogenous chicken c-myc protein. Fibroblast cultures expressing the various MyBP-H mutants were also analyzed by western blot using the anti-H antibody. Fig. 4. Specificity of the anti-H antibody and evidence for expression of the MyBP-H mutants in cell culture. (A) Culture of myotubes (myo), or fibroblasts transfected with plasmids without insert (-), or coding for MyBP-Hmyc were analyzed by SDS-PAGE after differentiation. The proteins were then transferred to a nitrocellulose membrane and reacted with the anti-H antibody. (B) Myoblasts transfected with plasmids without insert (-), or coding for MyBPHmyc, HU, H4, HU-3C10, or HU-1 were analyzed by SDS-PAGE after differentiation. The proteins were then transferred to a nitrocellulose membrane and reacted with mAbs specific for the myc epitope. Numbers at the left of each gel indicate the relative mobilities of molecular mass markers (kDa). All of the constructs were recognized specifically by this antibody except for HU-1 (data not shown). This observation indicates that the epitopes recognized by the anti-H antibody are mainly located within the unique sequence and motif I. Motifs I to IV encode the A-band localization domain of MyBP-H To test if recombinant MyBP-H would incorporate into the Abands of the cross-striated myofibrils, myoblasts were isolated from day-11 chicken embryos and transfected with plasmids encoding MyBP-Hmyc. Three to four days after transfection, the cells were fixed and processed for immunofluorescence microscopy. The distribution of MyBP-Hmyc was assessed by staining the cells with a mAb against the myc epitope and compared with myosin by double-labeling the cells with mAbs specific for myosin. MyBP-Hmyc distribution was also compared with endogenous MyBP-H by double-staining the cells with the anti-H antibody and mAbs specific for myosin. In differentiated myotubes, MyBP-Hmyc staining was confined to the A-band of myofibrils, co-staining this region of the sarcomere with mAbs against myosin (Fig. 5A,B). At the resolution of the light microscope, the distribution of MyBPHmyc was identical to that of endogenous MyBP-H (compare Fig. 5A,C). Both proteins stained as doublets, one fluorescent stripe in each half A-band (insets Fig. 5A,C). To identify those domains of MyBP-H containing the information required for localization of the protein to the Aband, myoblasts were transfected with the mutants of Fig. 1B. In well differentiated myotubes, HU was identified in fluorescent doublets (Fig. 6A,B), one stripe in each half Aband (inset, Fig. 6A). The distribution of HU and its signal intensity in the A-band was indistinguishable from that of MyBP-Hmyc. Thus, the unique N-terminal sequence of MyBPH is not required for efficient A-band localization of this protein. In well differentiated myotubes, HU-1 was also colocalized with myosin in the A-band. However, its signal intensity in the A-band was weaker than that of MyBP-Hmyc and a large fraction of HU-1 remained diffuse in the cytoplasm (Fig. 6C). At higher magnification, a faint and somewhat blurred doublet was observed (Fig. 6C, inset). These data indicate that HU-1 was incorporated less efficiently into the Aband than HU and that motifs II to IV do not compete very efficiently with endogenous MyBP-H for A-band localization. Therefore, motif I contains important targeting information and is needed for efficient localization of MyBP-H to the A-band. The results obtained with HU and HU-1 are similar to those obtained with C7C10 and C8C10, respectively. Mutant H4, which lacks motif IV at the C terminus, was not localized to the A-band at all. Instead, it remained diffusely distributed in the myotube sarcoplasm (Fig. 6E,F). Comparable results were obtained by deleting motif X of MyBP-C where the resulting mutant was not localized to the A-band (Gilbert et al., 1996). These observations suggest that the A-band targeting domain of these two MyBPs is similar and consists of the same four analogous motifs. To further investigate this point, we asked if motif X of MyBP-C could replace motif IV of MyBP-H. This question was answered by studying the distribution of HU3C10. This chimera was efficiently localized to the A-band of myofibrils (Fig. 6G,H). At higher magnification a clear doublet was apparent (inset, Fig. 6G). The distribution of HU-3C10 appeared identical to that of MyBP-Hmyc. Its signal intensity Myosin binding proteins 75 Fig. 5. Evidence for the incorporation of recombinant MyBP-H into A-bands. Myoblasts transfected with MyBP-Hmyc (A,B), or un-transfected myoblasts (C,D) were processed for immunofluorescence after differentiation. The cells were double immunostained with mAbs specific for the myc epitope and sarcomeric myosin (A,B), or with the anti-H antibody and mAbs specific for sarcomeric myosin (C,D). The left row of figures indicates the distribution of the recombinant (A) and the endogenous MyBP-H (C). The right row shows the distribution of myosin in the same cells. In mature myotubes, the recombinant and endogenous MyBP-H are localized to the A-bands of the cross-striated myofibrils. Insets: higher magnifications illustrating that both recombinant and endogenous MyBP-H are distributed as doublets within each A-band (arrows, A,C). Bars, 20 µm. in the A-band and the sharpness of its doublet was comparable to MyBP-Hmyc. This result indicates the C-terminal IgC2 motifs of both MyBP-C and MyBP-H possess comparable targeting properties and can be interchanged with impunity, at least in this assay. DISCUSSION In this report, we have identified the domains of MyBP-C and MyBP-H that specify their intracellular targeting to the Abands of skeletal muscle myofibrils. These domains are composed of motifs VII to X of MyBP-C and I to IV for MyBP-H, the last four globular segments of both proteins. We believe that these four motifs encode all of the targeting information needed for faithful A-band incorporation in vivo because mutants composed of these four motifs (mutants C710 and HU) were localized to the A-band in an identical manner to the respective full-length proteins. The localization domains of MyBP-C and MyBP-H possess very similar characteristics: (i) their dimensions are the same (in both cases the domains consist of two IgC2 and two FnIII motifs arranged in alternating sequence); (ii) deletion of the upstream FnIII motif in this domain reduces but does not abolish protein targeting to the A-band (mutants C8-10 and HU-1); (iii) deletion of the C-terminal motif in either domain completely abolishes protein localization to the A-band (mutants H4 and 10 in Gilbert et al., 1996); and (iv) the C-terminal IgC2 motif of MyBP-C can replace the C-terminal IgC2 motif of MyBPH without affecting localization of the chimeric protein to the A-band (mutant HU-3C10). Based on these data, we have now extended our previous model (Gilbert et al., 1996) concerning the organization of the MyBPs in the thick filament to include MyBP-H. We propose that both MyBP-C and MyBP-H make contact with, and are positioned on, specific thick filament sites by a localization domain consisting of two FnIII and two IgC2 motifs located at their C-termini (Fig. 7). In addition, because the N-terminal moiety of these two proteins (motifs I to VI for MyBP-C and the unique sequence of MyBP-H) are not localized to the A-band when expressed in muscles (Gilbert et al., 1996; Koshida et al., 1995), they may not be physically part of the thick filament or are held on the thick filament by the C-terminal domains of both proteins. It is not known if MyBP-C and MyBP-H bind to the same sites on the thick filament. Both proteins are localized at the same stripes in the C-zone of the A-band in chicken pectoralis 76 R. Gilbert and others Fig. 6. Distribution of the mutants of MyBP-H. Myoblasts transfected with mutants HU (A,B), HU-1 (C,D), H4 (E,F) and HU-3C10 (G,H) were processed for immunofluorescence after differentiation. The cells were double immunostained with mAbs specific for the myc epitope and sarcomeric myosin. The left row shows the distribution of the recombinant proteins (Texas Red channel), the right row indicates the distribution of myosin in the same cells (FITC channel). Mutants HU, HU-1, and HU-3C10 were localized to the A-bands of cross-striated myofibrils (AD,G,H), whereas the product of H4 remains diffuse in the cytoplasm (E) and was not incorporated into the A-bands (F). Insets: higher magnifications illustrating that HU, HU-1 and HU-3C10 are distributed as A-band doublets (arrows, A,C,G). Note that the signal of HU-1 in the A-band is not as sharp as that for HU and HU-3C10 and that a relatively large fraction of the protein remains diffuse in the cytoplasm. Bars, 20 µm. Myosin binding proteins Fig. 7. Model indicating the tentative positioning of MyBP-C and MyBP-H on the thick filament. The data present in this study suggest that MyBP-C and MyBP-H are associated with the thick filament through their last four C-terminal motifs. In this model, the Nterminal moiety of these two proteins does not bind to the thick or to the thin filament in resting muscle. The C-termini of MyBP-C and MyBP-H are depicted as lying on the surface of the thick filament, but none of our data preclude deeper embedment of these proteins in the thick filament. Roman numerals indicate the position of the IgC2 and FnIII motifs (see Fig. 1). muscle (Bahler et al., 1985b). As demonstrated in the present study, the last IgC2 motif of MyBP-C can replace the last IgC2 motif of MyBP-H without affecting its localization to the A-band. Furthermore, pure preparations of MyBP-H can displace bound MyBP-C from synthetic myosin filaments (Alyonycheva et al., 1997b). Taken together, these observations suggest that MyBP-C and MyBP-H bind to the same or closely associated sites. Subtle but specific binding properties must exist that distinguish these two proteins since MyBP-C and MyBP-H are distributed in the pectoralis muscle of chicken in 7 or 9 stripes, respectively, in each half A-band. The C-terminal IgC2 motif is highly conserved among the various members of the MyBP-C and MyBP-H families that have been examined. More than 60 out of 100 amino acids are conserved in birds, mouse and man. In addition, in vitro binding studies have demonstrated that the major myosin binding domain of MyBP-C and MyBP-H is located within the C-terminal IgC2 motif (Okagaki et al., 1993; Alyonycheva et al., 1997b). Because of these structural and functional similarities, it is not surprising that the C-terminal motifs of MyBP-C and -H can be interchanged without affecting the localization of the resultant protein to the A-band. Electron microscopy will be required to see if subtle differences exist in the localization of the recombinant protein to specific stripes in the C-zones of the A-bands. The biochemical steps involved in the targeting of these proteins to the A-band remain unknown. Using an in vitro binding assay, we have demonstrated that the myosin binding domains of MyBP-C and MyBP-H are located within the Cterminal IgC2 motif of both proteins (Okagaki et al., 1993; Alyonycheva et al., 1997b). However, the myosin binding domain of MyBP-C, although essential, is not sufficient for protein targeting to the A-band, suggesting that other biochemical interactions are required for this reaction (Gilbert et al., 1996). MyBP-C binds to titin, and its titin binding 77 domain has been mapped to motifs VIII to X (Freiburg and Gautel, 1996). A comparable titin binding domain has been identified in chicken MyBP-H (D. A. Fischman and M. Gautel, unpublished observations). The fact that the titin binding motifs are part of the A-band localization domains of both proteins suggests that an interaction with titin is as important as an interaction with myosin for proper localization of either protein to the A-band. Soteriou et al. (1993) have demonstrated, using an overlay assay, that MyBP-H of rabbit skeletal muscle does not bind to titin. Conceivably, there are species-specific differences or technical differences in the assays used. Future work will be required to iron out these discordant data. However, it should be appreciated that MyBPH of the rabbit is present in only one stripe in each half A-band (Bennett et al., 1986) whereas the chicken skeletal muscle protein, used in the present study, is distributed in nine 43 nm stripes in each half A-band (Bahler et al., 1985b). One of these stripes, the one closest to the M-line, corresponds to the stripe found in rabbit muscle. Conceivably, the incorporation of rabbit MyBP-H to this single stripe does not require titin binding, but association with titin may be required for the localization of the chicken protein to the additional 8 stripes in each half A-band avian muscle sarcomeres. Cross-species studies comparing the incorporation of both proteins into the corresponding muscle fibers now appear warranted. Evidence has been presented that suggest the involvement of MyBP-C in both myofibril assembly and the regulation of contraction (see Introduction). These two functions are not mutually exclusive and it is possible, as proposed earlier, that different protein domains serve distinct functions (Gilbert et al., 1996). It now seems clear that the last four C-terminal motifs of MyBP-C are involved in myofibril assembly and it is likely that these regions of the molecule are an integral part of the thick filament (see Fig. 7). The observation that mutants of MyBP-C with deletions at the C terminus inhibit myofibrillogenesis supports this hypothesis (Gilbert et al., 1996). The domains of MyBP-C involved in the regulation of contraction may be located near the N terminus where it could potentially interact with a neighboring thin filament or with an adjacent cross-bridge. The cardiac isoform of MyBP-C is phosphorylated, and there is a correlation between the extent of such phosphorylation and the rate of cardiac relaxation (Hartzell and Titus, 1982; Hartzell, 1984). Interestingly, the phosphorylated amino acids are located in the linker joining motifs I and II in this N-terminal region of MyBP-C where they could participate in this regulation of contraction (Gautel et al., 1995). Furthermore, phosphorylation of these residues by phosphokinase A increases the apparent order of crossbridges in isolated cardiac thick filaments, possibly affecting the regulation of crossbridge cycling during cardiac muscle contraction (Weisberg and Winegrad, 1996, 1998). The function of MyBP-H is unclear. It appears that the last four motifs of this protein serve the same function as the comparable motifs in MyBP-C, since both are necessary and sufficient for A-band targeting of the respective proteins. In support of this hypothesis is the observation that co-expression of MyBP-H or MyBP-C with sarcomeric myosin heavy chain in COS cells promotes the formation of long cable-like copolymers of both proteins. The C terminus of MyBP-H is involved in the formation of these myosin cables, for mutants of MyBP-H lacking the C terminus fail to form the cables when 78 R. Gilbert and others co-expressed with myosin heavy chain (Seiler et al., 1996). No function has been assigned to the N-terminal unique sequence of MyBP-H. Because of its proposed organization on the thick filament, this peptide could theoretically interact either with the N terminus of MyBP-C, an adjacent cross-bridge or some other components of the thick filament. It is unlikely that it could interact with the thin filament because of its short length. MyBP-H can inhibit the actin-activated skeletal muscle myosin ATPase in vitro (Yamamoto, 1984). With its cDNA now available, it will be feasible to test whether this unique sequence of MyBP-H is responsible for this ATPase inhibition. Mutations of the cardiac isoform of MyBP-C in man have been shown to cause familial hypertrophic cardiomyopathy (FHC). Most but not all of the known mutations of MyBP-C are characterized by deletions of their C terminus or by an insertion within the C-terminal IgC2 motif, which probably interferes with binding to myosin and/or titin (Watkins et al., 1995; Bonné et al., 1995; Niimura et al., 1998; Carrier et al., 1997; Yu et al., 1998). It is not clear how expression of these mutated proteins cause FHC. The results of the present and earlier studies (Gilbert et al., 1996; Koshida et al., 1995) suggest that the mutations of MyBP-C which have disrupted C-termini would have a lower association with the myofibrils and be found in the sarcoplasm or rapidly degraded. FHC could result from an insufficient quantity of MyBP-C in the thick filament (i.e. haplo-insufficiency) assuming there is no upregulation of the second, wild-type MyBP-C allele. Since many of these FHC patients have a mild phenotype it is also conceivable that up-regulation of MyBP-H could partially substitute for MyBP-C insufficiency. Expression of these mutants could also affect the regulation of contraction or the formation of myofibrils. Support for the latter view comes from the prior demonstration that mutants of skeletal-type MyBP-C, lacking portions of the C terminus, perturb myofibrillogenesis when expressed in cultured muscle (Gilbert et al., 1996). The authors express their sincere appreciation for the many helpful suggestions and criticisms of this research by T. Mikawa, F. Reinach and R. Welikson. Thanks are also expressed to F. E. StockDale (Stanford University, CA) for antibody F59. Ms L. Ong and Ms C. Siewert made important technical contributions to this study for which the authors are very appreciative. This work was supported by grants AR32147 and HL45458 from the NIH and generous private contributions by Dr David Cofrin. R. Gilbert was an American Heart Association postdoctoral fellow during the course of this study. REFERENCES Alyonycheva, T., Cohen-Gould, L., Siewert, C., Fischman, D. 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