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. 1996 May 2;133(4):819–830. doi: 10.1083/jcb.133.4.819

Identification of a small cytoplasmic ankyrin (AnkG119) in the kidney and muscle that binds beta I sigma spectrin and associates with the Golgi apparatus

PMCID: PMC2120834  PMID: 8666667

Abstract

Ankyrins are a family of large, membrane-associated proteins that mediate the linkage of the cytoskeleton to a variety of membrane transport and receptor proteins. A repetitive 33-residue motif characteristic of domain I of ankyrin has also been identified in proteins involved with cell cycle control and development. We have cloned and characterized a novel ankyrin isoform, AnkG119 (GenBank accession No. U43965), from the human kidney which lacks part of this repetitive domain and associates in MDCK cells with beta I sigma spectrin and the Golgi apparatus, but not the plasma membrane. Sequence comparison reveals this ankyrin to be an alternative transcript of AnkG, a much larger ankyrin recently cloned from brain. AnkG119 has a predicted size of 119,201 D, and contains a 47-kD domain I consisting of 13 ankyrin repeat units, a 67-kD domain II with a highly conserved spectrin-binding motif, and a truncated 5-kD putative regulatory domain. An AnkG119 cDNA probe hybridized to a 6.0-kb message in human and rat kidney, placenta, and skeletal muscle. An antibody raised to AnkG119 recognized an apparent 116-kD peptide in rat kidney cortical tissue and MDCK cell lysates, and did not react with larger isoforms of ankyrin at 190 and 210 kD in these tissues, nor in bovine brain, nor with ankyrin from human erythrocytes. AnkG119 remains extractable in 0.5% Triton X-100, and assumes a punctuate cytoplasmic distribution in mature MDCK cells, in contrast to the Triton-stable plasma membrane localization of all previously described renal ankyrins. AnkG119 immunocreativity in subconfluent MDCK cells distributes with the Golgi complex in a pattern coincident with beta -COP and beta I sigma spectrin immunoreactivity. A fusion peptide containing residues 669-860 of AnkG119 interacts with beta I sigma 1 spectrin in vitro with a Kd = 4.2 +/- 4.0 ( +/- 2 SD) nM, and avidly binds the beta spectrin in MDCK cell lysates. Collectively, these data identify AnkG119 as a novel small ankyrin that binds and colocalizes with beta I sigma spectrin in the ER and Golgi apparatus, and possible on a subset of endosomes during the early stages of polarity development. We hypothesize that AnkG119 and beta I spectrin form a vesicular Golgi-associated membrane skeleton, promote the organization of protein microdomains within the Golgi and trans-Golgi networks, and contribute to polarized vesicle transport.

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Selected References

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  1. Axton J. M., Shamanski F. L., Young L. M., Henderson D. S., Boyd J. B., Orr-Weaver T. L. The inhibitor of DNA replication encoded by the Drosophila gene plutonium is a small, ankyrin repeat protein. EMBO J. 1994 Jan 15;13(2):462–470. doi: 10.1002/j.1460-2075.1994.tb06281.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Beck K. A., Buchanan J. A., Malhotra V., Nelson W. J. Golgi spectrin: identification of an erythroid beta-spectrin homolog associated with the Golgi complex. J Cell Biol. 1994 Nov;127(3):707–723. doi: 10.1083/jcb.127.3.707. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bennett V. Ankyrins. Adaptors between diverse plasma membrane proteins and the cytoplasm. J Biol Chem. 1992 May 5;267(13):8703–8706. [PubMed] [Google Scholar]
  4. Bensadoun A., Weinstein D. Assay of proteins in the presence of interfering materials. Anal Biochem. 1976 Jan;70(1):241–250. doi: 10.1016/s0003-2697(76)80064-4. [DOI] [PubMed] [Google Scholar]
  5. Birkenmeier C. S., White R. A., Peters L. L., Hall E. J., Lux S. E., Barker J. E. Complex patterns of sequence variation and multiple 5' and 3' ends are found among transcripts of the erythroid ankyrin gene. J Biol Chem. 1993 May 5;268(13):9533–9540. [PubMed] [Google Scholar]
  6. Bork P. Hundreds of ankyrin-like repeats in functionally diverse proteins: mobile modules that cross phyla horizontally? Proteins. 1993 Dec;17(4):363–374. doi: 10.1002/prot.340170405. [DOI] [PubMed] [Google Scholar]
  7. Bourguignon L. Y., Walker G., Suchard S. J., Balazovich K. A lymphoma plasma membrane-associated protein with ankyrin-like properties. J Cell Biol. 1986 Jun;102(6):2115–2124. doi: 10.1083/jcb.102.6.2115. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Chan W., Kordeli E., Bennett V. 440-kD ankyrinB: structure of the major developmentally regulated domain and selective localization in unmyelinated axons. J Cell Biol. 1993 Dec;123(6 Pt 1):1463–1473. doi: 10.1083/jcb.123.6.1463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Chomczynski P., Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987 Apr;162(1):156–159. doi: 10.1006/abio.1987.9999. [DOI] [PubMed] [Google Scholar]
  10. Davis J. Q., Bennett V. Brain ankyrin. A membrane-associated protein with binding sites for spectrin, tubulin, and the cytoplasmic domain of the erythrocyte anion channel. J Biol Chem. 1984 Nov 10;259(21):13550–13559. [PubMed] [Google Scholar]
  11. Davis J. Q., Bennett V. The anion exchanger and Na+K(+)-ATPase interact with distinct sites on ankyrin in in vitro assays. J Biol Chem. 1990 Oct 5;265(28):17252–17256. [PubMed] [Google Scholar]
  12. Davis J., Davis L., Bennett V. Diversity in membrane binding sites of ankyrins. Brain ankyrin, erythrocyte ankyrin, and processed erythrocyte ankyrin associate with distinct sites in kidney microsomes. J Biol Chem. 1989 Apr 15;264(11):6417–6426. [PubMed] [Google Scholar]
  13. Davis L. H., Otto E., Bennett V. Specific 33-residue repeat(s) of erythrocyte ankyrin associate with the anion exchanger. J Biol Chem. 1991 Jun 15;266(17):11163–11169. [PubMed] [Google Scholar]
  14. Devarajan P., Scaramuzzino D. A., Morrow J. S. Ankyrin binds to two distinct cytoplasmic domains of Na,K-ATPase alpha subunit. Proc Natl Acad Sci U S A. 1994 Apr 12;91(8):2965–2969. doi: 10.1073/pnas.91.8.2965. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Devereux J., Haeberli P., Smithies O. A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 1984 Jan 11;12(1 Pt 1):387–395. doi: 10.1093/nar/12.1part1.387. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Diederich R. J., Matsuno K., Hing H., Artavanis-Tsakonas S. Cytosolic interaction between deltex and Notch ankyrin repeats implicates deltex in the Notch signaling pathway. Development. 1994 Mar;120(3):473–481. doi: 10.1242/dev.120.3.473. [DOI] [PubMed] [Google Scholar]
  17. Dubreuil R. R., Yu J. Ankyrin and beta-spectrin accumulate independently of alpha-spectrin in Drosophila. Proc Natl Acad Sci U S A. 1994 Oct 25;91(22):10285–10289. doi: 10.1073/pnas.91.22.10285. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Gallagher P. G., Tse W. T., Scarpa A. L., Lux S. E., Forget B. G. Large numbers of alternatively spliced isoforms of the regulatory region of human erythrocyte ankyrin. Trans Assoc Am Physicians. 1992;105:268–277. [PubMed] [Google Scholar]
  19. Griffiths G., Pepperkok R., Locker J. K., Kreis T. E. Immunocytochemical localization of beta-COP to the ER-Golgi boundary and the TGN. J Cell Sci. 1995 Aug;108(Pt 8):2839–2856. doi: 10.1242/jcs.108.8.2839. [DOI] [PubMed] [Google Scholar]
  20. Harris A. S., Anderson J. P., Yurchenco P. D., Green L. A., Ainger K. J., Morrow J. S. Mechanisms of cytoskeletal regulation: functional and antigenic diversity in human erythrocyte and brain beta spectrin. J Cell Biochem. 1986;30(1):51–69. doi: 10.1002/jcb.240300107. [DOI] [PubMed] [Google Scholar]
  21. Harris A. S., Croall D. E., Morrow J. S. Calmodulin regulates fodrin susceptibility to cleavage by calcium-dependent protease I. J Biol Chem. 1989 Oct 15;264(29):17401–17408. [PubMed] [Google Scholar]
  22. Kelly R. B. Secretory granule and synaptic vesicle formation. Curr Opin Cell Biol. 1991 Aug;3(4):654–660. doi: 10.1016/0955-0674(91)90037-y. [DOI] [PubMed] [Google Scholar]
  23. Kennedy S. P., Weed S. A., Forget B. G., Morrow J. S. A partial structural repeat forms the heterodimer self-association site of all beta-spectrins. J Biol Chem. 1994 Apr 15;269(15):11400–11408. [PubMed] [Google Scholar]
  24. Koob R., Zimmermann M., Schoner W., Drenckhahn D. Colocalization and coprecipitation of ankyrin and Na+,K+-ATPase in kidney epithelial cells. Eur J Cell Biol. 1988 Feb;45(2):230–237. [PubMed] [Google Scholar]
  25. Kordeli E., Bennett V. Distinct ankyrin isoforms at neuron cell bodies and nodes of Ranvier resolved using erythrocyte ankyrin-deficient mice. J Cell Biol. 1991 Sep;114(6):1243–1259. doi: 10.1083/jcb.114.6.1243. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Kordeli E., Davis J., Trapp B., Bennett V. An isoform of ankyrin is localized at nodes of Ranvier in myelinated axons of central and peripheral nerves. J Cell Biol. 1990 Apr;110(4):1341–1352. doi: 10.1083/jcb.110.4.1341. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Kordeli E., Lambert S., Bennett V. AnkyrinG. A new ankyrin gene with neural-specific isoforms localized at the axonal initial segment and node of Ranvier. J Biol Chem. 1995 Feb 3;270(5):2352–2359. doi: 10.1074/jbc.270.5.2352. [DOI] [PubMed] [Google Scholar]
  28. Kreis T. E., Pepperkok R. Coat proteins in intracellular membrane transport. Curr Opin Cell Biol. 1994 Aug;6(4):533–537. doi: 10.1016/0955-0674(94)90073-6. [DOI] [PubMed] [Google Scholar]
  29. Kunimoto M., Otto E., Bennett V. A new 440-kD isoform is the major ankyrin in neonatal rat brain. J Cell Biol. 1991 Dec;115(5):1319–1331. doi: 10.1083/jcb.115.5.1319. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. 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]
  31. Lambert S., Yu H., Prchal J. T., Lawler J., Ruff P., Speicher D., Cheung M. C., Kan Y. W., Palek J. cDNA sequence for human erythrocyte ankyrin. Proc Natl Acad Sci U S A. 1990 Mar;87(5):1730–1734. doi: 10.1073/pnas.87.5.1730. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Luna E. J., Hitt A. L. Cytoskeleton--plasma membrane interactions. Science. 1992 Nov 6;258(5084):955–964. doi: 10.1126/science.1439807. [DOI] [PubMed] [Google Scholar]
  33. Lux S. E., Tse W. T., Menninger J. C., John K. M., Harris P., Shalev O., Chilcote R. R., Marchesi S. L., Watkins P. C., Bennett V. Hereditary spherocytosis associated with deletion of human erythrocyte ankyrin gene on chromosome 8. Nature. 1990 Jun 21;345(6277):736–739. doi: 10.1038/345736a0. [DOI] [PubMed] [Google Scholar]
  34. Matter K., Mellman I. Mechanisms of cell polarity: sorting and transport in epithelial cells. Curr Opin Cell Biol. 1994 Aug;6(4):545–554. doi: 10.1016/0955-0674(94)90075-2. [DOI] [PubMed] [Google Scholar]
  35. Mays R. W., Siemers K. A., Fritz B. A., Lowe A. W., van Meer G., Nelson W. J. Hierarchy of mechanisms involved in generating Na/K-ATPase polarity in MDCK epithelial cells. J Cell Biol. 1995 Sep;130(5):1105–1115. doi: 10.1083/jcb.130.5.1105. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. McLean I. W., Nakane P. K. Periodate-lysine-paraformaldehyde fixative. A new fixation for immunoelectron microscopy. J Histochem Cytochem. 1974 Dec;22(12):1077–1083. doi: 10.1177/22.12.1077. [DOI] [PubMed] [Google Scholar]
  37. Michaely P., Bennett V. The membrane-binding domain of ankyrin contains four independently folded subdomains, each comprised of six ankyrin repeats. J Biol Chem. 1993 Oct 25;268(30):22703–22709. [PubMed] [Google Scholar]
  38. Molitoris B. A., Dahl R., Geerdes A. Cytoskeleton disruption and apical redistribution of proximal tubule Na(+)-K(+)-ATPase during ischemia. Am J Physiol. 1992 Sep;263(3 Pt 2):F488–F495. doi: 10.1152/ajprenal.1992.263.3.F488. [DOI] [PubMed] [Google Scholar]
  39. Morrow J. S., Cianci C. D., Ardito T., Mann A. S., Kashgarian M. Ankyrin links fodrin to the alpha subunit of Na,K-ATPase in Madin-Darby canine kidney cells and in intact renal tubule cells. J Cell Biol. 1989 Feb;108(2):455–465. doi: 10.1083/jcb.108.2.455. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Morrow J. S., Marchesi V. T. Self-assembly of spectrin oligomers in vitro: a basis for a dynamic cytoskeleton. J Cell Biol. 1981 Feb;88(2):463–468. doi: 10.1083/jcb.88.2.463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Nelson W. J., Veshnock P. J. Dynamics of membrane-skeleton (fodrin) organization during development of polarity in Madin-Darby canine kidney epithelial cells. J Cell Biol. 1986 Nov;103(5):1751–1765. doi: 10.1083/jcb.103.5.1751. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Otto E., Kunimoto M., McLaughlin T., Bennett V. Isolation and characterization of cDNAs encoding human brain ankyrins reveal a family of alternatively spliced genes. J Cell Biol. 1991 Jul;114(2):241–253. doi: 10.1083/jcb.114.2.241. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Pepperkok R., Scheel J., Horstmann H., Hauri H. P., Griffiths G., Kreis T. E. Beta-COP is essential for biosynthetic membrane transport from the endoplasmic reticulum to the Golgi complex in vivo. Cell. 1993 Jul 16;74(1):71–82. doi: 10.1016/0092-8674(93)90295-2. [DOI] [PubMed] [Google Scholar]
  44. Peters L. L., John K. M., Lu F. M., Eicher E. M., Higgins A., Yialamas M., Turtzo L. C., Otsuka A. J., Lux S. E. Ank3 (epithelial ankyrin), a widely distributed new member of the ankyrin gene family and the major ankyrin in kidney, is expressed in alternatively spliced forms, including forms that lack the repeat domain. J Cell Biol. 1995 Jul;130(2):313–330. doi: 10.1083/jcb.130.2.313. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Peterson G. L. Determination of total protein. Methods Enzymol. 1983;91:95–119. doi: 10.1016/s0076-6879(83)91014-5. [DOI] [PubMed] [Google Scholar]
  46. Platt O. S., Lux S. E., Falcone J. F. A highly conserved region of human erythrocyte ankyrin contains the capacity to bind spectrin. J Biol Chem. 1993 Nov 15;268(32):24421–24426. [PubMed] [Google Scholar]
  47. Robinson M. S. The role of clathrin, adaptors and dynamin in endocytosis. Curr Opin Cell Biol. 1994 Aug;6(4):538–544. doi: 10.1016/0955-0674(94)90074-4. [DOI] [PubMed] [Google Scholar]
  48. Smith D. B., Johnson K. S. Single-step purification of polypeptides expressed in Escherichia coli as fusions with glutathione S-transferase. Gene. 1988 Jul 15;67(1):31–40. doi: 10.1016/0378-1119(88)90005-4. [DOI] [PubMed] [Google Scholar]
  49. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Van Why S. K., Hildebrandt F., Ardito T., Mann A. S., Siegel N. J., Kashgarian M. Induction and intracellular localization of HSP-72 after renal ischemia. Am J Physiol. 1992 Nov;263(5 Pt 2):F769–F775. doi: 10.1152/ajprenal.1992.263.5.F769. [DOI] [PubMed] [Google Scholar]
  51. Weaver D. C., Marchesi V. T. The structural basis of ankyrin function. I. Identification of two structural domains. J Biol Chem. 1984 May 25;259(10):6165–6169. [PubMed] [Google Scholar]
  52. Weaver D. C., Pasternack G. R., Marchesi V. T. The structural basis of ankyrin function. II. Identification of two functional domains. J Biol Chem. 1984 May 25;259(10):6170–6175. [PubMed] [Google Scholar]
  53. Whitney J. A., Gomez M., Sheff D., Kreis T. E., Mellman I. Cytoplasmic coat proteins involved in endosome function. Cell. 1995 Dec 1;83(5):703–713. doi: 10.1016/0092-8674(95)90183-3. [DOI] [PubMed] [Google Scholar]

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