SUMMARY Mammalian cells reorganize their proteomes in response to nutrient stress via translational suppression and degradative mechanisms using the proteasome and autophagy systems 1 , 2 . Ribosomes are central targets of this response, as they are responsible for translation and subject to lysosomal turnover upon nutrient stress 3 – 5 . Ribosomal (r)-protein abundance (~6% of the proteome, ~10 7 copies/cell) 6 , 7 and their enrichment in arginine (Arg) and lysine (Lys) residues has led to the hypothesis that they are selectively used as a source of basic AAs during nutrient stress via autophagy 4 . However, the relative contributions of translational and degradative mechanisms to the control of r-protein abundance during acute stress responses is poorly understood, as is the extent to which r-proteins are employed to generate AAs when specific building blocks are limited 7 . Here, we integrate quantitative global translatome and degradome proteomics 8 with genetically encoded Ribo-Keima 5 and Ribo-Halo reporters to interrogate r-protein homeostasis with and without active autophagy. Upon acute nutrient stress, cells strongly suppress r-protein translation, but, remarkably, r-protein degradation occurs largely through non-autophagic pathways. Simultaneously, loss of r-protein abundance is compensated for by reduced dilution of pre-existing ribosomes and reduced cell volume, thereby maintaining ribosome density within single cells. Withdrawal of basic or hydrophobic AAs induces translational repression without differential induction of ribophagy, indicating that ribophagy is not used to selectively produce basic amino acids during acute nutrient stress. We present a quantitative framework describing the contributions of biosynthetic and degradative mechanisms to r-protein abundance and proteome remodeling during nutrient stress.

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