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Spatio-Temporal Manipulation of Small GTPase Activity at Subcellular Level and on Timescale of Seconds in Living Cells
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Role of Rab9 GTPase in facilitating receptor recruitment by TIP47

K S Carroll1, J Hanna, I Simon

  • 1Department of Biochemistry, Stanford University School of Medicine, Stanford, CA 94305-5307, USA.

Science (New York, N.Y.)
|May 19, 2001
PubMed

Insights

TIP47 binds to active Rab9 GTPase, enhancing its affinity for mannose 6-phosphate receptors (MPRs). This interaction is crucial for efficient endosome-to-Golgi transport of MPRs, linking cargo selection to Rab GTPase activity.

Area of Science:

  • Cell biology
  • Molecular and cell biology
  • Biochemistry

Background:

  • Mannose 6-phosphate receptors (MPRs) mediate the transport of lysosomal hydrolases from the Golgi apparatus to endosomes.
  • TIP47 is a known cargo-binding protein essential for the retrograde transport of MPRs from endosomes back to the Golgi.
  • The precise molecular mechanisms by which TIP47 facilitates this transport remain incompletely understood.

Purpose of the Study:

  • To investigate the interaction between TIP47 and Rab9 GTPase.
  • To determine the role of Rab9 in regulating TIP47's function in MPR transport.
  • To elucidate the molecular basis for the recruitment of cytosolic cargo selection machinery.

Main Methods:

  • Co-immunoprecipitation assays to detect protein-protein interactions.
  • In vitro binding assays to quantify binding affinities.
  • In vivo transport assays in cultured cells to assess functional consequences.
  • Site-directed mutagenesis to disrupt specific binding interactions.

Main Results:

  • TIP47 directly binds to the active, GTP-bound form of Rab9.
  • Rab9 binding significantly increases TIP47's affinity for its cargo, the MPR cytoplasmic domains.
  • A functional Rab9 binding site on TIP47 is essential for TIP47-mediated stimulation of MPR transport in vivo.
  • These findings suggest a mechanism for selective recruitment of cargo adaptors to organelles.

Conclusions:

  • Rab9 GTPase acts as a key regulator of TIP47 function in the endosome-to-Golgi transport pathway.
  • The interaction between Rab9 and TIP47 couples cargo selection to the activation state of Rab GTPases.
  • This study reveals a novel mechanism for organelle-specific vesicle budding mediated by Rab GTPase-dependent recruitment of cytosolic factors.

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