Target of rapamycin (TOR) is an evolutionarily conserved protein kinase that controls multiple cellular processes upon various intracellular and extracellular stimuli. Since its first discovery, extensive studies have been conducted both in yeast and animal species including humans. Those studies have revealed that TOR forms two structurally and physiologically distinct protein complexes; TOR complex 1 (TORC1) is ubiquitous among eukaryotes including animals, yeast, protozoa, and plants, while TOR complex 2 (TORC2) is conserved in diverse eukaryotic species other than plants. The studies have also identified two crucial regulators of mammalian TORC1 (mTORC1), Ras homolog enriched in brain (RHEB) and RAG GTPases. Of these, RAG regulates TORC1 in yeast as well and is conserved among eukaryotes with the green algae and land plants as apparent exceptions. RHEB is present in various eukaryotes but sporadically missing in multiple taxa. RHEB, in the budding yeast Saccharomyces cerevisiae, appears to be extremely divergent with concomitant loss of its function as a TORC1 regulator. In this review, we summarize the evolutionarily conserved functions of the key regulatory subunits of TORC1 and TORC2, namely RAPTOR, RICTOR, and SIN1. We also delve into the evolutionary conservation of RHEB and RAG and discuss the conserved roles of these GTPases in regulating TORC1.

The human Mre11/Rad50 complex is one of the key factors in genome maintenance pathways. Previous nanoscale imaging by atomic force microscopy (AFM) showed that the ring-like structure of the human Mre11/Rad50 complex transiently opens at the zinc hook of Rad50. However, imaging of the human Mre11/Rad50 complex by high-speed AFM shows that the Rad50 coiled-coil arms are consistently bridged by the dimerized hooks while the Mre11/Rad50 ring opens by disconnecting the head domains; resembling other SMC proteins such as cohesin or condensin. These architectural features are conserved in the yeast and bacterial Mre11/Rad50 complexes. Yeast strains harboring the chimeric Mre11/Rad50 complex containing the SMC hinge of bacterial condensin MukB instead of the RAD50 hook properly functions in DNA repair. We propose that the basic role of the Rad50 hook is similar to that of the SMC hinge, which serves as rather stable dimerization interface.

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) protein kinase forms multi-subunit TOR complex 1 (TORC1) and TOR complex 2 (TORC2), which exhibit distinct substrate specificities. Sin1 is one of the TORC2-specific subunit essential for phosphorylation and activation of certain AGC-family kinases. Here, we show that Sin1 is dispensable for the catalytic activity of TORC2, but its conserved region in the middle (Sin1CRIM) forms a discrete domain that specifically binds the TORC2 substrate kinases. Sin1CRIM fused to a different TORC2 subunit can recruit the TORC2 substrate Gad8 for phosphorylation even in the sin1 null mutant of fission yeast. The solution structure of Sin1CRIM shows a ubiquitin-like fold with a characteristic acidic loop, which is essential for interaction with the TORC2 substrates. The specific substrate-recognition function is conserved in human Sin1CRIM, which may represent a potential target for novel anticancer drugs that prevent activation of the mTORC2 substrates such as AKT.

Mammalian target of rapamycin complex 1 (TORC1) is controlled by the GATOR complex composed of the GATOR1 subcomplex and its inhibitor, the GATOR2 subcomplex, sensitive to amino acid starvation. Previously, we identified fission yeast GATOR1 that prevents deregulated activation of TORC1 (Chia et al., 2017). Here, we report identification and characterization of GATOR2 in fission yeast. Unexpectedly, the GATOR2 subunit Sea3, an ortholog of mammalian WDR59, is physically and functionally proximal to GATOR1, rather than GATOR2, attenuating TORC1 activity. The fission yeast GATOR complex is dispensable for TORC1 regulation in response to amino acid starvation, which instead activates the Gcn2 pathway to inhibit TORC1 and induce autophagy. On the other hand, nitrogen starvation suppresses TORC1 through the combined actions of the GATOR1-Sea3 complex, the Gcn2 pathway, and the TSC complex, another conserved TORC1 inhibitor. Thus, multiple, parallel signaling pathways implement negative regulation of TORC1 to ensure proper cellular starvation responses.

Saccharomyces cerevisiae sake yeast strain Kyokai no. 7 (K7) and its relatives carry a homozygous loss-of-function mutation in the RIM15 gene, which encodes a Greatwall family protein kinase. Disruption of RIM15 in nonsake yeast strains leads to improved alcoholic fermentation, indicating that the defect in Rim15p is associated with the enhanced fermentation performance of sake yeast cells. In order to understand how Rim15p mediates fermentation control, we here focused on target-of-rapamycin protein kinase complex 1 (TORC1) and protein phosphatase 2A with the B55δ regulatory subunit (PP2AB55δ), complexes that are known to act upstream and downstream of Rim15p, respectively. Several lines of evidence, including our previous transcriptomic analysis data, suggested enhanced TORC1 signaling in sake yeast cells during sake fermentation. Fermentation tests of the TORC1-related mutants using a laboratory strain revealed that TORC1 signaling positively regulates the initial fermentation rate in a Rim15p-dependent manner. Deletion of the CDC55 gene, encoding B55δ, abolished the high fermentation performance of Rim15p-deficient laboratory yeast and sake yeast cells, indicating that PP2AB55δ mediates the fermentation control by TORC1 and Rim15p. The TORC1-Greatwall-PP2AB55δ pathway similarly affected the fermentation rate in the fission yeast Schizosaccharomyces pombe, strongly suggesting that the evolutionarily conserved pathway governs alcoholic fermentation in yeasts. It is likely that elevated PP2AB55δ activity accounts for the high fermentation performance of sake yeast cells. Heterozygous loss-of-function mutations in CDC55 found in K7-related sake strains may indicate that the Rim15p-deficient phenotypes are disadvantageous to cell survival.IMPORTANCE The biochemical processes and enzymes responsible for glycolysis and alcoholic fermentation by the yeast S. cerevisiae have long been the subject of scientific research. Nevertheless, the factors determining fermentation performance in vivo are not fully understood. As a result, the industrial breeding of yeast strains has required empirical characterization of fermentation by screening numerous mutants through laborious fermentation tests. To establish a rational and efficient breeding strategy, key regulators of alcoholic fermentation need to be identified. In the present study, we focused on how sake yeast strains of S. cerevisiae have acquired high alcoholic fermentation performance. Our findings provide a rational molecular basis to design yeast strains with optimal fermentation performance for production of alcoholic beverages and bioethanol. In addition, as the evolutionarily conserved TORC1-Greatwall-PP2AB55δ pathway plays a major role in the glycolytic control, our work may contribute to research on carbohydrate metabolism in higher eukaryotes.