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The Hydrophilic Domain of Small Ankyrin-1 Interacts with the Two N-terminal Immunoglobulin Domains of Titin

2003; Elsevier BV; Volume: 278; Issue: 6 Linguagem: Inglês

10.1074/jbc.m209012200

ISSN

1083-351X

Autores

Aikaterini Kontrogianni‐Konstantopoulos, Robert J. Bloch,

Tópico(s)

Muscle Physiology and Disorders

Resumo

Little is known about the mechanisms that organize the internal membrane systems in eukaryotic cells. We are addressing this question in striated muscle, which contains two novel systems of internal membranes, the transverse tubules and the sarcoplasmic reticulum (SR). Small ankyrin-1 (sAnk1) is an ∼17-kDa transmembrane protein of the SR that concentrates around the Z-disks and M-lines of each sarcomere. We used the yeast two-hybrid assay to determine whether sAnk1 interacts with titin, a giant myofibrillar protein that organizes the sarcomere. We found that the hydrophilic cytoplasmic domain of sAnk1 interacted with the two most N-terminal Ig domains of titin, ZIg1 and ZIg2, which are present at the Z-line in situ. Both ZIg1 and ZIg2 were required for binding activity. sAnk1 did not interact with other sequences of titin that span the Z-disk or with Ig domains of titin near the M-line. Titin ZIg1/2 also bound T-cap/telethonin, a 19-kDa protein of the Z-line. We show that titin ZIg1/2 could form a three-way complex with sAnk1 and T-cap. Our results indicate that titin ZIg1/2 can bind sAnk1 in muscle homogenates and suggest a role for these proteins in organizing the SR around the contractile apparatus at the Z-line. Little is known about the mechanisms that organize the internal membrane systems in eukaryotic cells. We are addressing this question in striated muscle, which contains two novel systems of internal membranes, the transverse tubules and the sarcoplasmic reticulum (SR). Small ankyrin-1 (sAnk1) is an ∼17-kDa transmembrane protein of the SR that concentrates around the Z-disks and M-lines of each sarcomere. We used the yeast two-hybrid assay to determine whether sAnk1 interacts with titin, a giant myofibrillar protein that organizes the sarcomere. We found that the hydrophilic cytoplasmic domain of sAnk1 interacted with the two most N-terminal Ig domains of titin, ZIg1 and ZIg2, which are present at the Z-line in situ. Both ZIg1 and ZIg2 were required for binding activity. sAnk1 did not interact with other sequences of titin that span the Z-disk or with Ig domains of titin near the M-line. Titin ZIg1/2 also bound T-cap/telethonin, a 19-kDa protein of the Z-line. We show that titin ZIg1/2 could form a three-way complex with sAnk1 and T-cap. Our results indicate that titin ZIg1/2 can bind sAnk1 in muscle homogenates and suggest a role for these proteins in organizing the SR around the contractile apparatus at the Z-line. transverse tubules sarcoplasmic reticulum small ankyrin-1 glutathione S-transferase maltose-binding protein As striated muscle develops, the basic contractile unit, the sarcomere, is assembled before the transverse tubules (T-tubules)1 and the sarcoplasmic reticulum (SR) mature (1Franzini-Armstrong C. Peachey L.D. J. Cell Biol. 1981; 91: 166-186Crossref PubMed Scopus (33) Google Scholar, 2Flucher B.E. Dev. Biol. 1992; 154: 245-260Crossref PubMed Scopus (124) Google Scholar). The contractile cycle in striated muscle normally requires the spread of the action potential along the T-tubules into the interior of the muscle fiber, where depolarization induces the release of Ca2+ from the terminal cisternae of the SR, causing contraction (2Flucher B.E. Dev. Biol. 1992; 154: 245-260Crossref PubMed Scopus (124) Google Scholar, 3Flucher B.E. Franzini-Armstrong C. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 8101-8106Crossref PubMed Scopus (189) Google Scholar). Relaxation follows the re-uptake of Ca2+ into a distinct domain of the SR, which has been referred to as the longitudinal or network SR (4Franzini-Armstrong C. FASEB J. 1999; 13 Suppl. 2: S266-S270PubMed Google Scholar). Typically, the network SR is positioned around the Z-disks and M-lines of each sarcomere, but the structural elements that determine its location have not been determined. Early ultrastructural studies demonstrated the presence of numerous filaments joining the periphery of sarcomeric Z-disks to adjacent SR membranes (5Franzini-Armstrong C. Myology. 2nd Ed. McGraw-Hill Inc., New York1994: 176-199Google Scholar), but the molecular identity of these structures remains elusive. We have searched for protein partners of small ankyrin-1 (sAnk1), a muscle-specific isoform of the erythroid ankyrin-1 gene that is concentrated in the network SR of striated muscle fibers, surrounding the Z-disks and M-lines (6Zhou D. Birkenmeier C.S. Williams M.W. Sharp J.J. Barker J.E. Bloch R.J. J. Cell Biol. 1997; 136: 621-631Crossref PubMed Scopus (89) Google Scholar). Ankyrins are a family of proteins that possess binding sites for diverse integral membrane proteins as well as cytoskeletal components (7Bennett V. J. Biol. Chem. 1992; 267: 8703-8706Abstract Full Text PDF PubMed Google Scholar, 8De Matteis M.A. Morrow J.S. Curr. Opin. Cell Biol. 1998; 10: 542-549Crossref PubMed Scopus (119) Google Scholar, 9Bennett V. Chen L. Curr. Opin. Cell Biol. 2001; 13: 61-67Crossref PubMed Scopus (140) Google Scholar). To date, molecular cloning has identified three distinct ankyrin genes in mammals (Ank1,Ank2, and Ank3) that are expressed as tissue-specific, alternatively spliced isoforms (10Mohler P.J. Gramolini A.O. Bennett V. J. Cell Sci. 2002; 115: 1565-1566Crossref PubMed Google Scholar, 11Rubtsov A.M. Lopina O.D. FEBS Lett. 2000; 482: 1-5Crossref PubMed Scopus (110) Google Scholar, 12Sedgwick S.G. Smerdon S.J. Trends Biochem. Sci. 1999; 24: 311-316Abstract Full Text Full Text PDF PubMed Scopus (664) Google Scholar).Ank1 is expressed predominantly in erythroid cells, striated muscle, and brain (13Lux S.E. John K.M. Bennett V. Nature. 1990; 344: 36-42Crossref PubMed Scopus (408) Google Scholar, 14Birkenmeier C.S. White R.A. Peters L.L. Hall E.J. Lux S.E. Barker J.E. J. Biol. 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Chem. 1995; 270: 2352-2359Abstract Full Text Full Text PDF PubMed Scopus (428) Google Scholar, 21Peters L.L. John K.M., Lu, F.M. Eicher E.M. Higgins A. Yialamas M. Turtzo L.C. Otsuka A.J. Lux S.E. J. Cell Biol. 1995; 130: 313-330Crossref PubMed Scopus (130) Google Scholar, 22Devarajan P. Stabach P.R. Mann A.S. Ardito T. Kashgarian M. Morrow J.S. J. Cell Biol. 1996; 133: 819-830Crossref PubMed Scopus (161) Google Scholar, 23Gagelin C. Bruno C. Deprette C. Ludosky M.A. Recouvreur M. Cartaud J. Cognard J. Raymond G. Kordeli E. J. Biol. Chem. 2002; 277: 12978-12987Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). The large canonical ankyrins share a similar structure, consisting of an N-terminal ∼89-kDa membrane-binding domain, a central ∼62-kDa spectrin-binding domain, and a C-terminal ∼55-kDa regulatory domain (10Mohler P.J. Gramolini A.O. Bennett V. J. Cell Sci. 2002; 115: 1565-1566Crossref PubMed Google Scholar, 11Rubtsov A.M. Lopina O.D. FEBS Lett. 2000; 482: 1-5Crossref PubMed Scopus (110) Google Scholar). In striated muscle, the products of the Ank1 gene include the large (∼210 kDa) and small (∼17–19 kDa) ankyrin isoforms (6Zhou D. Birkenmeier C.S. Williams M.W. Sharp J.J. Barker J.E. Bloch R.J. J. Cell Biol. 1997; 136: 621-631Crossref PubMed Scopus (89) Google Scholar,15Gallagher P.G. Tse W.T. Scarpa A.L. Lux S.E. Forget B.G. J. Biol. Chem. 1997; 272: 19220-19228Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). sAnk1 lacks both the membrane- and spectrin-binding regions of the larger form and has a C-terminal domain that is significantly shortened (24Gallagher P.G. Forget B.G. J. Biol. Chem. 1998; 273: 1339-1348Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 25Birkenmeier C.S. Sharp J.J. Gifford E.J. Deveau S.A. Barker J.E. Genomics. 1998; 50: 79-88Crossref PubMed Scopus (38) Google Scholar). The N-terminal portion of sAnk1 contains a unique 73-amino acid segment, whereas the C-terminal 82 residues are identical to the C-terminal sequence of the large ∼210-kDa ankyrin-1. The first 29 residues of sAnk1 are highly hydrophobic and target the molecule to the SR membrane, whereas the remaining 126 amino acids extend into the myoplasm. 2N. Porter, W. Resneck, A. O'Neill, D. van Rossum, and R. J. Bloch, unpublished data. Thus, the hydrophilic tail of sAnk1 is appropriately oriented in the cytoplasm of striated muscle fibers to serve as a binding site for sarcomeric proteins. Here, we describe a direct and specific association between sAnk1 and titin, also known as connectin (25Birkenmeier C.S. Sharp J.J. Gifford E.J. Deveau S.A. Barker J.E. Genomics. 1998; 50: 79-88Crossref PubMed Scopus (38) Google Scholar, 26Labeit S. Gautel M. Lakey A. Trinick J. EMBO J. 1992; 11: 1711-1716Crossref PubMed Scopus (279) Google Scholar, 27Trinick J. Tskhovrebova L. 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The remaining ∼10% consists of unique non-repetitive sequence motifs, including phosphorylation sites, binding sites for muscle-specific calpain proteases, and C-terminal Ser/Thr kinase domains (30Labeit S. Kolmerer B. Linke W.A. Circ. Res. 1997; 80: 290-294Crossref PubMed Scopus (203) Google Scholar, 33Importa S. Krueger J.K. Gautel M. Atkinson R.A. Lefevre J.F. Moulton S. Trewhella J. Pastore A. J. Mol. Biol. 1998; 284: 761-777Crossref PubMed Scopus (74) Google Scholar, 34Muhle-Goll C. Pastore A. Nilges M. Structure. 1998; 6: 1291-1302Abstract Full Text Full Text PDF PubMed Google Scholar, 35Linke W.A. Stockmeier M.R. Ivemeyer M. Hosser H. Mundel P. J. Cell Sci. 1998; 111: 1567-1574PubMed Google Scholar). The C-terminal 2 MDa of titin are located within the A-band, where titin tightly associates with the myosin thick filaments and several A-band proteins such as C-protein, M-protein, and myomesin (36Houmeida A. Holt J. Tskhovrebova L. Trinick J. J. Cell Biol. 1995; 131: 1741-1781Crossref Scopus (87) Google Scholar, 37Obermann W.M. Gautel M. Steiner F. van der Ven P.F. Weber K. Furst D.O. J. Cell Biol. 1996; 134: 1411-1453Crossref PubMed Scopus (168) Google Scholar, 38Obermann W.M. Gautel M. Weber K. Furst D.O. EMBO J. 1997; 16: 211-220Crossref PubMed Scopus (194) Google Scholar). The most C-terminal end of the molecule (∼200 kDa), which is embedded in the M-line, contains a Ser/Thr kinase domain, which implicates titin in myofibrillar signal transduction pathways (37Obermann W.M. Gautel M. Steiner F. van der Ven P.F. Weber K. Furst D.O. J. Cell Biol. 1996; 134: 1411-1453Crossref PubMed Scopus (168) Google Scholar, 38Obermann W.M. Gautel M. Weber K. Furst D.O. EMBO J. 1997; 16: 211-220Crossref PubMed Scopus (194) Google Scholar, 39Kolmerer B. Olivieri N. Witt C.C. Herrmann B.G. Labeit S. J. Mol. Biol. 1996; 256: 556-563Crossref PubMed Scopus (85) Google Scholar, 40Mayans O. van der Ven P.F. Wilm M. Mues A. Young P. Furst D.O. Wilmanns M. Gautel M. Nature. 1998; 395: 863-869Crossref PubMed Scopus (314) Google Scholar). In the I-band, titin (∼800 kDa to 1.5 MDa) carries proline/glutamate/valine/lysine-rich sequences, which confer great extensibility to the titin filaments (35Linke W.A. Stockmeier M.R. Ivemeyer M. Hosser H. Mundel P. J. Cell Sci. 1998; 111: 1567-1574PubMed Google Scholar, 41Witt C.C. Olivieri N. Centner T. Kolmerer B. Millevoi S. Labeit D. Jockusch H. Pastore A. Labeit S J. Struct. Biol. 1998; 122: 1-10Crossref Scopus (45) Google Scholar, 42Tskhovrebova L. Trinick J. Sleep J.A. Simmons R.M. Nature. 1997; 387: 308-312Crossref PubMed Scopus (668) Google Scholar, 43Trombitas K. Greaser M. Labeit S. Jin J.-P Kellermayer M. Helmes M. Granzier H. J. Cell Biol. 1998; 140: 853-859Crossref PubMed Scopus (200) Google Scholar, 44Linke W.A. Ivemeyer M. Mundel P. Stockmeyer M.R. Kolmerer B. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8052-8057Crossref PubMed Scopus (206) Google Scholar), in addition to numerous Ig domains. At the junction of the I-band with the Z-disk, titin interacts with the actin thin filaments, although it is still unclear which titin motifs mediate this interaction (45Linke W.A. Ivemeyer M. Labeit S. Hinssen H. Ruegg J.C. Gautel M. Biophys. J. 1997; 73: 905-919Abstract Full Text PDF PubMed Scopus (146) Google Scholar, 46Granzier H. Kellermeyer M. Trombitas K. Biophys. J. 1997; 73: 2043-2053Abstract Full Text PDF PubMed Scopus (99) Google Scholar). The N-terminal 80-kDa region of titin spans the entire Z-disk (47Gregorio C.C. Trombitas K. Centner T. Kolmerer B. Stier G. Kunkle K. Suzuki K. Obermayr F. Herrmann B. Granzier H. Sorimachi H. Labeit S. J. Cell Biol. 1998; 143: 1013-1027Crossref PubMed Scopus (247) Google Scholar). Several copies of a 45-residue repeat, called the Z-repeat, bind α-actinin within the Z-disk (47Gregorio C.C. Trombitas K. Centner T. Kolmerer B. Stier G. Kunkle K. Suzuki K. Obermayr F. Herrmann B. Granzier H. Sorimachi H. Labeit S. J. Cell Biol. 1998; 143: 1013-1027Crossref PubMed Scopus (247) Google Scholar, 48Ohtsuka H. Yajima H. Kimura S. Maruyama K. FEBS Lett. 1997; 401: 65-67Crossref PubMed Scopus (72) Google Scholar, 49Sorimachi H. Freiburg A. Kolmerer B. Ishiura S. Stier G. Gregorio C.C. Labeit D. Linke W.A. Suzuki K. Labeit S. J. Mol. Biol. 1998; 270: 688-695Crossref Scopus (170) Google Scholar). The two most N-terminal Ig domains of titin, which are constitutively expressed in all titin isoforms and reside in the periphery of the Z-disk, bind a recently identified, 19-kDa protein of striated muscle, referred to as T-cap or telethonin (47Gregorio C.C. Trombitas K. Centner T. Kolmerer B. Stier G. Kunkle K. Suzuki K. Obermayr F. Herrmann B. Granzier H. Sorimachi H. Labeit S. J. Cell Biol. 1998; 143: 1013-1027Crossref PubMed Scopus (247) Google Scholar, 50Mues A. van der Ven P.F.M. Young P. Furst D.O. Gautel M. FEBS Lett. 1998; 428: 111-114Crossref PubMed Scopus (138) Google Scholar). Titin has two functions in striated muscle: as a “molecular blueprint” for sarcomeric protein assembly during myofibrillogenesis and as a “molecular spring” that maintains myofibrillar integrity during contraction, relaxation, and stretch (27Trinick J. Tskhovrebova L. Trends Cell Biol. 1999; 9: 377-380Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 30Labeit S. Kolmerer B. Linke W.A. Circ. Res. 1997; 80: 290-294Crossref PubMed Scopus (203) Google Scholar, 32Gregorio C.C. Granzier H.L. Sorimachi H. Labeit S. Curr. Opin. Cell Biol. 1999; 11: 18-25Crossref PubMed Scopus (275) Google Scholar). Our results show that, in addition to binding T-cap, the two N-terminal Ig domains of titin interact specifically with sAnk1, suggesting that titin also coordinates the assembly of the contractile apparatus with the network SR that surrounds the Z-disk. PCR amplification was used to obtain cDNAs encoding fragments of the Z-disk (∼80 kDa) (47Gregorio C.C. Trombitas K. Centner T. Kolmerer B. Stier G. Kunkle K. Suzuki K. Obermayr F. Herrmann B. Granzier H. Sorimachi H. Labeit S. J. Cell Biol. 1998; 143: 1013-1027Crossref PubMed Scopus (247) Google Scholar) and M-line (∼200 kDa) (37Obermann W.M. Gautel M. Steiner F. van der Ven P.F. Weber K. Furst D.O. J. Cell Biol. 1996; 134: 1411-1453Crossref PubMed Scopus (168) Google Scholar) portions of titin. cDNA from human cardiac muscle (Origene Technologies Inc., Rockville, MD) was used as template, and the following sets of custom oligonucleotide primers were generated, based on the sequence of human cardiac titin (GenBankTM/EBI accession number X90568). For amplification of ZIg1/2, the two most N-terminal Ig domains of titin, primers A (5′-ACGTGAATTCATGACAACTCAAGCACCG-3′, sense) and B (5′-ACGTCTCGAGAGGTACTTCTTCTTCACC-3′, antisense) were used. For ZIg1/2-A (including ZIg1 only), the sense primer A was used in combination with the antisense primer C (5′-ACGTCTCGAGAGCTTTCACGAGAAGCTC-3′). For generation of ZIg1/2-B (containing ZIg2 only), primer D (5′-ACGTGAATTCGAGACAGCACCACCCAAC-3′) was used along with the antisense primer B. For ZIg3, the Ig domain just C-terminal to ZIg1/2, primer E (5′-ACGTGAATTCGCTAAAAAGACAAAGACA-3′, sense) was utilized in combination with primer F (5′-ACGTCTCGAGCATTATTGCTTCTTGAGT-3′, antisense). For amplification of Zr (forZ-repeat), the region of titin that interacts with α-actinin, primer G (5′-ACGTGAATTCAAGGAAACTAGGAAAACA-3′, sense) was used with primer H (5′-ACGTCTCGAGGACAGTCACATTTTTTAA-3′, antisense). For ZIg4/5, immediately following the Z-repeat region, primers I (5′-ACGTGAATTCATAGAAGGTGAATCTGTC-3′, sense) and J (5′-ACGTCTCGAGTCCATGCTTATTGCGAAC-3′, (antisense) were used. For generation of MIg1/2, the first two Ig domains in the M-line region of titin, primer K (5′-ACGTGAATTCGGTGAAAATGTCCGGTT-3′, sense) was used with primer L (5′-ACGTCTCGAGCCCAGCTGTGTTAGT-3′, antisense). For MIg5/6, two additional Ig domains in the M-line region of titin, primer M (5′-ACGTGAATTCCTGACCTGTGTGGTTGAA-3′, sense) was utilized in combination with primer N (5′-ACGTCTCGAGTCCAGCTGAATTTTTTAC-3′, antisense). All sense primers carried an EcoRI recognition sequence, whereas all antisense primers contained an XhoI site for insertion into the yeast two-hybrid pB42AD prey vector (Clontech, Palo Alto, CA) and the pGEX4T-1 vector (Amersham Biosciences) for production of glutathioneS-transferase (GST) fusion proteins. The titin ZIg1/2 fragment was also introduced into the pMAL-c2X vector atEcoRI/SalI sites (New England Biolabs Inc., Beverly, MA) (XhoI and SalI sites have compatible ends). Similarly, a fragment encoding the C-terminal hydrophilic sequence of sAnk1-(29–155) (25Birkenmeier C.S. Sharp J.J. Gifford E.J. Deveau S.A. Barker J.E. Genomics. 1998; 50: 79-88Crossref PubMed Scopus (38) Google Scholar) was inserted into the yeast two-hybrid pLexA bait vector and the pGEX4T-1 vector atEcoRI/XhoI sites after PCR amplification with primers 1 (5′-ACTGGAATTCGTCAAGGGTTCCCTGTGC-3′, sense) and 2 (5′-ACTGCTCGAGCTGCTTGCCCCTTTT, antisense). An identical set of primers carrying EcoRI and SalI recognition sites was used for insertion into the pMAL-c2X vector to produce a maltose-binding protein (MBP) fusion peptide. Additional sets of primers were used for amplification of sAnk1 deletion constructs. For sAnk1-A, the sense primer 1 was used in combination with the antisense primer 3 (5′-ACTGCTCGAGTTGTTCCTCTGTCAC-3′). For generation of sAnk1-B, the sense primer 4 (5′-ACTGGAATTCTTCACAGACGAACAG-3′) was used with the antisense primer 2. For sAnk1-C, primer 5 (5′-ACTGGAATCATCTCCACCAGGGTG-3′, sense) was used with primer 6 (5′-ACTGCTCGAGTCCACTCCCTCTTAG-3′, antisense). For generation of MBP-T-cap fusion protein, the full-length pET9D-T-cap plasmid (a generous gift from Drs. S. Labeit (European Molecular Biology Laboratory, Heidelberg, Germany) and C. C. Gregorio (University of Arizona, Tucson, AZ)) was used as template to obtain a PCR fragment that contained amino acids 1–140 (47Gregorio C.C. Trombitas K. Centner T. Kolmerer B. Stier G. Kunkle K. Suzuki K. Obermayr F. Herrmann B. Granzier H. Sorimachi H. Labeit S. J. Cell Biol. 1998; 143: 1013-1027Crossref PubMed Scopus (247) Google Scholar). Primers 7 (5′-ACGTGAATTCATGGCTACCTCAGAGCTG-3′, sense) and 8 (5′-ACGTGTCGACTCATGTCTCCAGCGCCAG-3′, antisense), carryingEcoRI and SalI sites, respectively, were used for insertion into the pMAL-c2X vector. T-cap-(1–140) was also introduced into the pGEX4T-1 vector at EcoRI/XhoI sites (XhoI and SalI have compatible ends) to produce a GST fusion protein. The authenticity of the obtained constructs was verified by sequencing analysis. GST and MBP recombinant polypeptides were expressed by induction with 0.5 mmisopropyl-β-d-thiogalactopyranoside for 3 h and purified by affinity chromatography on glutathione-agarose (for GST fusion proteins) (Amersham Biosciences) or amylose resin (for MBP fusion proteins) (New England Biolabs, Inc.) columns following the manufacturers' instructions. The Matchmaker LexA two-hybrid system (Clontech) was used as recommended by the manufacturer. The pB42AD prey vector and the pLexA bait vector were used to express titin (i.e. ZIg1/2, ZIg1/2-A, ZIg1/2-B, Zr, ZIg3, ZIg4/5, MIg1/2, and MIg5/6) and sAnk1 (i.e. sAnk1-(29–155), sAnk1-A, sAnk1-B, and sAnk1-C) hybrid peptides, respectively, as described above. Saccharomyces cerevisiae strain EGY48 was sequentially transformed with reporter p8op-lacZ, bait, and prey plasmids. True transformants were selected by plating on induction medium (i.e. synthetic dropout Gal/Raf lacking Ura, His, Trp, and Leu) in the presence of 80 mg/liter 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside (X-gal). Liquid β-galactosidase assays were performed as described in the Clontech Yeast Protocols Handbook using chlorophenol red β-d-galactopyranoside as substrate. For each interaction tested, four independent colonies were assayed, and each experiment was repeated twice. Results represent average values. Homogenates of quadriceps muscle of adult Sprague-Dawley rats (Zivic-Miller Laboratories, Inc., Zelienople, PA) were prepared at room temperature for 2–3 min with a Brinkmann Polytron homogenizer at setting 3 (VWR, West Chester, PA) in 10 mm NaPO4 (pH 7.2), 2 mmEDTA, 10 mm NaN3, 120 mm NaCl, and 1% Nonidet P-40 supplemented with a mixture of protease inhibitors (Roche Molecular Biochemicals). Equal amounts of recombinant GST and GST-ZIg1/2, GST-Zr, GST-ZIg3, GST-ZIg4/5, GST-MIg1/2, and GST-MIg5/6 fusion proteins were bound to glutathione-Sepharose and mixed with 0.5 mg of quadriceps muscle homogenate at 4 °C for 16 h. Beads were washed in the cold with 10 mm NaPO4 (pH 7.2), 120 mm NaCl, 10 mm NaN3, and 0.1% Tween 20 and heated for 5 min at 90 °C in 2× SDS Laemmli sample buffer. The soluble fraction was analyzed by 12% SDS-PAGE, transferred to nitrocellulose, and probed with antibodies to sAnk1. Immunoreactive bands were visualized with a chemiluminescence detection kit (Tropix Inc., Bedford, MA). The blot overlay assays were performed as previously described with minor modifications (51Kontrogianni-Konstantopoulos A. Huang S.-C. Benz E.J., Jr. Mol. Biol. Cell. 2000; 11: 3805-3817Crossref PubMed Scopus (39) Google Scholar). Briefly, ∼2.5 μg of bacterially expressed, affinity-purified GST and GST-ZIg1/2 proteins were separated by 12% SDS-PAGE and transferred to nitrocellulose. Nonspecific sites on the nitrocellulose membranes were blocked in buffer A (50 mm Tris (pH 7.2), 120 mm NaCl, 3% bovine serum albumin, 2 mmdithiothreitol, 0.5% Nonidet P-40, and 0.1% Tween 20) plus protease inhibitors for 3 h at 25 °C and then incubated with 3 μg/ml MBP-sAnk1 fusion protein in the same buffer for 16 h at 4 °C. Blots were washed five times (15 min each) with buffer A and once with buffer B (1× phosphate-buffered saline (pH 7.2), 10 mmNaN3, and 0.1% Tween 20). Subsequently, they were incubated in buffer C (1× phosphate-buffered saline (pH 7.2), 10 mm NaN3, 0.1% Tween 20, and 3% dry milk) and probed with antibodies to sAnk1, diluted in buffer C. In a set of parallel assays, increasing concentrations of affinity-purified GST-ZIg1/2 fusion protein (i.e. 5 and 10 μg) were added to buffer A along with MBP-sAnk1, and blots were processed as just described. Equivalent amounts of GST protein, GST-sAnk1, and GST-T-cap bound to glutathione matrices were allowed to interact with 5 μg of recombinant MBP-ZIg1/2, MBP-sAnk1, or MBP-T-cap in 250 μl of binding buffer (50 mm Tris (pH 7.2), 120 mm NaCl, 10 mm NaN3, 2 mm dithiothreitol, 0.1% Tween 20, and 10 mmmaltose plus protease inhibitors) for 12 h at 4 °C. Subsequently, the supernatants were removed, and the glutathione beads were extensively washed with a solution containing 50 mmTris (pH 7.2), 120 mm NaCl, 10 mmNaN3, and 0.1% Tween 20. In a parallel set of experiments, GST-sAnk1 and GST-T-cap attached to glutathione matrices were initially allowed to interact with 5 μg of bacterially expressed MBP-ZIg1/2 for 12 h at 4 °C. Following removal of the supernatants, 5 μg of affinity-purified MBP-T-cap or MBP-sAnk1 were added to the GST-sAnk1·MBP-ZIg1/2 or GST-T-cap·MBP-ZIg1/2 complexes, respectively, and allowed to interact for another 12 h in the cold. At the end of the incubation period, the glutathione beads were washed four times (15 min each) and dissolved in 2× SDS Laemmli sample buffer. The soluble proteins were fractionated on 12% SDS-PAGE, transferred to nitrocellulose, and probed with the appropriate antibodies. Frozen longitudinal sections and cross-sections of quadriceps muscle of adult rats were prepared as previously described (52Williams M.W. Resneck W.G. Kaysser T. Ursitti J.A. Birkenmeier C.S. Barker J.E. Bloch R.J. J. Cell Sci. 2001; 114: 751-762Crossref PubMed Google Scholar). Sections were incubated in buffer D (1× phosphate-buffered saline, 5% normal donkey serum, and 10 mm NaN3) for 1–2 h at 25 °C. Primary antibodies, including rabbit anti-titin-x112/x113 (3 μg/ml; a generous gift from Dr. C. C. Gregorio), rabbit anti-sAnk1 (3 μg/ml) (6Zhou D. Birkenmeier C.S. Williams M.W. Sharp J.J. Barker J.E. Bloch R.J. J. Cell Biol. 1997; 136: 621-631Crossref PubMed Scopus (89) Google Scholar), goat anti-telethonin (N-20 or C-20, 6 μg/ml; Santa Cruz Biotechnology), and rabbit (3 μg/ml) or goat (6 μg/ml) ChromaPure IgG (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA), were diluted in buffer D and added to the sections for 12 h at 4 °C. Following extensive washing with the same buffer, samples were counterstained with the appropriate secondary antibodies, including Alexa-568 goat anti-rabbit IgG and Alexa-488 donkey anti-goat IgG (Molecular Probes, Inc., Eugene, OR), in buffer D at 1:100 dilution for 1 h at 25 °C. Subsequently, sections were washed three times (15 min each), mounted with Vectashield (Vector Laboratories, Inc., Burlingame, CA), and analyzed with a Zeiss 410 confocal laser scanning microscope (Carl Zeiss, Inc., Tarrytown, NY) equipped with a ×63 NA 1.4 objective. Unless otherwise noted, all reagents were from Sigma and were the highest grade available. sAnk1 is an ∼17–19-kDa integral membrane protein of the network SR that has a 126-amino acid sequence extending into the sarcoplasm surrounding Z-disks and M-lines (6Zhou D. Birkenmeier C.S. Williams M.W. Sharp J.J. Barker J.E. Bloch R.J. J. Cell Biol. 1997; 136: 621-631Crossref PubMed Scopus (89) Google Scholar).2 We used the yeast two-hybrid assay to test the idea that the hydrophilic sequence of sAnk1 (sAnk1-(29–155)) interacts with the giant myofibrillar protein titin, which spans each half-sarcomere from the Z-disk to the M-line. We inserted cDNA encoding the hydrophilic cytoplasmic domain of sAnk1 (sAnk1-(29–155)) (25Birkenmeier C.S. Sharp J.J. Gifford E.J. Deveau S.A. Barker J.E. Genomics. 1998; 50: 79-88Crossref PubMed Scopus (38) Google Scholar) into the yeast two-hybrid pLexA bait vector (Fig. 1 A) and assayed its ability to interact with the N-terminal ∼80-kDa portion of titin that resides in the Z-line (47Gregorio C.C. Trombitas K. Centner T. Kolmerer B. Stier G. Kunkle K. Suzuki K. Obermayr F. Herrmann B. Granzier H. Sorimachi H. Labeit S. J. Cell Biol. 1998; 143: 1013-1027Crossref PubMed Scopus (247) Google Scholar), expressed by a series of constructs inserted into the yeast two-hybrid pB42AD prey vector (Fig. 1 B). Specifically, the PCR products of titin we assayed were ZIg1/2 (amino acids 1–200), ZIg3 (amino acids 201–557), the Z-repeats or Zr domain (amino acids 558–910), and ZIg4/5 (amino acids 911–1118) (see “Experimental Procedures”). Yeast two-hybrid analysis followed by qualitative liquid β-galactosidase assays (Fig. 1 C) indicated that sAnk1-(29–155) specifically interacted with the two most N-terminal Ig domains of titin, ZIg1/2 (amino acids 1–200), which reside at the edge of the Z-disk (28Furst D.O. Osborn M. Nave R. Weber K. J. Cell Biol. 1988; 106: 1563-1572Crossref PubMed Scopus (516) Google Scholar, 47Gregorio C.C. Trombitas K. Centner T. Kolmerer B. Stier G. Kunkle K. Suzuki K. Obermayr F. Herrmann B. Granzier H. Sorimachi H. Labeit S. J. Cell Biol. 1998; 143: 1013-1027Crossref PubMed Scopus (247) Google Scholar). No specific association between sAnk1-(29–155) and the remaining ∼60-kDa portion of Z-disk titin could be detected (Fig. 1 C). In additional tests of the specificity of the interaction with ZIg1/2, we generated two additional “prey” constructs encoding tandem Ig domains that reside in the M-line region of titin (37Obermann W.M. Gautel M. Steiner F. van der Ven P.F. Weber K. Furst D.O. J. Cell Biol. 1996; 134: 1411-1453Crossref PubMed Scopus (168) Google Scholar, 38Obermann W.M. Gautel M. Weber K. Furst D.O. EMBO J. 1997; 16: 211-220Crossref PubMed Scopus (194) Google Scholar, 3

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