Abstract
The actin filament core within each microvillus of the intestinal epithelial cell is attached laterally to the plasma membrane by brush border (BB) myosin I, a protein-calmodulin complex belonging to the myosin I class of actin-based mechanoenzymes. In this report, the binding of BB myosin I to pure phospholipid vesicles was examined and characterized. BB myosin I demonstrated saturable binding to liposomes composed of anionic phospholipids, but did not associate with liposomes composed of only neutral phospholipids. The binding of BB myosin I to phosphatidylserine and phosphatidylglycerol vesicles reached saturation at 4-5 x 10(-3) nmol protein/nmol phospholipid, while the apparent dissociation constant was determined to be 1-3 x 10(-7) M. Similar to the free protein, membrane-associated BB myosin I bound F-actin in an ATP-sensitive manner and demonstrated actin-activated Mg-ATPase activity. Immunoblot analysis of peptides generated from controlled proteolysis of vesicle-bound BB myosin I provided structural information concerning the site responsible for the membrane interaction. Immunoblot staining with domain-specific mAbs revealed a series of COOH-terminal, liposome-associated peptides that were protected from digestion, suggesting that the membrane-binding domain is within the carboxy-terminal "tail" of the BB myosin I heavy chain.
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- Adams R. J., Pollard T. D. Binding of myosin I to membrane lipids. Nature. 1989 Aug 17;340(6234):565–568. doi: 10.1038/340565a0. [DOI] [PubMed] [Google Scholar]
- Adams R. J., Pollard T. D. Membrane-bound myosin-I provides new mechanisms in cell motility. Cell Motil Cytoskeleton. 1989;14(2):178–182. doi: 10.1002/cm.970140203. [DOI] [PubMed] [Google Scholar]
- Benfenati F., Greengard P., Brunner J., Bähler M. Electrostatic and hydrophobic interactions of synapsin I and synapsin I fragments with phospholipid bilayers. J Cell Biol. 1989 May;108(5):1851–1862. doi: 10.1083/jcb.108.5.1851. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Burgess W. H., Jemiolo D. K., Kretsinger R. H. Interaction of calcium and calmodulin in the presence of sodium dodecyl sulfate. Biochim Biophys Acta. 1980 Jun 26;623(2):257–270. doi: 10.1016/0005-2795(80)90254-8. [DOI] [PubMed] [Google Scholar]
- Carboni J. M., Conzelman K. A., Adams R. A., Kaiser D. A., Pollard T. D., Mooseker M. S. Structural and immunological characterization of the myosin-like 110-kD subunit of the intestinal microvillar 110K-calmodulin complex: evidence for discrete myosin head and calmodulin-binding domains. J Cell Biol. 1988 Nov;107(5):1749–1757. doi: 10.1083/jcb.107.5.1749. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Cohen A. M., Liu S. C., Lawler J., Derick L., Palek J. Identification of the protein 4.1 binding site to phosphatidylserine vesicles. Biochemistry. 1988 Jan 26;27(2):614–619. doi: 10.1021/bi00402a018. [DOI] [PubMed] [Google Scholar]
- Collins J. H., Borysenko C. W. The 110,000-dalton actin- and calmodulin-binding protein from intestinal brush border is a myosin-like ATPase. J Biol Chem. 1984 Nov 25;259(22):14128–14135. [PubMed] [Google Scholar]
- Coluccio L. M., Bretscher A. Calcium-regulated cooperative binding of the microvillar 110K-calmodulin complex to F-actin: formation of decorated filaments. J Cell Biol. 1987 Jul;105(1):325–333. doi: 10.1083/jcb.105.1.325. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Coluccio L. M., Bretscher A. Mapping of the microvillar 110K-calmodulin complex: calmodulin-associated or -free fragments of the 110-kD polypeptide bind F-actin and retain ATPase activity. J Cell Biol. 1988 Feb;106(2):367–373. doi: 10.1083/jcb.106.2.367. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Coluccio L. M., Bretscher A. Reassociation of microvillar core proteins: making a microvillar core in vitro. J Cell Biol. 1989 Feb;108(2):495–502. doi: 10.1083/jcb.108.2.495. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Conzelman K. A., Mooseker M. S. The 110-kD protein-calmodulin complex of the intestinal microvillus is an actin-activated MgATPase. J Cell Biol. 1987 Jul;105(1):313–324. doi: 10.1083/jcb.105.1.313. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Coudrier E., Reggio H., Louvard D. Characterization of an integral membrane glycoprotein associated with the microfilaments of pig intestinal microvilli. EMBO J. 1983;2(3):469–475. doi: 10.1002/j.1460-2075.1983.tb01446.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- De Lozanne A., Spudich J. A. Disruption of the Dictyostelium myosin heavy chain gene by homologous recombination. Science. 1987 May 29;236(4805):1086–1091. doi: 10.1126/science.3576222. [DOI] [PubMed] [Google Scholar]
- Fukushima D., Yokoyama S., Kézdy F. J., Kaiser E. T. Binding of amphiphilic peptides to phospholipid/cholesterol unilamellar vesicles: a model for protein--cholesterol interaction. Proc Natl Acad Sci U S A. 1981 May;78(5):2732–2736. doi: 10.1073/pnas.78.5.2732. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Garcia A., Coudrier E., Carboni J., Anderson J., Vandekerkhove J., Mooseker M., Louvard D., Arpin M. Partial deduced sequence of the 110-kD-calmodulin complex of the avian intestinal microvillus shows that this mechanoenzyme is a member of the myosin I family. J Cell Biol. 1989 Dec;109(6 Pt 1):2895–2903. doi: 10.1083/jcb.109.6.2895. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hoshimaru M., Fujio Y., Sobue K., Sugimoto T., Nakanishi S. Immunochemical evidence that myosin I heavy chain-like protein is identical to the 110-kilodalton brush-border protein. J Biochem. 1989 Sep;106(3):455–459. doi: 10.1093/oxfordjournals.jbchem.a122873. [DOI] [PubMed] [Google Scholar]
- Hoshimaru M., Nakanishi S. Identification of a new type of mammalian myosin heavy chain by molecular cloning. Overlap of its mRNA with preprotachykinin B mRNA. J Biol Chem. 1987 Oct 25;262(30):14625–14632. [PubMed] [Google Scholar]
- Jung G., Schmidt C. J., Hammer J. A., 3rd Myosin I heavy-chain genes of Acanthamoeba castellanii: cloning of a second gene and evidence for the existence of a third isoform. Gene. 1989 Oct 30;82(2):269–280. doi: 10.1016/0378-1119(89)90052-8. [DOI] [PubMed] [Google Scholar]
- Kaiser E. T., Kézdy F. J. Amphiphilic secondary structure: design of peptide hormones. Science. 1984 Jan 20;223(4633):249–255. doi: 10.1126/science.6322295. [DOI] [PubMed] [Google Scholar]
- Keller T. C., 3rd, Mooseker M. S. Ca++-calmodulin-dependent phosphorylation of myosin, and its role in brush border contraction in vitro. J Cell Biol. 1982 Dec;95(3):943–959. doi: 10.1083/jcb.95.3.943. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Knecht D. A., Loomis W. F. Antisense RNA inactivation of myosin heavy chain gene expression in Dictyostelium discoideum. Science. 1987 May 29;236(4805):1081–1086. doi: 10.1126/science.3576221. [DOI] [PubMed] [Google Scholar]
- Knecht D. A., Loomis W. F. Developmental consequences of the lack of myosin heavy chain in Dictyostelium discoideum. Dev Biol. 1988 Jul;128(1):178–184. doi: 10.1016/0012-1606(88)90280-1. [DOI] [PubMed] [Google Scholar]
- Korn E. D., Hammer J. A., 3rd Myosins of nonmuscle cells. Annu Rev Biophys Biophys Chem. 1988;17:23–45. doi: 10.1146/annurev.bb.17.060188.000323. [DOI] [PubMed] [Google Scholar]
- Krizek J., Coluccio L. M., Bretscher A. ATPase activity of the microvillar 110 kDa polypeptide-calmodulin complex is activated in Mg2+ and inhibited in K+-EDTA by F-actin. FEBS Lett. 1987 Dec 10;225(1-2):269–272. doi: 10.1016/0014-5793(87)81172-9. [DOI] [PubMed] [Google Scholar]
- Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
- Lau S. H., Rivier J., Vale W., Kaiser E. T., Kézdy F. J. Surface properties of an amphiphilic peptide hormone and of its analog: corticotropin-releasing factor and sauvagine. Proc Natl Acad Sci U S A. 1983 Dec;80(23):7070–7074. doi: 10.1073/pnas.80.23.7070. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Matsudaira P. T., Burgess D. R. SDS microslab linear gradient polyacrylamide gel electrophoresis. Anal Biochem. 1978 Jul 1;87(2):386–396. doi: 10.1016/0003-2697(78)90688-7. [DOI] [PubMed] [Google Scholar]
- Mimms L. T., Zampighi G., Nozaki Y., Tanford C., Reynolds J. A. Phospholipid vesicle formation and transmembrane protein incorporation using octyl glucoside. Biochemistry. 1981 Feb 17;20(4):833–840. doi: 10.1021/bi00507a028. [DOI] [PubMed] [Google Scholar]
- Miyata H., Bowers B., Korn E. D. Plasma membrane association of Acanthamoeba myosin I. J Cell Biol. 1989 Oct;109(4 Pt 1):1519–1528. doi: 10.1083/jcb.109.4.1519. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Mooseker M. S., Coleman T. R. The 110-kD protein-calmodulin complex of the intestinal microvillus (brush border myosin I) is a mechanoenzyme. J Cell Biol. 1989 Jun;108(6):2395–2400. doi: 10.1083/jcb.108.6.2395. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Mooseker M. S., Conzelman K. A., Coleman T. R., Heuser J. E., Sheetz M. P. Characterization of intestinal microvillar membrane disks: detergent-resistant membrane sheets enriched in associated brush border myosin I (110K-calmodulin). J Cell Biol. 1989 Sep;109(3):1153–1161. doi: 10.1083/jcb.109.3.1153. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Pardee J. D., Spudich J. A. Purification of muscle actin. Methods Cell Biol. 1982;24:271–289. doi: 10.1016/s0091-679x(08)60661-5. [DOI] [PubMed] [Google Scholar]
- Rodaway A. R., Sternberg M. J., Bentley D. L. Similarity in membrane proteins. Nature. 1989 Dec 7;342(6250):624–624. doi: 10.1038/342624a0. [DOI] [PubMed] [Google Scholar]
- Shibayama T., Carboni J. M., Mooseker M. S. Assembly of the intestinal brush border: appearance and redistribution of microvillar core proteins in developing chick enterocytes. J Cell Biol. 1987 Jul;105(1):335–344. doi: 10.1083/jcb.105.1.335. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Szoka F., Jr, Papahadjopoulos D. Comparative properties and methods of preparation of lipid vesicles (liposomes). Annu Rev Biophys Bioeng. 1980;9:467–508. doi: 10.1146/annurev.bb.09.060180.002343. [DOI] [PubMed] [Google Scholar]
- TAUSSKY H. H., SHORR E. A microcolorimetric method for the determination of inorganic phosphorus. J Biol Chem. 1953 Jun;202(2):675–685. [PubMed] [Google Scholar]
- Vorherr T., James P., Krebs J., Enyedi A., McCormick D. J., Penniston J. T., Carafoli E. Interaction of calmodulin with the calmodulin binding domain of the plasma membrane Ca2+ pump. Biochemistry. 1990 Jan 16;29(2):355–365. doi: 10.1021/bi00454a008. [DOI] [PubMed] [Google Scholar]
- Wessels D., Soll D. R., Knecht D., Loomis W. F., De Lozanne A., Spudich J. Cell motility and chemotaxis in Dictyostelium amebae lacking myosin heavy chain. Dev Biol. 1988 Jul;128(1):164–177. doi: 10.1016/0012-1606(88)90279-5. [DOI] [PubMed] [Google Scholar]