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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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FKBP12-rapamycin target TOR2 is a vacuolar protein with an associated phosphatidylinositol-4 kinase activity

M E Cardenas1, J Heitman

  • 1Department of Genetics, Durham, NC 27710, USA.

The EMBO Journal
|December 1, 1995
PubMed

Insights

Rapamycin, in complex with FKBP12, targets TOR2, a yeast vacuole-associated protein kinase. This interaction delocalizes TOR2, impacting cell cycle progression and vacuolar function, revealing a conserved signaling pathway.

Area of Science:

  • Cell Biology
  • Molecular Biology
  • Biochemistry

Background:

  • Rapamycin inhibits cell cycle progression by targeting FKBP12-rapamycin complexes.
  • Genetic studies implicated TOR1 and TOR2 proteins as targets of FKBP12-rapamycin.
  • TOR2's role in cell cycle regulation and its localization were previously unclear.

Purpose of the Study:

  • To investigate the localization and function of the TOR2 protein in yeast.
  • To determine the effect of the FKBP12-rapamycin complex on TOR2 activity and localization.
  • To elucidate the role of TOR2 in cell cycle progression and vacuolar dynamics.

Main Methods:

  • Immunoprecipitation of TOR2 protein.
  • Assay of phosphatidylinositol-4 (PI-4) kinase activity associated with TOR2.
  • Analysis of FKBP12 binding to wild-type and mutant TOR2 in the presence of rapamycin.
  • Microscopy to assess TOR2 localization and vacuolar morphology.

Main Results:

  • TOR2 protein is membrane-associated and localized to the yeast vacuole surface.
  • Immunoprecipitated TOR2 exhibits PI-4 kinase activity.
  • Rapamycin stimulates FKBP12 binding to wild-type TOR2, causing its delocalization from the vacuole.
  • Delocalization, not inhibition of kinase activity, appears to be the key consequence of rapamycin binding.
  • TOR2 localization is crucial for its function and influences vacuolar morphology.

Conclusions:

  • TOR2 is an essential, conserved protein involved in cell cycle regulation.
  • Vacuolar localization of TOR2 is critical for its function in signal transduction.
  • Rapamycin-mediated delocalization of TOR2 from the vacuole disrupts its signaling pathway.
  • TOR2 plays a role in modulating vacuolar morphology and segregation.

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