Inhibition of target of rapamycin signaling by rapamycin in the unicellular green alga Chlamydomonas reinhardtii

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.

Figure 1.

Rapamycin inhibits growth of Chlamydomonas. Wild-type Chlamydomonas cells were subjected to 10-fold serial dilutions and spotted onto TAP plates containing the indicated concentrations of rapamycin. Plates were incubated at 25°C under continuous illumination.

Figure 2.

Phylogenetic analysis of Chlamydomonas FKB12. A, Amino acid sequence of Chlamydomonas FKB12 compared to the FKBP12 protein from other representative organisms. Identical residues in at least nine of the 16 sequences are shaded in gray. Asterisks indicate residues from human FKBP12 that interact with rapamycin (Choi et al., 1996). B, Neighbor-Joining tree of FKBP12s. Bootstrap values from 1,000 replicates are shown. The scale bar corresponds to 0.1 estimated amino acid substitutions per site.

Figure 3.

TOR is conserved in Chlamydomonas. A, Structure of CrTOR gene predicted from the Chlamydomonas nuclear genome. The intron and exon positions were deduced by comparison of genomic and partial cDNA sequences, and homology analysis with other TOR proteins. The black rectangles indicate the position of the protein-coding regions. B, Comparison of the CrTOR protein sequence to AtTOR. Values indicate the percentage of identity with the corresponding domain sequence of CrTOR. FAT, FRB, kinase, and FATC domains correspond to residues 1,374 to 1,924; 1,961 to 2,055; 2,124 to 2,372; and 2,493 to 2,523, respectively. C, Phylogenetic relationship of TOR from Chlamydomonas and representative organisms. The phylogenetic tree was constructed with full-length TOR amino acid sequences and aligned using the ClustalW program. The bootstrap values represent 1,000 replications. Sc, S. cerevisiae; Sp, Schizosaccharomyces pombe; Os, O. sativa; Zm, Zea mays; At, Arabidopsis; Cr, C. reinhardtii; Dm, Drosophila melanogaster; Aa, Anopheles aedes; Hs, Homo sapiens; Gg, Gallus gallus; Ce, Caenorhabditis elegans.

Figure 4.

Characterization of the Chlamydomonas rap2 mutant strain. A, rap2 mutant cells are rapamycin insensitive. Serial dilutions of wild-type, rap2, and rap2 cells transformed with the pJC20 plasmid on TAP plates containing 500 nm rapamycin (500 nm) or drug vehicle (no rap). Growth was recorded after 4 d incubation at 25°C under continuous illumination. B, Structure of the FKB12 gene from Chlamydomonas. The closed rectangles and numbers (1–4) indicate the four exons of the FKB12 gene. Primers used for the PCR analysis of the FKB12 gene (see D) are represented by arrows and letters. Restriction sites for NcoI and PvuII restriction enzymes are shown by dotted lines. C, Southern-blot analysis of genomic DNA from the rap2 mutant compared to the wild-type strain. Ten micrograms of DNA were digested with NcoI or PvuII. The blot was hybridized with an FKB12 probe obtained by PCR (see B). D, PCR analysis of the FKB12 gene from wild-type and rap2 cells. Letters indicate the different set of primers used for PCRs reactions (see B for the position of different primers). E, RT-PCR analysis of FKB12 in wild-type and rap2 mutant cells. Semiquantitative RT-PCR was terminated after 20 cycles for FKB12 and 24 cycles for GS1. The cytosolic gene, GS1, whose expression is not expected to change in the rap2 mutant, was used as an internal control for RNA level. F, Western blot of FKB12 from wild-type and rap2 strains. Fifteen micrograms of total protein obtained from wild-type and rap2 cells were resolved on 15% polyacrylamide gels, blotted, and incubated with anti-FKB12 antibodies.

Figure 5.

Chlamydomonas FKB12 functionally complements a yeast FKBP12 mutant. Wild-type JK9-3da cells were transformed with empty vector (wt). The fpr1 mutant strain lacking the FKBP12 protein was transformed with empty vector (fpr1) or with plasmids expressing wild-type Chlamydomonas FKB12 or M52V E54Q and M52K G53Q mutants. Cultures were normalized, subjected to 10-fold serial dilutions, and spotted onto SD or SGal-Leu plates without rapamycin or containing 200 nm rapamycin. Plates were incubated at 30°C for 2 or 5 d.

Figure 6.

Modeling analysis of Chlamydomonas wild-type and mutant FKB12s. A, Amino acid substitutions performed in the M52V E54Q and M52K G53Q mutants compared to wild-type FKB12. Schematic diagrams of wild-type FKB12 (B), and the M52V E54Q (C) and M52K G53Q (D) mutants showing protein-rapamycin interactions. Data were generated using the LIGPLOT program (Wallace et al., 1995).

Figure 7.

Expression of mutant FKB12s in Chlamydomonas partially increases the sensitivity to rapamycin. A, Rapamycin sensitivity of wild-type and rap2 cells compared to rap2 cells expressing wild-type or mutant FKB12s. Serial dilutions were spotted onto TAP plates supplemented or not with 500 nm rapamycin, and incubated at 25°C under continuous illumination. Growth was recorded after 4 or 8 d. B, Levels of FKB12 protein detected by western blot. About 15 μg of total protein obtained from wild-type (wt) or rap2 mutant cells transformed with the indicated constructs were resolved in 15% polyacrylamide gels, blotted, and incubated with anti-FKB12 antibodies.

Figure 8.

The FRB domain of CrTOR interacts with FKB12 in the presence of rapamycin. GST pull-down assays to test the interaction of FKB12 with the FRB domain of CrTOR. Five micrograms of purified GST fusion protein were incubated with 0.5 mg of Chlamydomonas total extract in the presence of 4 μm rapamycin (+rap) or a similar concentration of drug vehicle (−rap). FKB12 and GST fusion protein were detected by immunoblotting using anti-FKB12 antibody and Coomassie staining, respectively.

Figure 9.

Rapamycin-induced vacuolization in Chlamydomonas. A, Nomarski micrographs of wild-type and rap2 mutant living cells treated with 500 nm rapamycin (+rap) or drug vehicle (control) for 24 h. Cultures were synchronized by alternating light and dark (12 h/12 h) cycles before rapamycin addition. B, Nomarski and CLSM images of a wild-type living cell treated with rapamycin (+rap) or drug vehicle (control) for 24 h. Cells were incubated with the pH-sensitive dye LysoSensor Green DND-189, which produces fluorescent foci mainly in the region of the cell containing vacuoles.