Characterization of Ubiquitinated Lysosomal Membrane Proteins in Acanthamieba castellanii

  • Oh, Sekyung (School of Biological Sciences, Seoul National University) ;
  • Ahn, Tae-In (School of Biological Sciences, Seoul National University)
  • Published : 2000.06.01

Abstract

Ubiquitinated proteins in lysosomes were characterized by using two monoclonal antibodies (mAbs): LYS8-1, a mAb to lysosomal proteins, and NYA124, a mAb to ubiquitin. LYS8-1 stained lysosome-like vesicles in immunofluorescence microscopy of Amoeba proteus and Acanthamoeba castellanii. In immunoblotting, LYS8-1's antigens (LYS proteins) were detected as 68-kDa and 77-kDa proteins in A. proteus, and as 30-kDa and 39-kDa proteins in A. castellanii. In immunoprecipitation of A. castellanii, at least four distinct LYS proteins, LVS35p, LyS39p, LyS42p, and LYS46p, were detected and accumulated upon inhibition of lysosome functions but not upon that of 26S proteasome functions. They were all found to be ubiquitinated, and were recovered in the lysosome fractions in subcellular fractionation experiments. In chemical fractionation analyses, LYS35p and LYS39p were demonstrated to be peripherally associated with lysosome membrane, while LYS42p and LYS46p tightly bound to the membrane. These results suggest that the LYS proteins become associated to lysosomal membrane upon ubiquitination.

Keywords

References

  1. Anathan J, Goldberg AL, and Voellmy R (1986) Abnormal proteins serve as eukaryotic stress signals and trigger the activation of heat shock genes. Science 232: 522-524 https://doi.org/10.1126/science.3083508
  2. Beers EP, Moreno TN, and Callis J (1992) Subcellular localization of ubiquitin and ubiquitinated proteins in Arabidopsis thaliana. J Biol Chem 267: 15432-15439
  3. Bond U and Schlesinger MJ (1985) Ubiquitin is a heat shock protein in chicken embryo fibroblasts. Mol Cell Biol 5: 949-956
  4. Bonifacino JS and Weissman AM (1998) Ubiputin and the control of protein fate in the cecretory and endocytic pathqats. Annu Rev Cell Dev Biol 14: 19-57 https://doi.org/10.1146/annurev.cellbio.14.1.19
  5. Bordier C (1981) Phase sepaeation of integral membrane proteins in triton X-114 Solution. J Biol Chem 256: 1604-1607
  6. Crane DI, Kalish JE, and Gould SJ (1994) The Pichia pastoris PAS4 gene encodes a ubiquitin-conjugates enzyme required for peroxisome assembly. J Biol Chem 269: 21835-21844
  7. Doherty FJ, Osborn NU, Wassell JA, Heggie PE, Laszlo L, and Mayer RJ (1989) Ubiquitin-Protein conjugates accumlate in proteinase inhibitors. Biochem J 263: 47-55
  8. Goldstein L and Ko C (1976) A method for the mass culturing of large free-living amoebas. Meth Cell Biol 13: 239-246
  9. Gropper R, Brandt RA, Elias S, Bearer CF, Mayer A, Schwartz AL, and Ciechanover A (1991) The ubiquitin-activating enzyme,E1,is required for stress-induced lysosomal degradation of cellular proteins. J Biol Chem 266: 3602-3610
  10. Guaarino LA, Smith G and Dong W (1995) Ubiquitin is attached to membranes of baculovirus particles by a novel type of phospholipid anchor. Cell 80: 301-309 https://doi.org/10.1016/0092-8674(95)90413-1
  11. Harlow E and Lane D (1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York
  12. Hershko A and Ciechanover A (1988) The ubiquitin system, Annu Rev Biochem 67: 425-479
  13. Hicke L (1997) Ubiquitin-dependent internalization and downregulation of plasma membrane proteins. FASEB J 11: 1215-1226
  14. Hingamp PM, Leyland ML, Webb J, Twigger S, Mayer RJ, and Dixon LK (1995) Characterization of a ubiquitinated protein which is externally located in African swine fever virus. J Virol 69: 1785-1793
  15. Hochatrasser M (1996) Protein degradation or regulation: Ub the judge. Cell 84, 813-815 https://doi.org/10.1016/S0092-8674(00)81058-2
  16. Jeon KW and Jeon MS (1975) Cytoplasmic filaments and cellular wound healing in Amoeba proteus. J Cell Biol 67: 243-249 https://doi.org/10.1083/jcb.67.1.243
  17. Kim KJ, Choi EY, and Jeon KW (1992) Use of antibodies as probes in the study of amoebae In: Lee JJ and Soldo AT(ed), Protocols in Protozoology, The Society of Protozoolpgists, pp D-5.1-D.5.3
  18. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head bacteriophage T4. Nature 227: 680-685 https://doi.org/10.1038/227680a0
  19. Laszol L, Doherty FJ, Watson A, Self T, Landon M, Lowe J, and Mayer RJ (1991) Immunogold localization of ubiquitinprotein conjugates in primary (azurophilic) granules of polymorphonuclear neutrophils. FEBS Lett 279: 175-178 https://doi.org/10.1016/0014-5793(91)80142-P
  20. Lee DH and Gold AL (1998) Proteasome inhibitors:valuable new tools for cell biologists. Trends Cell Biol 8: 397-403 https://doi.org/10.1016/S0962-8924(98)01346-4
  21. Lee JE, Ahn EY, and Ahn TI (1998a) Development of a plasmid vector for overproduction of ${\beta}$-galactosidease in Escherichia coli by using genetic components of groEx from symbiotic bacteria in Amoeba proteus. J Micobiol Biotech 8: 509-516
  22. Lee SY, Kim HJ, Yoo SY, and Ahn TI (1998b) Characterization of a monoclonal antibody and a cDNA for polyubiquitin of Amoeba proteus. J Euk Microbiol 45: 431-438 https://doi.org/10.1111/j.1550-7408.1998.tb05095.x
  23. Low P, Doherty FJ, Sass M, Kovacs J, Mayer RJ, and Laszlo L (1993) Immunogold localization of ubiquitin protein conjugates in Sf9 insect cells:implications for the biogenesis of lysosome-related organelles. FEBS Lett 316: 152-156 https://doi.org/10.1016/0014-5793(93)81205-E
  24. Lowry OL, Rosenberg NJ, Farr AL and Randall RT (1951) Proteins measurement with phenol reagent. J Biol Chem 193: 265-275
  25. Mahajan R, Delphin C, Guan T, Gerace L, and Melchior F (1997) A small ubiquitin-related polypetide involved in targeting RanGAP1 to nuclear pore complex protein RanBP2. Cell 88: 97-107 https://doi.org/10.1016/S0092-8674(00)81862-0
  26. Matunis MJ, Coutavas E, and Blobel G (1996) A novel ubiquitin-like modification modulates the partitoning of the Ran-GTPase-activating protein RanGAP1 between the cytosol and the nuclear pore complex. J Cell Biol 135: 1457-1470 https://doi.org/10.1083/jcb.135.6.1457
  27. Paolini R and Kinet JP (1993) Cell surface control of the multiubiquitination and deubiquitination of high-affinity immunoglobulin E receptors. EMBO J 12: 779-786
  28. Simeon A, van Der Klei IJ, Veenhuis M, and Wolf DH (1992) Ubiquitin, a central component of selective cytoplasmic proteolysis, is linked to proteins residing at the locus of nonselective proteolysis, the vacuole. FEBS Lett 301: 231-235 https://doi.org/10.1016/0014-5793(92)81254-J
  29. Sternsdorf T, Jensen K, and Will H (1997) Evidence for covalent modification of the nuclear dot-associated proteins PML and Sp 100 by PIC1/SUMO-1. J Cell Biol 139: 1621-1634 https://doi.org/10.1083/jcb.139.7.1621
  30. Strous G, van Kerkhof P, Govers R, Ciechanover A, and Schwartz AL (1996) The ubiquitin conjugation system is required for ligand-induced endocytosis and degradation of the growth hormone receptor. EMBO J 15: 3806-3812
  31. Towbin H, Staehelin T, and Gordon J (1979) Electrophoretic transfer of proteins from polyasrylamide gels to nitrocellulose sheets; procedures and some applications. Proc Natl Acad Sci USA 76: 4350-4354 https://doi.org/10.1073/pnas.76.9.4350
  32. Wiebel FF and Kunau WH (1992) The Pas2 protein seeential for peroxisome biogenesis is related to ubiquitin-conjugating enzymes. Nature 359: 73-76 https://doi.org/10.1038/359073a0
  33. Yoo SY, Choi JY, Kim H, and Ahn TI (1996) Lysosomes are in subpopulations in Amoeba proteus. Mol Cells 6: 316-324