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An effector domain mutant of Arf6 implicates phospholipase D in endosomal membrane recycling

Olivera A Jovanovic1, Fraser D Brown, Julie G Donaldson

  • 1Laboratory of Cell Biology, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892, USA.

Molecular Biology of the Cell
|November 11, 2005
PubMed

Insights

Phospholipase D (PLD) is essential for Arf6 protein function in cells, regulating membrane recycling and cell protrusions. Inhibiting PLD blocks these processes, while increasing phosphatidic acid levels reverses the effects.

Area of Science:

  • Cell Biology
  • Molecular Biology
  • Biochemistry

Background:

  • Arf6 is a small GTPase involved in membrane trafficking.
  • Phospholipase D (PLD) produces phosphatidic acid (PA), a key signaling lipid.
  • The precise role of PLD in Arf6-mediated cellular processes remains unclear.

Purpose of the Study:

  • To investigate the role of phospholipase D (PLD) in mediating Arf6 function.
  • To determine if PLD activity is necessary for Arf6-mediated membrane recycling and protrusion formation.

Main Methods:

  • Expression of wild-type and mutant Arf6 proteins in cells.
  • Inhibition of PLD activity using 1-butanol.
  • Assessment of membrane trafficking, endosomal morphology, and cell protrusion formation.
  • Recruitment assays for Arf6 GTPase activating protein (GAP) ACAP1.

Main Results:

  • Arf6 mutants defective in PLD activation inhibited membrane recycling and caused endosomal accumulation.
  • These Arf6 mutants failed to generate protrusions or recruit ACAP1.
  • Inhibition of PLD with 1-butanol mimicked the Arf6 mutant phenotypes.
  • Elevating phosphatidic acid levels reversed the observed defects.

Conclusions:

  • Arf6 stimulation of PLD is required for efficient endosomal membrane recycling to the plasma membrane.
  • PLD activity is essential for Arf6-mediated cell protrusion formation and ACAP1 recruitment.
  • Phosphatidic acid is a critical downstream effector of Arf6 in regulating membrane dynamics.

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