Immunolocalisation of phospholipase D1 on tubular vesicular membranes of endocytic and secretory origin

John Lucocq, Maria Manifava, Kun Bi, Michael G. Roth, Nicholas T. Ktistakis

Research output: Contribution to journalArticle

26 Citations (Scopus)

Abstract

We have examined the localisation of overexpressed phospholipase D1 (PLD1) using antibodies against its amino- and carboxyl-terminal domains. PLD1 overexpressed in COS-7 cells showed variable distribution by immunofluorescence but was mainly in punctate structures in the perinuclear region and at the plasma membrane. Downregulation by an anti-sense plasmid resulted in almost exclusively perinuclear distribution in punctate structures that contained immunoreactivity for the endogenous KDEL receptor and the early endosomal antigen EEA1 protein. Influenza haemagglutinin (HA) and HA-derived mutants designed to locate primarily to secretory or endocytic membranes were present in PLD1-positive membranes. Immunofluorescence analysis in permanent CHO cell lines that express PLD1 inducibly confirmed the presence of PLD1 on both endocytic and secretory membranes. Analysis of PLD1 distribution by immunocytochemistry and electron microscopy of intact CHO cells and of isolated membranes revealed that PLD1 was present in tubulovesicular elements and multivesicular bodies. Some of these were close to the Golgi region whereas others stained positive for endocytic cargo proteins. Morphometric analysis assigned the majority of PLD1 immunoreactivity on endosomal membranes and a smaller amount on membranes of secretory origin. PLD1, via signals that are currently not understood, is capable of localising in tubulovesicular membranes of both endocytic and secretory origin.

Original languageEnglish (US)
Pages (from-to)508-520
Number of pages13
JournalEuropean Journal of Cell Biology
Volume80
Issue number8
StatePublished - 2001

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Membranes
CHO Cells
Hemagglutinins
Fluorescent Antibody Technique
Cell Membrane
Multivesicular Bodies
phospholipase D1
COS Cells
Human Influenza
Electron Microscopy
Proteins
Plasmids
Down-Regulation
Immunohistochemistry
Antigens
Cell Line
Antibodies

Keywords

  • Endocytosis
  • Localisation
  • Morphometry
  • PLD1
  • Secretion

ASJC Scopus subject areas

  • Anatomy
  • Cell Biology

Cite this

Immunolocalisation of phospholipase D1 on tubular vesicular membranes of endocytic and secretory origin. / Lucocq, John; Manifava, Maria; Bi, Kun; Roth, Michael G.; Ktistakis, Nicholas T.

In: European Journal of Cell Biology, Vol. 80, No. 8, 2001, p. 508-520.

Research output: Contribution to journalArticle

Lucocq, John ; Manifava, Maria ; Bi, Kun ; Roth, Michael G. ; Ktistakis, Nicholas T. / Immunolocalisation of phospholipase D1 on tubular vesicular membranes of endocytic and secretory origin. In: European Journal of Cell Biology. 2001 ; Vol. 80, No. 8. pp. 508-520.
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