TOR complex 1 (TORC1) is a multi-subunit protein kinase complex that controls cellular growth in response to environmental cues. The regulatory subunits of mammalian TORC1 (mTORC1) include RAPTOR (also known as RPTOR), which recruits mTORC1 substrates, such as S6K1 (also known as RPS6KB1) and 4EBP1 (EIF4EBP1), by interacting with their TOR signaling (TOS) motif. Despite the evolutionary conservation of TORC1, no TOS motif has been described in lower eukaryotes. In the present study, we show that the fission yeast S6 kinase Psk1 contains a TOS motif that interacts with Mip1, a RAPTOR ortholog. The TOS motif in Psk1 resembles those in mammals, including the conserved phenylalanine and aspartic acid residues essential for the Mip1 interaction and TORC1-dependent phosphorylation of Psk1. The binding of the TOS motif to Mip1 is dependent on Mip1 Tyr-533, whose equivalent in RAPTOR is known to interact with the TOS motif in their co-crystals. Furthermore, we utilized the mip1-Y533A mutation to screen the known TORC1 substrates in fission yeast and successfully identified Atg13 as a novel TOS-motif-containing substrate. These results strongly suggest that the TOS motif represents an evolutionarily conserved mechanism of the substrate recognition by TORC1.
The target of rapamycin (TOR) kinase, a member of the phosphoinositide 3-kinase-related kinase (PIKK) family, is highly conserved among diverse eukaryotes as a master regulator of cell growth and metabolism (Soulard et al., 2009; Wullschleger et al., 2006). TOR forms two distinct multi-subunit complexes termed TOR complex 1 (TORC1) and TOR complex 2 (TORC2), each of which phosphorylates specific sets of cellular substrates in response to diverse stimuli, such as nutrients and growth factors (Laplante and Sabatini, 2012; Saxton and Sabatini, 2017; Zoncu et al., 2011). Mammalian TORC1 (mTORC1), whose regulatory subunits include RAPTOR (also known as RPTOR) (Hara et al., 2002; Kim et al., 2002) and mLST8 (Kim et al., 2003), promotes protein synthesis and other anabolic processes, while suppressing cellular catabolic processes such as autophagy. Accumulating evidence indicates that deregulated activation of mTORC1 is associated with a variety of human diseases, including cancers, diabetes and neurodegenerative disorders (Laplante and Sabatini, 2012; Saxton and Sabatini, 2017; Zoncu et al., 2011).
As the mTOR kinase serves as a catalytic subunit in both mTORC1 and mTORC2, the distinct substrate specificities of the two complexes are likely to be determined by the regulatory subunits unique to each complex. mTORC1 has been proposed to recruit its substrates through the mTORC1-specific subunit RAPTOR. RAPTOR physically interacts with an AGC-family kinase known as ribosomal protein S6 kinase 1 (S6K1, also known as RPS6KB1), as well as eukaryotic initiation factor 4E (eIF4E)-binding protein 1 (4EBP1, also known as EIF4EBP1), two of the best-characterized mTORC1 substrates (Hara et al., 2002; Nojima et al., 2003). The N-terminus of S6K1 and the C-terminus of 4EBP1 contain a consensus sequence of approximately five amino acid residues called the TOR signaling (TOS) motif that binds to RAPTOR. The TOS motif–RAPTOR interaction is essential for the phosphorylation of S6K1 and 4EBP1 by mTORC1 both in vitro and in vivo (Choi et al., 2003; Nojima et al., 2003; Schalm and Blenis, 2002; Schalm et al., 2003). The proline-rich AKT substrate of 40 kDa (PRAS40, also known as AKT1S1) (Vander Haar et al., 2007; Sancak et al., 2007) also carries the TOS motif, which contributes to the inhibitory interaction of PRAS40 with mTORC1 (Oshiro et al., 2007; Wang et al., 2007). A recent structural study of the co-crystals of RAPTOR and the TOS motif peptides as well as the mTORC1–4EBP1 complex has revealed how the TOS motif interacts with the RAPTOR subunit of mTORC1 (Yang et al., 2017); the TOS motif binds to a groove formed between the RAPTOR N-terminal conserved (RNC) domain and the first several helices of the α-solenoid HEAT repeat in the middle of RAPTOR. On the other hand, no apparent TOS motif is present in many of the known mTORC1 substrates, including MAF1, a negative regulator of RNA polymerase III (Michels et al., 2010) and the autophagy regulators ULK1 and ATG13 (Ganley et al., 2009; Hosokawa et al., 2009; Jung et al., 2009). In contrast to the TOS-motif-dependent recruitment of substrates to mTORC1, very little is known about how mTORC1 recognizes substrates with no discernible TOS motif.
The TORC1 signaling pathway is also conserved in the fission yeast Schizosaccharomyces pombe that has been utilized as an excellent model system for the study of diverse cellular processes. Unlike mammalian cells, this unicellular eukaryote has two TOR paralogs named Tor1 and Tor2, which form TORC2 and TORC1, respectively (Morozumi and Shiozaki, 2021; Otsubo and Yamamato, 2008). The regulatory subunits of fission yeast TORC1 include Mip1, a RAPTOR ortholog, as well as the mLST8 equivalent Wat1 (also known as Pop3) (Álvarez and Moreno, 2006; Hayashi et al., 2007; Matsuo et al., 2007). Under nitrogen-replete conditions, activated TORC1 promotes vegetative growth and suppresses sexual differentiation (Álvarez and Moreno, 2006; Matsuo et al., 2007; Uritani et al., 2006). One of the confirmed substrates of fission yeast TORC1 is Psk1, an AGC-family protein kinase responsible for the phosphorylation of the ribosomal S6 proteins in fission yeast (Nakashima et al., 2012); thus, the TORC1-dependent regulation of the S6 protein phosphorylation appears to be conserved from yeast to humans. Similar to the activation of S6K1 by mTORC1 (Magnuson et al., 2012), fission yeast Psk1 is activated by TORC1 through phosphorylation of Thr-392 in the turn motif as well as Thr-415 in the hydrophobic motif (Nakashima et al., 2012). However, no TOS-like motif has been identified in Psk1 or any known TORC1 substrates in lower eukaryotes.
In the present study, we have characterized the physical interaction between Psk1 and the TORC1 subunit Mip1. The RAPTOR ortholog Mip1 interacts with the N-terminus of Psk1, and this interaction is indispensable for the Psk1 phosphorylation by TORC1. Interestingly, the N-terminal region of Psk1 contains an amino acid stretch that resembles the mammalian TOS motif, including the phenylalanine and aspartic acid residues essential for the interaction with RAPTOR. Moreover, the amino acid residues in RAPTOR that are implicated in the direct interaction with the TOS motif are conserved in fission yeast Mip1, and a mutation to one of those residues, mip1-Y533A, disrupts its interaction with Psk1. By screening the known TORC1 substrates in the mip1-Y533A background, we have successfully identified fission yeast Atg13 as a novel TOS-motif-containing substrate. These results strongly suggest that the mechanism of the TOS-motif-mediated recruitment of the TORC1 substrates is conserved between fission yeast and humans. As far as we know, this study reports the first examples of the TOS motif in lower eukaryotes.
A TOS-motif-like sequence in the Psk1 kinase is required for its interaction with Mip1
Because the RAPTOR subunit of mTORC1 binds and recruits S6K1 for its phosphorylation and activation (see the Introduction), we set out to examine if Mip1, a fission yeast ortholog of RAPTOR, also physically interacts with the TORC1 substrate Psk1. Using Psk1 and Mip1 as bait and prey, respectively, yeast two-hybrid assays were carried out, which successfully detected interaction between the two proteins (Fig. 1A; Fig. S1A). To narrow down the Mip1-binding region within Psk1, yeast two-hybrid assays were repeated with the Psk1 N-terminal fragment (amino acids 1–90), the kinase catalytic domain (amino acids 83–369) and the C-terminal fragment (amino acids 343–436). Among those Psk1 fragments, only the N-terminal fragment of Psk1 exhibited interaction with Mip1 (Fig. 1A; Fig. S1B), and even more strikingly, we found that the N-terminal 10 amino acid residues of Psk1 were sufficient for the interaction (Fig. 1A; Fig. S1C). Consistently, the Psk1 fragment lacking the N-terminal 10 residues (amino acids 11–436) failed to interact with Mip1, indicating that the N-terminal 10 amino acid residues of Psk1 are indispensable for the Psk1–Mip1 interaction.
Next, we assessed each of the Psk1 N-terminal 10 residues for the Mip1 interaction by alanine substitutions of the individual residues followed by yeast two-hybrid assays. Mutations to Val-3, Phe-4, Phe-6, and Asp-7 in Psk1 drastically abolished the interaction with Mip1, whereas those to Met-5, Glu-8 and Asp-10 only partially compromised the Psk1–Mip1 interaction. On the other hand, alanine substitutions of Pro-2 and His-9 showed no apparent impact on the interaction (Fig. 1B; Fig. S1D). We noticed that this N-terminal region of Psk1 is somewhat reminiscent of the TOS motifs in human S6K1, 4EBP1 and PRAS40, with the spaced phenylalanine and aspartic acid residues essential for the interaction with RAPTOR or Mip1 (Fig. 1C; Nojima et al., 2003; Oshiro et al., 2007; Schalm and Blenis, 2002; Schalm et al., 2003; Wang et al., 2007; Yang et al., 2017). Taken together, these results suggest that fission yeast Psk1 has a TOS-motif-like sequence necessary and sufficient for the interaction with the RAPTOR ortholog Mip1.
The N-terminal TOS motif of Psk1 is essential for its phosphorylation by TORC1
Next, we examined whether the TOS-motif-like stretch found in the N-terminus of Psk1 is required for the TORC1-dependent phosphorylation of Psk1. The psk1 null (Δpsk1) strain was transformed with a plasmid that expresses the full-length or N-terminally truncated (amino acids 11–436) Psk1 with the FLAG epitope tag, followed by immunoblot analysis. As reported previously (Chia et al., 2017; Nakashima et al., 2012), the TORC1-dependent phosphorylation of Thr-415 was readily detected with the full-length Psk1 using the antibodies against phosphorylated S6K1 (‘pPsk1’ in Fig. 2A). Anti-FLAG immunoblotting also showed a slow-migrating band, which corresponds to the phosphorylated form of Psk1 (Chia et al., 2017; Nakashima et al., 2012), confirming the phosphorylation of full-length Psk1. In contrast, these analyses failed to detect phosphorylation of Psk1(11–436) lacking the N-terminal 10 residues (Fig. 2A). Similar experiments were carried out using plasmids that express Psk1 with an alanine substitution of the individual N-terminal residues. We found that the Psk1 phosphorylation was markedly reduced by the V3A, F4A, F6A, D7A and D10A substitutions (Fig. 2B). Consistent results were also obtained when the N-terminal truncation or substitution mutations were introduced to the chromosomal psk1 gene (Fig. 2C). Moreover, in those mutant strains defective in the Psk1 phosphorylation, the Psk1-dependent phosphorylation of the ribosomal S6 proteins (pRps6 in Fig. 2C) was significantly compromised. These observations are consistent with the earlier report that the TORC1-dependent phosphorylation activates Psk1, which in turn phosphorylates Rps6 (Nakashima et al., 2012). Importantly, those Psk1 mutants with reduced phosphorylation also showed compromised interaction with Mip1 in the yeast two-hybrid assays (Fig. 1A,B); therefore, the Psk1 N-terminal sequence identified by these experiments appears to serve as a TOS motif, which mediates the interaction with the Mip1 subunit of TORC1 as well as the TORC1-dependent phosphorylation and activation of Psk1.
Mip1 Y533A mutation impairs the TOS-motif-dependent recognition of TORC1 substrates
Based on the crystal structure of RAPTOR bound to the S6K1 TOS motif peptide, it has been proposed that Arg-54, Arg-335, Arg-446 and Tyr-475 in human RAPTOR directly interact with the TOS motif (Fig. 3A; Yang et al., 2017). Interestingly, all of the four residues are conserved in fission yeast Mip1 (Arg-113, Arg-358, Arg-504 and Tyr-533, respectively; Fig. 3B). To test whether these conserved residues of Mip1 contribute to the interaction with Psk1, we constructed strains whose chromosomal mip1 gene carries an alanine substitution to each of those residues. The growth rate of all the constructed mip1 mutants was comparable to those of the wild-type strain (Fig. S2A). We also assessed sexual differentiation of mip1 mutants; mating of the homothallic (h90) haploid mip1 mutants was induced by nitrogen starvation (Fig. S2B), while being suppressed completely on rich yeast extract medium (data not shown), as in the wild-type strain. On the other hand, these mip1 mutants were sensitive to rapamycin, a specific inhibitor of TORC1, with mip1-R113A exhibiting a milder phenotype (Fig. 3C), implying that the TORC1 function is somewhat compromised in these mip1 mutants. Indeed, the phosphorylation of Psk1 was drastically reduced in these mutant strains with the exception of mip1-R113A (Fig. 3D).
To examine whether the compromised Psk1 phosphorylation in the mip1-R358A, -R504A, and -Y533A mutants is caused by impaired Mip1–Psk1 interaction, we carried out yeast two-hybrid assays using Mip1 carrying those mutations as prey. As expected, Mip1-R504A and Mip1-Y533A hardly interacted with full-length Psk1 as well as its N-terminal fragment of 10 residues (Fig. 4A and Fig. S3). On the other hand, no obvious defect was observed in the interaction between Mip1-R358A and Psk1. One likely possibility is that Mip1-R358A can interact with Psk1 but the mutation affects another function of Mip1, such as its ability to bind the Tor2 kinase for TORC1 assembly. To test the Mip1–Tor2 interaction, we constructed fission yeast strains in which the chromosomal tor2+ gene is tagged with the FLAG epitope sequence and the wild-type or mutant Mip1 proteins are expressed with the myc epitope from the mip1 locus. Immunoprecipitation of FLAG-tagged Tor2 co-purified comparable amounts of the wild-type and Y533A mutant Mip1 proteins, but less of Mip1-R358A and Mip1-R504A (Fig. 4B). Together, these results suggest that the R358A and R504A substitutions affect the integrity of TORC1, whereas the Y533A mutation more specifically interferes with the recognition of Psk1 by Mip1. We also confirmed that those mip1 mutations have little impact on the cellular localization of Mip1, by fluorescence microscopy of the wild-type and mip1 mutant strains with their mip1 loci tagged with the sequence encoding GFP (Fig. 4C). It was previously reported that fission yeast TORC1 localizes to the surface of vacuoles, the lysosome equivalent in yeasts (Chia et al., 2017; Valbuena et al., 2012); we observed similar vacuolar localization of Mip1-GFP as well as those with the R358A, R504A or Y533A mutations.
Our experiments described above strongly suggest that the impaired phosphorylation (Fig. 3D) and Mip1 interaction (Fig. 4A) of Psk1 in the mip1-Y533A mutant are caused by a defect in the evolutionarily conserved mechanism of TOS-motif recognition. To corroborate the defective TOS-motif recognition by Mip1-Y533A, we examined whether TORC1 carrying the Mip1-Y533A subunit fails to phosphorylate the canonical TOS-motif-containing substrate 4EBP1 (Nojima et al., 2003; Schalm et al., 2003). Although 4EBP1 is not conserved in fission yeast, TORC1 isolated from fission yeast cells can phosphorylate rat 4EBP1 in vitro (Fig. 4D; Takahara and Maeda, 2012). Importantly, 4EBP1 harboring an alanine substitution of Phe-113 within its TOS motif was not phosphorylated by TORC1 (‘F113A’ in Fig. 4D), demonstrating that fission yeast TORC1 phosphorylates mammalian 4EBP1 in a TOS-motif-dependent manner. As expected, the in vitro kinase assay found that TORC1 immunopurified from mip1-Y533A cells had significantly compromised activity toward 4EBP1 (Fig. 4E), consistent with the impaired ability of Mip1-Y533A to recognize the TOS motif.
Taken together, these data strongly suggest that the TOS-motif-dependent phosphorylation of TORC1 substrates is conserved between fission yeast and mammals and that the Y533A mutation undermines the TOS-motif recognition by the Mip1 subunit.
atg13-F538A mutation impairs TORC1-dependent phosphorylation of Atg13
As shown in Fig. 5A, neither the Δpsk1 strain nor the psk1(11-436) strain defective in Psk1 phosphorylation (Fig. 2) showed any growth defect in the presence or absence of rapamycin. Thus, the TORC1-dependent phosphorylation and activation of the Psk1 kinase (Nakashima et al., 2012) does not appear to be critical to fission yeast cell growth. Therefore, the rapamycin-sensitive phenotype of the mip1-Y533A mutant (Figs 3C and 5A) might indicate that phosphorylation of additional TORC1 substrates other than Psk1 is compromised in this mutant. Thus, in mip1-Y533A cells we examined the phosphorylation status of Sck1, Sck2, Maf1 and Atg13, the previously reported TORC1 substrates in fission yeast (Du et al., 2012; Nakashima et al., 2012; Otsubo et al., 2017, 2018; Shetty et al., 2020). The FLAG-tagged Sck1 protein phosphorylated by TORC1 is detectable as a band with less electrophoretic mobility (Chia et al., 2017), which disappeared in the presence of rapamycin but remained detectable in the mip1-Y533A mutant (Fig. 5B). Similarly, the mip1-Y533A mutation exhibited little impact on the slow-migrating, rapamycin-sensitive bands of the Sck2-FLAG (Fig. 5C) and Maf1-FLAG (Fig. 5D) proteins. These observations indicate that the mip1 mutation does not affect the TORC1-dependent phosphorylation of Sck1, Sck2 and Maf1.
On the other hand, the electrophoretic mobility of Atg13-myc increased in wild-type cells treated with rapamycin as well as in the mip1-Y533A mutant (Fig. 5E), suggesting impaired TORC1-dependent phosphorylation of Atg13 in the mutant strain. Thus, in addition to Psk1, Atg13 also appears to be phosphorylated by TORC1 in a manner dependent on Mip1 Tyr-533, a residue critical for the TOS-motif-mediated recruitment of TORC1 substrates. Therefore, we carefully scanned the amino acid sequence of Atg13 and found a sequence (FDIDT, amino acids 538–542) with significant similarity to the TOS motif in human S6K1 (Fig. S4). To examine whether this sequence contributes to the TORC1-dependent phosphorylation of Atg13, we constructed a strain whose chromosomal atg13 gene carries a mutation that substitutes Phe-538 with alanine. Importantly, the electrophoretic mobility of Atg13-F538A protein was found to be comparable to that of the wild-type Atg13 protein in mip1-Y533A:myc cells (Fig. 5F). These results strongly suggest that FDIDT in fission yeast Atg13 serves as a TOS motif that mediates the TORC1-dependent phosphorylation of Atg13.
Under nutrient-replete conditions, TORC1 inhibits the initiation of autophagy by phosphorylating the regulatory components of the autophagy pathway, such as Atg13 (Kohda et al., 2007; Otsubo et al., 2017, 2018). As Atg13 phosphorylation is compromised in the mip1-Y533A mutant (Fig. 5E), we assessed the induction of autophagy in this mutant by monitoring the proteolytic cleavage of the GFP-Atg8 fusion protein (Mukaiyama et al., 2009). Nitrogen starvation induced the autophagic degradation of GFP-Atg8, releasing free GFP both in wild-type and mip1-Y533A cells (‘-N’ in Fig. S5), but not before starvation, suggesting that the mip1-Y533A mutation is not enough to induce autophagy. A small amount of the free GFP was detectable in the mip1-Y533A mutant treated by rapamycin, whereas rapamycin did not induce autophagy at all in wild-type cells as reported previously (Mukaiyama et al., 2009; Takahara and Maeda, 2012). In addition, the F538A mutation within the TOS-like motif of Atg13 did not affect autophagy induction both in the presence and absence of rapamycin (data not shown). It is likely that the lack of the TOS-motif-dependent phosphorylation of Atg13 per se is insufficient for the autophagy initiation, probably because there are other TORC1 substrates in the negative regulation of autophagy.
In mammals, the RAPTOR subunit serves as a substrate-binding subunit of mTORC1, interacting with the TOS motif in some of the mTORC1 substrates, such as S6K1 and 4EBP1 (Nojima et al., 2003; Schalm and Blenis, 2002). Despite the evolutionary conservation of RAPTOR (Tatebe and Shiozaki, 2017) and the extensive studies of TORC1 signaling in yeast species, there has been no report of the TOS motif in lower eukaryotes. The reported TOS motifs are 5 amino acid residues in length with limited consensus among them, handicapping the sequence-based search for TOS motifs.
In the present study, we have demonstrated that, like S6K1, the fission yeast S6 kinase Psk1 contains a TOS motif in its N-terminus. This short sequence in Psk1 is essential for its interaction with the RAPTOR ortholog Mip1 as well as for its phosphorylation and activation by TORC1. Importantly, the phenylalanine and aspartic acid residues common among the known TOS motifs in the mTORC1 substrates are also conserved in the TOS-motif-like sequence found in Psk1 (Phe-4 and Asp-7; Fig. 1C), and these residues are indispensable for the interaction of Psk1 with Mip1. In the budding yeast Saccharomyces cerevisiae, Ypk3 has been identified as an AGC-family kinase that phosphorylates ribosomal protein S6 (González et al., 2015; Yerlikaya et al., 2016). Like human S6K1 and fission yeast Psk1, Ypk3 is phosphorylated and activated by TORC1; however, it remains unknown whether Ypk3 also has a TOS motif. By examining the amino acid sequence of the N-terminal non-catalytic domain of Ypk3, we have found FSLDE (amino acids 3–7), a short sequence that resembles the mammalian TOS motifs as well as the one found in Psk1 (Fig. S4). Although experimental validation is required, we propose that this N-terminal segment of Ypk3 is a strong candidate for the first example of the TOS motif in S. cerevisiae.
A co-crystal structural study identified the residues within RAPTOR that directly interact with the TOS motif (Yang et al., 2017), although the contribution of those residues to the substrate recognition by TORC1 in vivo has not been evaluated. By mutating the equivalent residues in fission yeast Mip1, we demonstrated that Mip1 Tyr-533, which corresponds to Tyr-475 in RAPTOR, is critical to the interaction of Mip1 with Psk1 as well as the TORC1-dependent phosphorylation of Psk1 in vivo. Furthermore, we utilized the mip1-Y533A mutation to search for additional TORC1 substrates with a TOS motif and successfully identified Atg13, which indeed contains a TOS-motif-like sequence (FDIDT, amino acids 538–542) essential for its phosphorylation (Fig. S4). We propose that the mip1-Y533A mutation characterized in this study and equivalent mutations in RAPTOR and its orthologs can serve as a new tool to identify TORC1 substrates with a TOS motif. Interestingly, Arabidopsis thaliana ATG13 possesses a TOS-motif-like sequence (FSDIF, amino acids 189–193), which is important for its interaction with RAPTOR and TORC1-dependent phosphorylation (Fig. S4; Son et al., 2018). On the other hand, we failed to find a TOS-motif-like sequence in human ATG13, possibly because of the short length and limited homology of the TOS motif. Alternatively, human ATG13 may have no TOS motif, being recruited to mTORC1 through a TOS-motif-independent mechanism. It has been reported that ATG13 forms a stable protein complex with ULK1, which is also an mTORC1 substrate that interacts with RAPTOR (Hosokawa et al., 2009). Thus, it is likely that human ATG13 is recruited to mTORC1 by forming a complex with ULK1.
In addition to Atg13, we examined the phosphorylation of Sck1, Sck2 and Maf1, the known TORC1 substrates in fission yeast (Nakashima et al., 2012; Otsubo et al., 2017; Shetty et al., 2020), without detectable effect of the mip1-Y533A mutation. It should be noted that budding yeast Maf1 is phosphorylated by both TORC1 (Lee et al., 2009; Wei et al., 2009) and Sch9, an AGC-family kinase activated by TORC1 (Urban et al., 2007). Thus, the rapamycin-sensitive electrophoretic mobility of fission yeast Maf1 (Fig. 5D) may also reflect phosphorylation by not only TORC1 but also an additional kinase(s), making it more difficult to evaluate the effect of mip1-Y533A on the Maf1 phosphorylation. In contrast, a previous study demonstrated the TORC1-dependent phosphorylation of Sck1 and Sck2 in fission yeast as well as in vitro phosphorylation of purified recombinant Sck1 and Sck2 by TORC1 (Nakashima et al., 2012). Therefore, these AGC-family kinases are likely to be direct substrates of TORC1 but, unlike Psk1, they both failed to interact with Mip1 in our yeast two-hybrid assays (data not shown). We speculate that TORC1 recognizes Sck1 and Sck2 by a TOS-motif-independent mechanism. Indeed, the majority of the known mTORC1 and TORC1 substrates have no apparent TOS motif, and very little is known about how those substrates are specifically recognized by mTORC1 and TORC1.
It has been established that mTORC2 and TORC2 contain the regulatory subunit Sin1, whose conserved region in the middle (CRIM) domain specifically binds and recruits particular AGC-family kinases for phosphorylation and activation (Cameron et al., 2011; Liao and Chen, 2012; Tatebe et al., 2017). Thus, the substrate specificity of the TOR kinase appears to be determined by its evolutionarily conserved regulatory subunits, although the kinase itself is likely to recognize and phosphorylate specific sequences within the recruited substrates. Further studies are required to fully understand the substrate specificity of mTORC1 and TORC1 at a molecular level, particularly the roles of RAPTOR or Mip1 and the other regulatory subunits in the TOS-motif-independent recognition of the substrates.
MATERIALS AND METHODS
Fission yeast strains and general methods
S. pombe strains used in this study are listed in Table S1. Growth media and basic techniques for S. pombe have been described previously (Moreno et al., 1991; Shiozaki and Russell, 1997). For the strain constructions, the PCR-based method was applied to introduce alanine substitutions and the epitope tag sequences to chromosomal genes as reported previously (Bähler et al., 1998; Erdeniz et al., 1997). Each alanine substitution was confirmed by genomic PCR followed by Sanger DNA sequencing.
Construction of gene expression plasmids
For the plasmids used in yeast two-hybrid assay, various Psk1 and Mip1 fragments were subcloned in the pGBT8 and pGAD GH vectors (Clontech Laboratories), respectively. For the plasmids used in the Psk1 expression experiments in fission yeast, various Psk1 fragments were subcloned in a derivative of pREP1 vector whose Pnmt1 promoter was replaced with the psk1+ promoter. The PrimeSTAR Mutagenesis Basal Kit (Takara Bio, Inc.) was used for site-directed mutagenesis.
Yeast two-hybrid assay
Yeast two-hybrid assays were performed as described previously (Morigasaki et al., 2019). Briefly, the Y2HGold budding yeast strain (Clontech Laboratories) was used as host, and interaction was determined by adenine and histidine auxotrophic markers.
S. pombe growth assay
Fission yeast cells were grown in YES liquid medium, and the cultures were adjusted to the cell concentration equivalent to an optical density at 600 nm (OD600) of 1.0. Serial dilutions of the adjusted cultures were spotted onto agar solid media. Images were captured by the LAS-4000 system (Fujifilm, Japan).
Homothallic haploid h90 cells were spotted onto a synthetic sporulation agar (SSA) plate (Egel et al., 1974). The cells were analyzed by microscopy after 48 h incubation at 25°C, and the mating efficiency was calculated as described previously (Kunitomo et al., 1995). At least 300 cells were counted for each strain.
Crude cell lysates were prepared using trichloroacetic acid (TCA) as described previously (Tatebe and Shiozaki, 2003). Proteins were separated by SDS-PAGE, transferred to a nitrocellulose membrane, and probed with primary antibodies diluted as follows; anti-phospho-p70 S6K (1:5000; cat. no. 9206, Cell Signaling Technology) for phospho-Psk1 (Thr-415) detection, anti-phospho-Akt substrate (1:4000; cat. no. 9614, Cell Signaling Technology) for phosphorylated Rps6 detection (Nakashima et al., 2010), anti-Psk1 (1:5000; Chia et al., 2017), anti-Spc1 (1:10 000; Tatebe and Shiozaki, 2003), anti-FLAG (1:5000; M2, Sigma-Aldrich), anti-myc (1:5000; 9E10, Covance), anti-phospho-4EBP1 (T37/46) (1:2500; cat. no. 2855, Cell Signaling Technology), anti-4EBP1 (1:2500; cat. no. 9452, Cell Signaling Technology), anti-GFP (1:2500; cat. no. 04404, Nacalai Tesque). Anti-rabbit IgG (H+L) HRP-conjugated (1:10 000; cat. no. W4011, Promega), anti-mouse IgG (H+L) HRP-conjugated (1:10 000; cat. no. W4021, Promega) and anti-rat IgG (H+L) HRP-conjugated (1:10 000; cat. no. 112-035-003, Jackson ImmunoResearch) were used as secondary antibodies. For the detection of the phosphorylated form of Maf1, proteins were separated by Phos-tag SDS-PAGE using an 8% SDS polyacrylamide gel containing 50 µM Phos-tag and 100 µM MnCl2.
Preparation of recombinant 4EBP1 expressed in Escherichia coli
pET14b expression vector carrying rat 4EBP1 (Addgene #15679; Choi et al., 2003) was transformed into the BL21 codon(+)RIL E. coli strain. The cells were grown in LB medium at 37°C until the OD600 reached 0.4–0.6. Expression of 4EBP1 was then induced by the addition of 0.1 mM isopropyl-β-D-1-thiogalactopyranoside, and the cells were further cultured at 18°C overnight. The cultures were harvested, resuspended in lysis buffer (50 mM Tris-HCl, pH 7.5, 500 mM NaCl, 2 mM 2-mercaptoethanol and 10% glycerol) and disrupted by sonication (for 10 min with on-time of 1 s and off-time of 1 s at 4°C). The cell debris was removed by centrifugation for 20 min at 10 000 g, and the supernatant was mixed gently with 1 ml of Ni-NTA agarose beads (Fujifilm Wako) at 4°C for 1 h. The protein-bound beads were packed into an Econo-Column (Bio-Rad Laboratories) and washed with lysis buffer. The proteins were eluted with lysis buffer containing 500 mM imidazole and dialyzed against storage buffer (20 mM Tris-HCl, pH 7.5, 200 mM NaCl, 2 mM 2-mercaptoethanol and 10% glycerol). The samples were flash-frozen in liquid nitrogen and stored at −80°C.
Immunoprecipitation and in vitro kinase assay
Yeast cells were disrupted in lysis buffer (20 mM HEPES-NaOH, pH 7.5, 150 mM sodium glutamate, 10% glycerol, 0.25% Tween-20, 10 mM sodium fluoride, 10 mM p-nitrophenylphosphate, 10 mM sodium pyrophosphate, 10 mM β-glycerophosphate and 0.1 mM sodium orthovanadate) containing 1 mM phenylmethylsulfonyl fluoride and protease inhibitor cocktail (P8849, Sigma-Aldrich) with glass beads using a Multi-beads Shocker (Yasui Kikai). Cell lysate was recovered by centrifugation for 15 min at 17 700 g and the total protein concentrations of cell lysates were determined by Bradford assay. For interaction between FLAG-tagged protein and myc-tagged protein, the recovered cell lysates were incubated with anti-FLAG M2-affinity gel (Sigma-Aldrich) for 2 h at 4°C, followed by extensive washing with lysis buffer. Resultant samples were subjected to immunoblotting.
For the in vitro kinase assay, cell lysates prepared from a fission yeast mip1:myc strain were incubated with anti-c-myc antibody beads (Fujifilm Wako) at 4°C for 2 h. The beads were washed with lysis buffer followed by additional washing with kinase wash buffer [20 mM HEPES-NaOH, pH 7.5, 150 mM NaCl and 1 mM dithiothreitol (DTT)]. The beads were then mixed with 250 ng of rat 4EBP1 in 20 μl of reaction buffer (20 mM HEPES-NaOH, pH 7.5, 1 mM MgCl2, 1 mM DTT and 100 mM NaCl) and reactions were initiated by the addition of ATP (1 mM). After incubation at 30°C for 30 min, the reactions were stopped by addition of Laemmli sample buffer, and the phosphorylation of 4EBP1 was monitored by immunoblotting.
Fluorescence microscopic analysis was performed using a DeltaVision Elite Microscopy System (GE Healthcare) as described previously (Chia et al., 2017; Morigasaki et al., 2019). Briefly, cells grown exponentially in EMM liquid were stained with FM4-64 fluorescent dye (Fujifilm Wako) for vacuole visualization and mounted on a thin layer of EMM agar. Z-axial images were taken at 0.4 μm with a 60× objective lens. Deconvolution of images was performed using DeltaVision SoftWoRx software.
We thank M. Yanagida for strains, K. Pan and A. Higuchi for technical assistance, and T. Toda for his advice.
Conceptualization: Y.M., H.T., K.S.; Methodology: Y.M., H.T.; Validation: Y.M., A.H., S.F., F.S., H.T.; Formal analysis: Y.M.; Investigation: Y.M., A.H., S.F., F.S., H.T.; Resources: Y.M., H.T.; Data curation: Y.M.; Writing - original draft: Y.M., K.S.; Writing - review & editing: K.S.; Visualization: Y.M.; Supervision: Y.M., H.T., K.S.; Project administration: Y.M., H.T., K.S.; Funding acquisition: Y.M., F.S., H.T., K.S.
This study was supported in part by the Japan Society for the Promotion of Science (JSPS) KAKENHI grants (17H06870 and 19K06564 to Y.M., 25440086 to H.T., 26291024 and 19H03224 to K.S.) and research grants to K.S. from the Ohsumi Frontier Science Foundation and Takeda Science Foundation. F.S. was supported by Graduate Student Scholarships from Panasonic Corporation and Sato Yo International Scholarship Foundation. Deposited in PMC for immediate release.
Peer review history
The peer review history is available online at https://journals.biologists.com/jcs/article-lookup/doi/10.1242/jcs.258865
The authors declare no competing or financial interests.