Continuous translocation of Rac2 and the NADPH oxidase component p67(phox) during phagocytosis

J Biol Chem. 2004 Mar 5;279(10):9097-102. doi: 10.1074/jbc.M309284200. Epub 2003 Nov 17.

Abstract

In this study, the translocation of the NADPH oxidase components p67(phox) and Rac2 was studied during phagocytosis in living cells. For this purpose, green fluorescent protein (GFP)-tagged versions of these proteins were expressed in the myeloid cell line PLB-985. First, the correct localization of p67GFP and GFP-Rac2 was shown during phagocytosis of serum-treated zymosan by wild-type PLB-985 cells and PLB-985 X-CGD (chronic granulomatous disease) cells, which lack expression of flavocytochrome b(558). Subsequently, these constructs were used for fluorescence recovery after photobleaching studies to elucidate the turnover of these proteins on the phagosomal membrane. The turnover of p67GFP and GFP-Rac2 proved to be very high, indicating a continuous exchange of flavocytochrome b(558)-bound p67GFP and GFP-Rac2 for cytosolic, free p67GFP and GFP-Rac2. Furthermore, the importance of an intact actin cytoskeleton for correct localization of these proteins was investigated by disrupting the actin cytoskeleton with cytochalasin B. However, cytochalasin B treatment of PLB-985 cells did not alter the localization of p67GFP and GFP-Rac2 once phagocytosis was initiated. In addition, the continuous exchange of flavocytochrome b(558)-bound p67GFP and GFP-Rac2 for cytosolic p67GFP and GFP-Rac2 was still intact in cytochalasin B-treated cells, indicating that the translocation of these proteins does not depend on a rearrangement of the actin cytoskeleton.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Actins / metabolism
  • Cytochalasin B / pharmacology
  • Cytoskeleton / metabolism
  • Green Fluorescent Proteins
  • Humans
  • K562 Cells
  • Luminescent Proteins
  • NADPH Oxidases / metabolism
  • Phagocytosis* / drug effects
  • Phosphoproteins / metabolism*
  • Protein Transport
  • RAC2 GTP-Binding Protein
  • rac GTP-Binding Proteins / metabolism*

Substances

  • Actins
  • Luminescent Proteins
  • Phosphoproteins
  • neutrophil cytosol factor 67K
  • Green Fluorescent Proteins
  • Cytochalasin B
  • NADPH Oxidases
  • rac GTP-Binding Proteins