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Visualizing RNA Localization in Xenopus Oocytes
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Sequential actions of Rab5 and Rab7 regulate endocytosis in the Xenopus oocyte

A Mukhopadhyay1, A M Barbieri, K Funato

  • 1Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 63110, USA.

The Journal of Cell Biology
|March 24, 1997
PubMed

Insights

Guanine nucleotide-binding proteins (GTPases) and N-ethylmaleimide-sensitive factor (NSF) are crucial for endocytosis in Xenopus oocytes. Rab7 plays a rate-limiting role downstream of Rab5 in this process.

Area of Science:

  • Cell Biology
  • Molecular Biology
  • Endocytosis Research

Background:

  • Endocytosis is a fundamental cellular process for nutrient uptake and signal transduction.
  • Guanine nucleotide-binding proteins (GTPases), particularly Rab proteins, are known regulators of vesicular transport.
  • The specific roles of GTPases and associated factors like NSF in oocyte endocytosis require further elucidation.

Purpose of the Study:

  • To investigate the involvement of GTPases in endocytosis using a Xenopus oocyte assay.
  • To determine the specific roles of Rab5 and Rab7 in regulating endocytic uptake and cargo processing.
  • To confirm the requirement for N-ethylmaleimide-sensitive factor (NSF) in the endocytic pathway.

Main Methods:

  • Development of an assay in Xenopus oocytes to track fluid-phase marker (HRP) uptake.
  • Injection of various recombinant GTPases (Rab5, Rab7) and mutants, as well as NSF and its mutants.
  • Inhibition studies using GTPgammaS and aluminum fluoride to assess general GTPase function.

Main Results:

  • GTPgammaS and aluminum fluoride inhibited HRP uptake, indicating a general role for GTPases.
  • Rab5 and constitutively active Rab5 (Rab5:Q79L) enhanced HRP uptake, while dominant-negative Rab5 (Rab5:S34N) inhibited it.
  • Rab7 enhanced both HRP uptake and degradation, functioning downstream of Rab5 in a monensin-sensitive pathway.

Conclusions:

  • GTPases, including Rab5 and Rab7, are essential regulators of endocytosis in Xenopus oocytes.
  • NSF is confirmed to be required for endocytosis, acting in conjunction with Rab5.
  • Rab7 appears to be a rate-limiting factor for endocytosis, acting downstream of Rab5.

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