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ARF-induced lysosomal lysis in vitro

K Arai1, T Matsuda, Y Sai

  • 1Faculty of Pharmaceutical Sciences, Kanazawa University, Kanazawa 920-0934.

Journal of Biochemistry
|May 21, 1998
PubMed
Summary

Guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS) disintegrates lysosomes via a GTP-binding protein (G-protein). This study identifies ADP-ribosylation factor 1 (ARF1) as the key protein mediating GTPgammaS-induced lysosomal lysis.

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Area of Science:

  • Cell Biology
  • Molecular Biology
  • Biochemistry

Background:

  • Lysosomes are crucial for cellular degradation.
  • GTP-binding proteins (G-proteins) regulate various cellular processes.
  • The specific role of G-proteins in lysosomal integrity was unclear.

Purpose of the Study:

  • To investigate the mechanism of lysosomal disintegration induced by GTPgammaS.
  • To identify the specific GTP-binding protein involved in lysosomal lysis.
  • To elucidate the role of this protein in lysosomal membrane association.

Main Methods:

  • Lysosomal integrity assay using fluorescein isothiocyanate-dextran release.
  • Cytosol treatment with guanosine 5 -O-(3-thiotriphosphate) (GTPgammaS).
  • Gel filtration, GTP-ligand blotting, and protein purification (ARF1).
  • Reconstitution assays with purified ARF1.
  • Analysis of ARF1 binding to lysosomal membranes.

Main Results:

  • GTPgammaS caused dose-dependent lysosomal disintegration.
  • GTP or GDP suppressed the GTPgammaS effect, indicating G-protein involvement.
  • A small G-protein, identified as ARF1, was found to participate in lysosomal lysis.
  • GTPgammaS stimulated ARF1 binding to lysosomal membranes.
  • Reconstitution of lysis with purified ARF1 confirmed its role.

Conclusions:

  • ARF1 mediates GTPgammaS-induced lysosomal lysis.
  • ARF1 transfer from cytosol to lysosomal membrane is essential for GTPgammaS-stimulated lysis.
  • This finding reveals a novel mechanism for regulating lysosomal integrity.

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