Localization of a sequence motif complementary to the nuclear localization signal in proteasomes from Thermoplasma acidophilum by immunoelectron microscopy

J Struct Biol. Sep-Oct 1992;109(2):168-75. doi: 10.1016/1047-8477(92)90048-f.

Abstract

A sequence motif complementary to the nuclear localization signal (NLS) has been localized in proteasomes from Thermoplasma acidophilum by immunoelectron microscopy using sequence-specific antibodies. The antibodies were generated in two different ways: by immunization with a carrier-coupled peptide and by isolation of the sequence-specific antibody from an immune serum against native proteasomes using a peptide-affinity column. The sequence specificity of the isolated antibody was confirmed by a PEPSCAN-ELISA performed on overlapping nonapeptides deduced from the sequence of the alpha-subunit of the Thermoplasma proteasome. Compared to the antibody induced by the carrier-coupled peptide this antibody fraction showed a much higher affinity for native proteasomes. The attachment site of the Fab portion of the antibody to the proteasome was mapped by electron microscopy in conjunction with image processing. The antibody was found to bind to the periphery of the two outer "disks" of the proteasome complex formed by the alpha-subunits.

MeSH terms

  • Amino Acid Sequence
  • Antibodies, Bacterial / immunology
  • Bacterial Proteins / chemistry*
  • Bacterial Proteins / immunology
  • Bacterial Proteins / ultrastructure
  • Cysteine Endopeptidases / chemistry*
  • Cysteine Endopeptidases / immunology
  • Cysteine Endopeptidases / ultrastructure
  • Image Processing, Computer-Assisted
  • Microscopy, Immunoelectron*
  • Molecular Sequence Data
  • Multienzyme Complexes / chemistry*
  • Multienzyme Complexes / immunology
  • Multienzyme Complexes / ultrastructure
  • Peptide Fragments / immunology
  • Proteasome Endopeptidase Complex
  • Protein Sorting Signals / chemistry*
  • Protein Sorting Signals / immunology
  • Thermoplasma / chemistry*

Substances

  • Antibodies, Bacterial
  • Bacterial Proteins
  • Multienzyme Complexes
  • Peptide Fragments
  • Protein Sorting Signals
  • Cysteine Endopeptidases
  • Proteasome Endopeptidase Complex