# Experimental Approaches in Delineating mTOR Signaling
## 2 Overview of mTOR Signaling  
mTOR is present in two structurally distinctive mTOR protein complexes, namely, mTOR1 and mTORC2. Both complexes share common subunits mTOR and GBL, whereas Raptor depicts mTOR1 and Rictor/Sin1 uniquely earmark mTORC2 [8]. Activation of mTORC2 is largely induced by growth signaling, and mTOR1 responds to both growth stimulations from mTORC2/Akt/TSC2/Rheb signaling and availability of amino acids through Rag signaling [5; 6]. Given to its pivotal roles in controlling essential cellular processes and physiological functions, activation of mTORC1 and mTORC2 is tightly controlled in both tempo and spatial manners [4]. This is achieved by a hierarchy of protein cascade-mediated mTOR1 activity control mechanisms on lysosome (detailed in later sections in this review) or by a variety of post-translational modifications (such as phosphorylation [9] and ubiquitination [10]) to acutely respond to cellular and environmental cues. Due to the large entity of all mTOR-related research articles published in the past and limited space in this review, we will only focus on discoveries that lay the foundation to shape up the mTOR signaling pathway, although important and disease-related, transcriptional, translational, and post-translational regulations of the mTOR signaling will not be discussed in this review. We also apologize to colleagues whose important and fabulous work could not be mentioned in this review due to page limits. Identification of _TOR_ Genes by Yeast Genetics and Biochemical Purification Approaches as Rapamycin Effects  
Distinct from kinases such as p44 MAPK [11], whose discovery was initiated by cloning of the kinase gene, the journey of mTOR signaling starts from isolation of an antifungal compound rapamycin from soil on Easter Island in 1975 by [PERSON] [12]. Rapamycin demonstrated an activity against _Candida albicans_ but not Gram-positive and -negative bacteria. Although earlier work revealed that rapamycin was immunosuppressive [13] and impeded cancer growth [14], the underlying mechanisms for rapamycin function was halted until rapamycin was broadly available to researchers. Rapamycin was observed to arrest eukaryotic cells at the G1 phase in cell cycle. A genetic search for mutations that confer to rapamycin resistance in budding yeast led to identification of _TOR1_ and _TOR2_ genes as rapamycin effectors [15]. Specifically, spontaneous independent mutants resistant to rapamycin (0.1 ug/mL) were isolated from a haploid derivative of yeast strain JK9-3d. Genetic crosses between rapamycin-resistant and -sensitive parental strains demonstrated that the majority of the mutations (15 of 18) were fully recessive (diploids were rapamycin sensitive). Two mutations, labeled as _tor1-1_ and _tor2-1_ for target of rapamycin, were recessive at 24 \({}^{\circ}\)C, while were partially dominant at 30 \({}^{\circ}\)C or 37 \({}^{\circ}\)C. The one fully dominant mutant, _tor1-2_, was isolated from the strain JK9-3d. Examination of crosses between the _tor1-1_ and _tor2-1_ mutants initially suggested that these two mutations were nonallelic because they failed to complement; a _tor1-1\({}^{\text{\text{\text{\text{\textasteraster}}}}}\)tor2-1_ diploid was resistant to rapamycin (10 ug/mL) under conditions where the two mutations were recessive (24 \({}^{\circ}\)C). However, the two mutations segregated independently in meiosis [15], therefore they were defined as two independent genes (_TOR1_ and _TOR2_). Two years later, an independent study isolating dominant rapamycin-resistant mutants using yeast genetics also pinpointed _DRR1/DRR2_ (which are _TORs_) as targets mediating the toxicity activity of rapamycin in yeast [16]. Subsequently, the connection between rapamycin and TORs was revealed by both biochemical and genetic approaches. Biochemically, the cellular binding partner(s) for rapamycin was identified as FKBP12, which is shared by FK506 [17], a related immunosuppressive compound that also blocks helper T cell activation.