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CALNUC (nucleobindin) is localized in the Golgi apparatus in insect cells

J Kawano1, T Kotani, Y Ogata

  • 1Department of Anatomy, Miyazaki Medical College, Japan.

European Journal of Cell Biology
|April 25, 2000
PubMed

Insights

Researchers identified a novel calcium-binding Golgi protein, CALNUC, in insect cells, demonstrating its conserved function and widespread presence across the animal kingdom. This finding highlights the essential role of CALNUC in cellular processes.

Area of Science:

  • Cell Biology
  • Molecular Biology
  • Biochemistry

Background:

  • The Golgi apparatus is crucial for protein modification and transport.
  • CALNUC (nucleobindin) is a calcium-binding protein found in the Golgi of mammalian cells, involved in DNA binding.
  • Homologues of CALNUC and related proteins like NEFA exist, but their evolutionary relationships and functions in non-mammalian species are less understood.

Purpose of the Study:

  • To identify and characterize Golgi-specific proteins in insect cells.
  • To determine if insect cells possess a homologue of the mammalian CALNUC protein.
  • To investigate the evolutionary origins and functional conservation of CALNUC.

Main Methods:

  • Generation of a mouse monoclonal antibody against insect cell Golgi fractions.
  • Immunoaffinity chromatography for antigen purification.
  • N-terminal and internal amino acid sequencing.
  • cDNA cloning and sequencing.
  • Sequence homology analysis.
  • Electron microscopic immunoperoxidase staining.
  • Biochemical assays (proteinase K digestion, carbonate extraction, Triton X-114 extraction) to determine protein localization and properties.
  • Phylogenetic tree analysis.

Main Results:

  • A Golgi-specific antigen was purified from Sf21 insect cells.
  • The insect protein shares homology with mammalian CALNUC and NEFA, possessing EF-hand calcium-binding motifs but lacking a leucine zipper.
  • Immunoelectron microscopy localized the insect protein to the cis-Golgi cisternae and networks, similar to mammalian CALNUC.
  • Biochemical assays confirmed the insect protein is a soluble, membrane-associated protein with calcium-binding activity, consistent with CALNUC.
  • Phylogenetic analysis suggests NEFA evolved from CALNUC after mammalian and insect lineages diverged.

Conclusions:

  • The identified insect protein is a functional homologue of mammalian CALNUC.
  • CALNUC is conserved in insect cells, indicating its essential role as a calcium-binding Golgi protein across a broad range of the animal kingdom.
  • The evolutionary trajectory suggests CALNUC is an ancient protein, with NEFA arising later from it.

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