Systematic quantitative analysis of ribosome inventory during nutrient stress

Abstract

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.

Ribosome recycling is not critical for translational coupling in Escherichia coli

“…Levels of protein synthesis were measured by HPG incorporation ( Sherratt et al, 2017 ; An et al, 2020 ). Cultures were grown at 37°C in M9 media containing 1x M9 salt (49 mM Na 2 HPO 4, 22 mM KH 2 PO 4, 8.6 mM NaCl, 18.7 mM NH 4 Cl), 100 μM CaCl 2 , 1 mM MgSO 4 , 1x minimal essential medium (MEM) vitamin solution, 1x methionine biosynthesis inhibition amino acids (L-lysine (100 μg/ml), L-threonine (100 μg/ml), L-phenylalanine (100 μg/ml), L-isoleucine (50 μg/ml), L-leucine (50 μg/ml), and L-valine (50 μg/ml)), and 0.2% carbon source.…”

“…Levels of protein synthesis were measured by HPG incorporation (Sherratt et al, 2017;An et al, 2020).…”

iRQC, a surveillance pathway for 40S ribosomal quality control during mRNA translation initiation

“…An autophagic mechanism seemed most plausible given that previous studies in S. cerevisiae have demonstrated that starvation conditions that inhibit mTOR signaling and stimulate autophagic flux result in enhanced ribosomal turnover by the autophagy pathway (Kraft et al, 2008). While mTOR-dependent degradation of ribosomes via autophagy does not appear to play a large role in regulating ribosomal abundance in mammalian cells (An and Harper, 2018;An et al, 2020), we directly evaluated if uS3 or uS5 ubiquitylation resulted in autophagy-dependent degradation of 40S ribosomal subunits. We first examined uS3 and uS5 protein levels upon RNF10 overexpression in parental or autophagy-deficient cells that are devoid of the critical ULK1 complex member, RB1CC1 (FIP200) (An et al, 2019).…”

Selectivity of mRNA degradation by autophagy in yeast

“…While the clear sequestration of ribosomes within vacuoles has been reported 26 , whether mammalian ribophagy occurs is currently a point of contention. While one study reported containing ribosomes within autophagosomes and attributed their sequestration into autophagosomes to the receptor protein NUFIP1 39 , another showed only limited degradation of ribosomes by autophagy during nutrient stress 40 . Meanwhile, the proportion of ribosomal protein in yeast is an order of magnitude higher than that of mammalian cells 41 .…”