Target of Rapamycin and LST8 Proteins Associate with Membranes from the Endoplasmic Reticulum in the Unicellular Green Alga Chlamydomonas reinhardtii

Abstract

The highly conserved target of rapamycin (TOR) kinase is a central controller of cell growth in all eukaryotes. TOR exists in two functionally and structurally distinct complexes, termed TOR complex 1 (TORC1) and TORC2. LST8 is a TOR-interacting protein that is present in both TORC1 and TORC2. Here we report the identification and characterization of TOR and LST8 in large protein complexes in the model photosynthetic green alga Chlamydomonas reinhardtii. We demonstrate that Chlamydomonas LST8 is part of a rapamycin-sensitive TOR complex in this green alga. Biochemical fractionation and indirect immunofluorescence microscopy studies indicate that TOR and LST8 exist in high-molecular-mass complexes that associate with microsomal membranes and are particularly abundant in the peri-basal body region in Chlamydomonas cells. A Saccharomyces cerevisiae complementation assay demonstrates that Chlamydomonas LST8 is able to functionally and structurally replace endogenous yeast LST8 and allows us to propose that binding of LST8 to TOR is essential for cell growth.The Ser/Thr kinase target of rapamycin (TOR) is a central regulator of cell growth and metabolism in all eukaryotes (recently reviewed by Wullschleger et al. [54]). Studies of TOR signaling in yeast and mammals have demonstrated that TOR controls cell growth in response to nutrients and different stresses (10, 54). The TOR kinases are large (about 270 kDa) proteins that assemble into two structurally and functionally distinct multisubunit complexes of 1.5 to 2.0 MDa termed TOR complex 1 (TORC1) and TORC2. The similar composition of TORCs in widely divergent kingdoms such as metazoans and fungi suggests that these complexes are broadly conserved across all eukaryotes. The two TORCs were initially identified in yeast (34, 52) and subsequently in mammals (18,23,26,44). In yeast, TORC1 contains either TOR1 or TOR2, KOG1, TCO89, and LST8, whereas TORC2 includes TOR2 (but not TOR1), LST8, BIT61, AVO1, AVO2, and AVO3 (34, 41, 52). Mammalian TOR (mTOR) associates with raptor (homologue of yeast KOG1) and mLST8 to constitute mTORC1, while mTORC2 consists of mLST8, rictor (homologue of yeast AVO3), hSIN1 (homologue of yeast AVO1), and mTOR (15,18,22,23,27,34,44,57). With the exception of the yeast proteins TCO89, BIT61, and AVO2, all TOR partners are essential for growth of yeast and mammalian cells (16,34,42).Studies performed in yeast and mammalian cells demonstrate that TORC1 mediates the rapamycin-sensitive signaling branch that regulates translation, ribosome biogenesis, autophagy, and nitrogen and carbon degradative pathways (54). Some of these readouts are mediated via the AGC kinases S6K1 (mammals) and Sch9 (yeast), which are phosphorylated and thus activated by TORC1 (4, 21, 50). TORC2, on the other hand, is insensitive to rapamycin and controls actin cytoskeleton organization (23,34,44), presumably via its direct AGC kinase substrates Akt/PKB (mammals) and YPK1/2 (yeast) (24,36,45). Recently, it has been reported that TORC1 and TORC2 are multimeric supercomplexes...

Autophagy in protists

“…Interestingly, the rapamycin-sensitive TOR-LST8 complex implicated in the regulation of autophagy initiation in Chlamydomonas 104,105 localizes to the peri-basal body region of the alga. 106 The overlapping localization of TOR and LST8 homologues in C. reinhardtii within the vicinity of the flagellar basal bodies not only strengthens existing data linking flagellum function and intracellular cell signalling in this alga (reviewed in ref. 107), but also draws to mind the increase in mTOR activity in renal epithelial cells from human patients with polycystic kidney disease or mouse models of polycystic kidney disease where intracellular signalling is perturbed due to defects or loss in function of immotile primary (or sensory) cilia.…”

Inhibition of Protein Synthesis by TOR Inactivation Revealed a Conserved Regulatory Mechanism of the BiP Chaperone in Chlamydomonas

“…Besides KOG1/raptor and TOR proteins, two putative LST8 homologs can be identified in the Arabidopsis genome (Moreau et al, 2010;Dobrenel et al, 2011). In Chlamydomonas, it has been demonstrated that CrTOR localizes in high-molecularmass complexes that include a highly conserved LST8 homolog (Díaz-Troya et al, 2008a). These findings indicate that TORC1 might be functionally and structurally conserved in photosynthetic eukaryotes.…”

Inhibition of Target of Rapamycin Signaling and Stress Activate Autophagy in Chlamydomonas reinhardtii

“…Wild-type Chlamydomonas cells were fixed and stained for IF microscopy as described previously (Cole et al, 1998;Diaz-Troya et al, 2008a). The primary antibody used was rabbit polyclonal anti-CrATG8 (1:500).…”

“…The different fractions were processed and subjected to western-blot analysis with the anti-CrATG8 antibody. Suc density gradient analysis was performed as described previously (Diaz-Troya et al, 2008a).…”

“…More recently, Chlamydomonas has also been proposed as a useful system for the characterization of the TOR signaling pathway in photosynthetic eukaryotes based on the finding that, unlike plants, Chlamydomonas cell growth is sensitive to rapamycin (Crespo et al, 2005;Diaz-Troya et al, 2008a). Treatment of Chlamydomonas cells with rapamycin results in a pronounced increase of vacuole size that resembles autophagy-like processes (Crespo et al, 2005).…”