Amino acid availability controls mTORC1 activity via a heterodimeric Rag GTPase complex that functions as a scaffold at the lysosomal surface, bringing together mTORC1 with its activators and effectors. Mammalian cells express four Rag proteins (RagA–D) that form dimers composed of RagA/B bound to RagC/D. Traditionally, the Rag paralogue pairs (RagA/B and RagC/D) are referred to as functionally redundant, with the four dimer combinations used interchangeably in most studies. Here, by using genetically modified cell lines that express single Rag heterodimers, we uncover a Rag dimer code that determines how amino acids regulate mTORC1. First, RagC/D differentially define the substrate specificity downstream of mTORC1, with RagD promoting phosphorylation of its lysosomal substrates TFEB/TFE3, while both Rags are involved in the phosphorylation of non-lysosomal substrates such as S6K. Mechanistically, RagD recruits mTORC1 more potently to lysosomes through increased affinity to the anchoring LAMTOR complex. Furthermore, RagA/B specify the signalling response to amino acid removal, with RagB-expressing cells maintaining lysosomal and active mTORC1 even upon starvation. Overall, our findings reveal key qualitative differences between Rag paralogues in the regulation of mTORC1, and underscore Rag gene duplication and diversification as a potentially impactful event in mammalian evolution.

Rapamycin is a macrolide antibiotic that functions as an immunosuppressive and anti-cancer agent, and displays robust anti-ageing effects in multiple organisms including humans. Importantly, rapamycin analogs (rapalogs) are of clinical importance against certain cancer types and neurodevelopmental diseases. Although rapamycin is widely perceived as an allosteric inhibitor of mTOR (mechanistic target of rapamycin), the master regulator of cellular and organismal physiology, its specificity has not been thoroughly evaluated so far. In fact, previous studies in cells and in mice suggested that rapamycin may be also acting independently from mTOR to influence various cellular functions. Here, we generated a gene-edited cell line, that expresses a rapamycin-resistant mTOR mutant (mTORRR), and assessed the effects of rapamycin treatment on the transcriptome and proteome of control or mTORRR-expressing cells. Our data reveal a striking specificity of rapamycin towards mTOR, demonstrated by virtually no changes in mRNA or protein levels in rapamycin-treated mTORRR cells, even following prolonged drug treatment. Overall, this study provides the first comprehensive and conclusive assessment of the specificity of rapamycin, with important potential implications for ageing research and human therapeutics.

Rapamycin is a macrolide antibiotic that functions as an immunosuppressive and anti‐cancer agent, and displays robust anti‐ageing effects in multiple organisms including humans. Importantly, rapamycin analogues (rapalogs) are of clinical importance against certain cancer types and neurodevelopmental diseases. Although rapamycin is widely perceived as an allosteric inhibitor of mTOR (mechanistic target of rapamycin), the master regulator of cellular and organismal physiology, its specificity has not been thoroughly evaluated so far. In fact, previous studies in cells and in mice hinted that rapamycin may be also acting independently from mTOR to influence various cellular processes. Here, we generated a gene‐edited cell line that expresses a rapamycin‐resistant mTOR mutant (mTORRR) and assessed the effects of rapamycin treatment on the transcriptome and proteome of control or mTORRR‐expressing cells. Our data reveal a striking specificity of rapamycin towards mTOR, demonstrated by virtually no changes in mRNA or protein levels in rapamycin‐treated mTORRR cells, even following prolonged drug treatment. Overall, this study provides the first unbiased and conclusive assessment of rapamycin's specificity, with potential implications for ageing research and human therapeutics.

The authors noticed a mistake in Supplementary Table 1 after publication. The sequences listed in rows 44-46 for oligos sgRagC_5′_rev, sgRagC_3′_for and sgRagC_3′_rev were not correct, due to copy-paste errors introduced during file preparation. The correct sequences for these oligos are now presented in rows 44-46 in the corrected Supplementary Table 1 online. All other oligo sequences were double-checked and remain accurate. The respective plasmid constructs used to knock out RagC as well as the resulting cell lines are also correct. The authors sincerely apologize for this error.