Inhibition of Target of Rapamycin Signaling by Rapamycin in the Unicellular Green Alga Chlamydomonas reinhardtii

José L. Crespo,Francisco J. Florencio,Sandra Díaz-Troya

doi:10.1104/pp.105.070847

Abstract

The macrolide rapamycin specifically binds the 12-kD FK506-binding protein (FKBP12), and this complex potently inhibits the target of rapamycin (TOR) kinase. The identification of TOR in Arabidopsis (Arabidopsis thaliana) revealed that TOR is conserved in photosynthetic eukaryotes. However, research on TOR signaling in plants has been hampered by the natural resistance of plants to rapamycin. Here, we report TOR inactivation by rapamycin treatment in a photosynthetic organism. We identified and characterized TOR and FKBP12 homologs in the unicellular green alga Chlamydomonas reinhardtii. Whereas growth of wild-type Chlamydomonas cells is sensitive to rapamycin, cells lacking FKBP12 are fully resistant to the drug, indicating that this protein mediates rapamycin action to inhibit cell growth. Unlike its plant homolog, Chlamydomonas FKBP12 exhibits high affinity to rapamycin in vivo, which was increased by mutation of conserved residues in the drug-binding pocket. Furthermore, pull-down assays demonstrated that TOR binds FKBP12 in the presence of rapamycin. Finally, rapamycin treatment resulted in a pronounced increase of vacuole size that resembled autophagic-like processes. Thus, our findings suggest that Chlamydomonas cell growth is positively controlled by a conserved TOR kinase and establish this unicellular alga as a useful model system for studying TOR signaling in photosynthetic eukaryotes.

Highlights

The macrolide rapamycin binds the 12-kD FK506-binding protein (FKBP12), and this complex potently inhibits the target of rapamycin (TOR) kinase
Our results show that rapamycin inhibits growth of a photosynthetic organism and strongly suggest the presence of a TOR signaling cascade in Chlamydomonas
Plants have been reported to express TOR and FKBP12 proteins (Xu et al, 1998; Menand et al, 2002), in contrast to other eukaryotes, land plants are naturally insensitive to rapamycin likely due to the inability of plant FKBP12 to bind rapamycin (Xu et al, 1998)

Summary

Introduction

The macrolide rapamycin binds the 12-kD FK506-binding protein (FKBP12), and this complex potently inhibits the target of rapamycin (TOR) kinase. Our findings suggest that Chlamydomonas cell growth is positively controlled by a conserved TOR kinase and establish this unicellular alga as a useful model system for studying TOR signaling in photosynthetic eukaryotes. Studies performed in yeasts uncovered the unique mechanism of action of rapamycin Both receptor and functional target were initially identified in S. cerevisiae (Heitman et al, 1991). FKBP12 is a member of the FK506- and rapamycin-binding protein (FKBP) family This group [OA] Open Access articles can be viewed online without a subscription. The findings that dominant mutations in either TOR1 or TOR2 confer complete resistance to the growth-inhibitory properties of

Methods

Results

Conclusion