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Mechanisms of In Vivo Ribosome Maintenance Change in Response to Nutrient Signals*

Open AccessPublished:December 08, 2016DOI:https://doi.org/10.1074/mcp.M116.063255

      Abstract

      Control of protein homeostasis is fundamental to the health and longevity of all organisms. Because the rate of protein synthesis by ribosomes is a central control point in this process, regulation, and maintenance of ribosome function could have amplified importance in the overall regulatory circuit. Indeed, ribosomal defects are commonly associated with loss of protein homeostasis, aging, and disease (
      • Ben-Zvi A.
      • Miller E.A.
      • Morimoto R.I.
      Collapse of proteostasis represents an early molecular event in Caenorhabditis elegans aging.
      ,
      • Conn C.S.
      • Qian S.B.
      Nutrient signaling in protein homeostasis: An increase in quantity at the expense of quality.
      ,
      • Choesmel V.
      • Bacqueville D.
      • Rouquette J.
      • Noaillac-Depeyre J.
      • Fribourg S.
      • Crétien A.
      • Leblanc T.
      • Tchernia G.
      • Da Costa L.
      • Gleizes P.E.
      Impaired ribosome biogenesis in Diamond–Blackfan anemia.
      ,
      • Xue S.
      • Barna M.
      Specialized ribosomes: A new frontier in gene regulation and organismal biology.
      ), whereas improved protein homeostasis, implying optimal ribosomal function, is associated with disease resistance and increased lifespan (
      • Karunadharma P.P.
      • Basisty N.
      • Dai D.F.
      • Chiao Y.A.
      • Quarles E.K.
      • Hsieh E.J.
      • Crispin D.
      • Bielas J.H.
      • Ericson N.G.
      • Beyer R.P.
      • MacKay V.L.
      • MacCoss M.J.
      • Rabinovitch P.S.
      Subacute calorie restriction and rapamycin discordantly alter mouse liver proteome homeostasis and reverse aging effects.
      ,
      • Price J.C.
      • Khambatta C.F.
      • Li K.W.
      • Bruss M.D.
      • Shankaran M.
      • Dalidd M.
      • Floreani N.A.
      • Roberts L.S.
      • Turner S.M.
      • Holmes W.E.
      • Hellerstein M.K.
      The effect of long term calorie restriction on in vivo hepatic proteostatis: A novel combination of dynamic and quantitative proteomics.
      ,
      • van der Goot A.T.
      • Zhu W.
      • Vázquez-Manrique R.P.
      • Seinstra R.I.
      • Dettmer K.
      • Michels H.
      • Farina F.
      • Krijnen J.
      • Melki R.
      • Buijsman R.C.
      • Ruiz Silva M.
      • Thijssen K.L.
      • Kema I.P.
      • Neri C.
      • Oefner P.J.
      • Nollen E.A.
      Delaying aging and the aging-associated decline in protein homeostasis by inhibition of tryptophan degradation.
      ). To maintain a high-quality ribosome population within the cell, dysfunctional ribosomes are targeted for autophagic degradation. It is not known if complete degradation is the only mechanism for eukaryotic ribosome maintenance or if they might also be repaired by replacement of defective components. We used stable-isotope feeding and protein mass spectrometry to measure the kinetics of turnover of ribosomal RNA (rRNA) and 71 ribosomal proteins (r-proteins) in mice. The results indicate that exchange of individual proteins and whole ribosome degradation both contribute to ribosome maintenance in vivo. In general, peripheral r-proteins and those with more direct roles in peptide-bond formation are replaced multiple times during the lifespan of the assembled structure, presumably by exchange with a free cytoplasmic pool, whereas the majority of r-proteins are stably incorporated for the lifetime of the ribosome. Dietary signals impact the rates of both new ribosome assembly and component exchange. Signal-specific modulation of ribosomal repair and degradation could provide a mechanistic link in the frequently observed associations among diminished rates of protein synthesis, increased autophagy, and greater longevity (
      • Karunadharma P.P.
      • Basisty N.
      • Dai D.F.
      • Chiao Y.A.
      • Quarles E.K.
      • Hsieh E.J.
      • Crispin D.
      • Bielas J.H.
      • Ericson N.G.
      • Beyer R.P.
      • MacKay V.L.
      • MacCoss M.J.
      • Rabinovitch P.S.
      Subacute calorie restriction and rapamycin discordantly alter mouse liver proteome homeostasis and reverse aging effects.
      ,
      • Price J.C.
      • Khambatta C.F.
      • Li K.W.
      • Bruss M.D.
      • Shankaran M.
      • Dalidd M.
      • Floreani N.A.
      • Roberts L.S.
      • Turner S.M.
      • Holmes W.E.
      • Hellerstein M.K.
      The effect of long term calorie restriction on in vivo hepatic proteostatis: A novel combination of dynamic and quantitative proteomics.
      ,
      • Houtkooper R.H.
      • Mouchiroud L.
      • Ryu D.
      • Moullan N.
      • Katsyuba E.
      • Knott G.
      • Williams R.W.
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      Mitonuclear protein imbalance as a conserved longevity mechanism.
      ,
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      • Irvine E.
      • Lingard S.J.
      • Choudhury A.I.
      • Claret M.
      • Al-Qassab H.
      • Carmignac D.
      • Ramadani F.
      • Woods A.
      • Robinson I.C.
      • Schuster E.
      • Batterham R.L.
      • Kozma S.C.
      • Thomas G.
      • Carling D.
      • Okkenhaug K.
      • Thornton J.M.
      • Partridge L.
      • Gems D.
      • Withers D.J.
      Ribosomal protein S6 kinase 1 signaling regulates mammalian life span.
      ).
      Cells in the body achieve protein homeostasis (proteostasis)
      The abbreviations used are: Proteostasis, protein homeostasis; rRNA, ribosomal RNA; r-proteins, ribosomal proteins; DR, dietary restriction; AL, Ad Libitium or unrestricted diet; RQC, ribosome quality control; ribophagy, directed autophagy of ribosomes; M0, mono isotopic mass spectral peak; M1, mass spectral peak with 1 extra neutron; M2, mass spectral peak with 2 extra neutrons; RCR, respiratory control ratio; mTOR, mechanistic target of rapamycin.
      1The abbreviations used are: Proteostasis, protein homeostasis; rRNA, ribosomal RNA; r-proteins, ribosomal proteins; DR, dietary restriction; AL, Ad Libitium or unrestricted diet; RQC, ribosome quality control; ribophagy, directed autophagy of ribosomes; M0, mono isotopic mass spectral peak; M1, mass spectral peak with 1 extra neutron; M2, mass spectral peak with 2 extra neutrons; RCR, respiratory control ratio; mTOR, mechanistic target of rapamycin.
      by carefully balancing the synthesis and folding of each protein against the protein degradation and cellular proliferation rates. Controlled shifts in proteostasis occur during cell differentiation, and in response to stimuli (
      • Ben-Zvi A.
      • Miller E.A.
      • Morimoto R.I.
      Collapse of proteostasis represents an early molecular event in Caenorhabditis elegans aging.
      ,
      • Xue S.
      • Barna M.
      Specialized ribosomes: A new frontier in gene regulation and organismal biology.
      ), while uncontrolled changes promote neurodegenerative disease (
      • Kristiansen M.
      • Deriziotis P.
      • Dimcheff D.E.
      • Jackson G.S.
      • Ovaa H.
      • Naumann H.
      • Clarke A.R.
      • van Leeuwen F.W.
      • Menéndez-Benito V.
      • Dantuma N.P.
      • Portis J.L.
      • Collinge J.
      • Tabrizi S.J.
      Disease-associated prion protein oligomers inhibit the 26S proteasome.
      ,
      • Repetto E.
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      • Zheng H.
      • Kang D.E.
      Presenilin 1 regulates epidermal growth factor receptor turnover and signaling in the endosomal-lysosomal pathway.
      ,
      • Safar J.G.
      • DeArmond S.J.
      • Kociuba K.
      • Deering C.
      • Didorenko S.
      • Bouzamondo-Bernstein E.
      • Prusiner S.B.
      • Tremblay P.
      Prion clearance in bigenic mice.
      ,
      • Lee S.
      • Notterpek L.
      Dietary restriction supports peripheral nerve health by enhancing endogenous protein quality control mechanisms.
      ), cancer (
      • Leprivier G.
      • Rotblat B.
      • Khan D.
      • Jan E.
      • Sorensen P.H.
      Stress-mediated translational control in cancer cells.
      ,
      • Montanaro L.
      • Trere D.
      • Derenzini M.
      Nucleolus, ribosomes, and cancer.
      ,
      • Kolch W.
      • Pitt A.
      Functional proteomics to dissect tyrosine kinase signalling pathways in cancer.
      ), and aging (
      • Ben-Zvi A.
      • Miller E.A.
      • Morimoto R.I.
      Collapse of proteostasis represents an early molecular event in Caenorhabditis elegans aging.
      ,
      • Karunadharma P.P.
      • Basisty N.
      • Dai D.F.
      • Chiao Y.A.
      • Quarles E.K.
      • Hsieh E.J.
      • Crispin D.
      • Bielas J.H.
      • Ericson N.G.
      • Beyer R.P.
      • MacKay V.L.
      • MacCoss M.J.
      • Rabinovitch P.S.
      Subacute calorie restriction and rapamycin discordantly alter mouse liver proteome homeostasis and reverse aging effects.
      ,
      • van der Goot A.T.
      • Zhu W.
      • Vázquez-Manrique R.P.
      • Seinstra R.I.
      • Dettmer K.
      • Michels H.
      • Farina F.
      • Krijnen J.
      • Melki R.
      • Buijsman R.C.
      • Ruiz Silva M.
      • Thijssen K.L.
      • Kema I.P.
      • Neri C.
      • Oefner P.J.
      • Nollen E.A.
      Delaying aging and the aging-associated decline in protein homeostasis by inhibition of tryptophan degradation.
      ,
      • Dai D.F.
      • Karunadharma P.P.
      • Chiao Y.A.
      • Basisty N.
      • Crispin D.
      • Hsieh E.J.
      • Chen T.
      • Gu H.
      • Djukovic D.
      • Raftery D.
      • Beyer R.P.
      • MacCoss M.J.
      • Rabinovitch P.S.
      Altered proteome turnover and remodeling by short-term caloric restriction or rapamycin rejuvenate the aging heart.
      ,
      • Miller B.F.
      • Drake J.C.
      • Naylor B.
      • Price J.C.
      • Hamilton K.L.
      The measurement of protein synthesis for assessing proteostasis in studies of slowed aging.
      ). This suggests that each cell coordinately regulates the ribosome, proteasome, and other key structures of protein metabolism to achieve proteostasis. However, the regulatory mechanisms for coordination and how the proteome is remodeled to achieve a new proteostasis are poorly understood.
      Diets restricting calories or amino acids protect against aging and the diseases of aging in model organisms (
      • McCay C.M.
      • Crowell M.F.
      • Maynard L.A.
      The effect of retarded growth upon the length of life span and upon the ultimate body size.
      ,
      • Anderson R.M.
      • Shanmuganayagam D.
      • Weindruch R.
      Caloric restriction and aging: Studies in mice and monkeys.
      ). Low calorie diets have been shown to reduce rates of protein synthesis and degradation for much of the observed proteome (
      • Karunadharma P.P.
      • Basisty N.
      • Dai D.F.
      • Chiao Y.A.
      • Quarles E.K.
      • Hsieh E.J.
      • Crispin D.
      • Bielas J.H.
      • Ericson N.G.
      • Beyer R.P.
      • MacKay V.L.
      • MacCoss M.J.
      • Rabinovitch P.S.
      Subacute calorie restriction and rapamycin discordantly alter mouse liver proteome homeostasis and reverse aging effects.
      ,
      • Price J.C.
      • Khambatta C.F.
      • Li K.W.
      • Bruss M.D.
      • Shankaran M.
      • Dalidd M.
      • Floreani N.A.
      • Roberts L.S.
      • Turner S.M.
      • Holmes W.E.
      • Hellerstein M.K.
      The effect of long term calorie restriction on in vivo hepatic proteostatis: A novel combination of dynamic and quantitative proteomics.
      ,
      • Dai D.F.
      • Karunadharma P.P.
      • Chiao Y.A.
      • Basisty N.
      • Crispin D.
      • Hsieh E.J.
      • Chen T.
      • Gu H.
      • Djukovic D.
      • Raftery D.
      • Beyer R.P.
      • MacCoss M.J.
      • Rabinovitch P.S.
      Altered proteome turnover and remodeling by short-term caloric restriction or rapamycin rejuvenate the aging heart.
      ). Recent reports suggest that high protein synthesis demand is associated with reduced ribosomal accuracy (
      • Conn C.S.
      • Qian S.B.
      Nutrient signaling in protein homeostasis: An increase in quantity at the expense of quality.
      ) and efficiency (
      • Borkowski O.
      • Goelzer A.
      • Schaffer M.
      • Calabre M.
      • Mäder U.
      • Aymerich S.
      • Jules M.
      • Fromion V.
      Translation elicits a growth rate-dependent, genome-wide, differential protein production in Bacillus subtilis.
      ). This suggests that maintaining ribosome quality is an intricate task that requires constant cellular effort.
      Translation rate is a metric of ribosome quality (
      • Brandman O.
      • Stewart-Ornstein J.
      • Wong D.
      • Larson A.
      • Williams C.C.
      • Li G.W.
      • Zhou S.
      • King D.
      • Shen P.S.
      • Weibezahn J.
      • Dunn J.G.
      • Rouskin S.
      • Inada T.
      • Frost A.
      • Weissman J.S.
      A ribosome-bound quality control complex triggers degradation of nascent peptides and signals translation stress.
      ). Ribosomes that stall during protein production are immediately tested by the ribosome quality control (RQC) complex (
      • Brandman O.
      • Stewart-Ornstein J.
      • Wong D.
      • Larson A.
      • Williams C.C.
      • Li G.W.
      • Zhou S.
      • King D.
      • Shen P.S.
      • Weibezahn J.
      • Dunn J.G.
      • Rouskin S.
      • Inada T.
      • Frost A.
      • Weissman J.S.
      A ribosome-bound quality control complex triggers degradation of nascent peptides and signals translation stress.
      ). The RQC specifically tests the large subunit for activity (
      • Shen P.S.
      • Park J.
      • Qin Y.
      • Li X.
      • Parsawar K.
      • Larson M.H.
      • Cox J.
      • Cheng Y.
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      • Weissman J.S.
      • Brandman O.
      • Frost A.
      Protein synthesis. Rqc2p and 60S ribosomal subunits mediate mRNA-independent elongation of nascent chains.
      ) and presumably sequesters inactive ribosomes. Degradation of unnecessary or damaged ribosomes often occurs through directed autophagy (ribophagy) (
      • Ossareh-Nazari B.
      • Niño C.A.
      • Bengtson M.H.
      • Lee J.W.
      • Joazeiro C.A.
      • Dargemont C.
      Ubiquitylation by the Ltn1 E3 ligase protects 60S ribosomes from starvation-induced selective autophagy.
      ). Ribophagy is presumably one of the primary roles for autophagy; indeed, the earliest descriptions of autophagic vesicles show ribosomes in the interior (
      • Ashford T.P.
      • Porter K.R.
      Cytoplasmic components in hepatic cell lysosomes.
      ). An important open question though is: Can dysfunctional ribosomes be repaired, or is ribophagy the only option?
      Exchange of damaged ribosomal components could allow the cell to repair faulty ribosomes instead of degrading the entire structure. The formation of new ribosomes is extremely costly and has been estimated to account for 15% of the protein synthesis budget (
      • Schleif R.
      Control of production of ribosomal protein.
      ). In yeast, protein synthesis accounts for at least 90% of the energy usage (
      • Warner J.R.
      The economics of ribosome biosynthesis in yeast.
      ). Damaged ribosomes in Escherichia coli have been shown to regain activity after exchange of r-proteins for undamaged copies (
      • Pulk A.
      • Liiv A.
      • Peil L.
      • Maiväli U.
      • Nierhaus K.
      • Remme J.
      Ribosome reactivation by replacement of damaged proteins.
      ). Although it has never been demonstrated in eukaryotes, exchange of damaged protein components could reduce the total energy expenditure to maintain active ribosomes.
      Here, we show that exchange of r-proteins is occurring in vivo. Using metabolic labeling (Fig. 1A) and a kinetic model, we calculate exchange rates between assembled and free pools (Fig. 1B). Further, we show that r-protein exchange and ribophagy rates change with dietary signals. We compare mice fed an ad libitum (AL) versus a restricted diet (dietary restriction, DR) and observe that kinetically there are three groups of proteins in the assembled ribosome. One group is never exchanged and is degraded via ribophagy with the rRNA. The second group is exchanged multiple times with cytosolic copies and has members with either fast or slow cytosolic turnover. A third group of proteins alternates between the first two groups. We find that both ribophagy and r-protein exchange are modulated by dietary signaling. Our observations offer insight into the connection between reduced protein synthesis (
      • Karunadharma P.P.
      • Basisty N.
      • Dai D.F.
      • Chiao Y.A.
      • Quarles E.K.
      • Hsieh E.J.
      • Crispin D.
      • Bielas J.H.
      • Ericson N.G.
      • Beyer R.P.
      • MacKay V.L.
      • MacCoss M.J.
      • Rabinovitch P.S.
      Subacute calorie restriction and rapamycin discordantly alter mouse liver proteome homeostasis and reverse aging effects.
      ,
      • Price J.C.
      • Khambatta C.F.
      • Li K.W.
      • Bruss M.D.
      • Shankaran M.
      • Dalidd M.
      • Floreani N.A.
      • Roberts L.S.
      • Turner S.M.
      • Holmes W.E.
      • Hellerstein M.K.
      The effect of long term calorie restriction on in vivo hepatic proteostatis: A novel combination of dynamic and quantitative proteomics.
      ,
      • Selman C.
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      • Wieser D.
      • Irvine E.
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      • Schuster E.
      • Batterham R.L.
      • Kozma S.C.
      • Thomas G.
      • Carling D.
      • Okkenhaug K.
      • Thornton J.M.
      • Partridge L.
      • Gems D.
      • Withers D.J.
      Ribosomal protein S6 kinase 1 signaling regulates mammalian life span.
      ,
      • Dai D.F.
      • Karunadharma P.P.
      • Chiao Y.A.
      • Basisty N.
      • Crispin D.
      • Hsieh E.J.
      • Chen T.
      • Gu H.
      • Djukovic D.
      • Raftery D.
      • Beyer R.P.
      • MacCoss M.J.
      • Rabinovitch P.S.
      Altered proteome turnover and remodeling by short-term caloric restriction or rapamycin rejuvenate the aging heart.
      ), and increased autophagy (
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      Mechanisms of amino acid-mediated lifespan extension in Caenorhabditis elegans.
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      ,
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      Regulation of the aging process by autophagy.
      ) with increased health and longevity.
      Figure thumbnail gr1
      Fig. 1Experimental Overview: Workflow for heavy isotope labeling, analyte isolation, and measurement of turnover rates (A). Kinetic model for utilizing turnover measurements to describe ribosome maintenance and turnover (B). Under these experimental conditions opposing rates are equal maintaining homeostasis.

      Discussion

      The ribosome is a multimegadalton complex of RNA and protein that synthesizes most proteins in the cell. High demand for protein synthesis reduces ribosome efficiency (
      • Borkowski O.
      • Goelzer A.
      • Schaffer M.
      • Calabre M.
      • Mäder U.
      • Aymerich S.
      • Jules M.
      • Fromion V.
      Translation elicits a growth rate-dependent, genome-wide, differential protein production in Bacillus subtilis.
      ) and accuracy (
      • Conn C.S.
      • Qian S.B.
      Nutrient signaling in protein homeostasis: An increase in quantity at the expense of quality.
      ). In bacteria, damaged ribosomes can regain activity by replacement of damaged r-proteins in the assembled structure with undamaged cytosolic copies (
      • Pulk A.
      • Liiv A.
      • Peil L.
      • Maiväli U.
      • Nierhaus K.
      • Remme J.
      Ribosome reactivation by replacement of damaged proteins.
      ). Exchange of r-proteins may also be important to the functional integrity of the ribosome in eukaryotes (
      • Xue S.
      • Barna M.
      Specialized ribosomes: A new frontier in gene regulation and organismal biology.
      ), although this idea is controversial. Metabolic labeling affords a means to evaluate this hypothesis by testing directly for the replacement (turnover) of ribosomal components in vivo. We used metabolic deuterium incorporation rates to compare turnover of rRNA and the individual r-proteins in the assembled ribosome in mice (Fig. 1A). We also tested whether DR, which has previously been shown to modulate rates of ribosome biogenesis, assembly, and activity in cells (
      • Conn C.S.
      • Qian S.B.
      Nutrient signaling in protein homeostasis: An increase in quantity at the expense of quality.
      ,
      • Molin S.
      • Von Meyenburg K.
      • Maaloe O.
      • Hansen M.T.
      • Pato M.L.
      Control of ribosome synthesis in Escherichia coli: Analysis of an energy source shift-down.
      ) and mice (
      • Karunadharma P.P.
      • Basisty N.
      • Dai D.F.
      • Chiao Y.A.
      • Quarles E.K.
      • Hsieh E.J.
      • Crispin D.
      • Bielas J.H.
      • Ericson N.G.
      • Beyer R.P.
      • MacKay V.L.
      • MacCoss M.J.
      • Rabinovitch P.S.
      Subacute calorie restriction and rapamycin discordantly alter mouse liver proteome homeostasis and reverse aging effects.
      ,
      • Price J.C.
      • Khambatta C.F.
      • Li K.W.
      • Bruss M.D.
      • Shankaran M.
      • Dalidd M.
      • Floreani N.A.
      • Roberts L.S.
      • Turner S.M.
      • Holmes W.E.
      • Hellerstein M.K.
      The effect of long term calorie restriction on in vivo hepatic proteostatis: A novel combination of dynamic and quantitative proteomics.
      ), can impact r-protein exchange rates.
      Components of the ribosome reside in two kinetically distinct pools (Fig. 1B), with different synthesis and degradation rates for the assembled complex and its individual constituents (
      • Chen S.S.
      • Sperling E.
      • Silverman J.M.
      • Davis J.H.
      • Williamson J.R.
      Measuring the dynamics of E. coli ribosome biogenesis using pulse-labeling and quantitative mass spectrometry.
      ,
      • Leick V.
      • Andersen S.B.
      Pools and turnover rates of nuclear ribosomal RNA in Tetrahymena pyriformis.
      ). At homeostasis, the rates of opposing steps in the model (e.g. assembly and ribophagy) should be equal (Fig. 1B). Physiological and biochemical metrics of the DR effect verified that the mice used in these experiments were at homeostasis (Fig. 2) prior to metabolic labeling, similar to previous studies (
      • Karunadharma P.P.
      • Basisty N.
      • Dai D.F.
      • Chiao Y.A.
      • Quarles E.K.
      • Hsieh E.J.
      • Crispin D.
      • Bielas J.H.
      • Ericson N.G.
      • Beyer R.P.
      • MacKay V.L.
      • MacCoss M.J.
      • Rabinovitch P.S.
      Subacute calorie restriction and rapamycin discordantly alter mouse liver proteome homeostasis and reverse aging effects.
      ,
      • Price J.C.
      • Khambatta C.F.
      • Li K.W.
      • Bruss M.D.
      • Shankaran M.
      • Dalidd M.
      • Floreani N.A.
      • Roberts L.S.
      • Turner S.M.
      • Holmes W.E.
      • Hellerstein M.K.
      The effect of long term calorie restriction on in vivo hepatic proteostatis: A novel combination of dynamic and quantitative proteomics.
      ). The model also assumes that free rRNA, without r-proteins, is degraded rapidly relative to the turnover of the assembled structure, as observed previously (
      • Fujii K.
      • Kitabatake M.
      • Sakata T.
      • Ohno M.
      40S subunit dissociation and proteasome-dependent RNA degradation in nonfunctional 25S rRNA decay.
      ,
      • Williamson R.
      • Lanyon G.
      • Paul J.
      Preferential degradation of “messenger RNA” in reticulocytes by ribonuclease treatment and sonication of polysomes.
      ). Under this assumption, turnover of rRNA reflects only turnover of the assembled ribosomes (Equation 3). Turnover of r-proteins in the assembled pool would depend on the kinetics of both assembly and exchange (Equation 2, kassemble + kadd, note that kassemble = kribophagy and kadd = kremove due to homeostasis). We therefore calculated the exchange rate (kadd and kremove) as the absolute value of the difference in turnover rates of individual r-proteins (Pi) in the assembled pool and the rRNA (Equation 5: Fig. 1B).
      Assembled ribosomes isolated from the liver tissue of two animals at each of eight time points after introduction of the metabolic label were separated into two samples for analysis of rRNA and r-protein turnover (Fig. 1A). The amount of new rRNA increased exponentially with time (Fig. 4) and could be modeled by assumption of a single pool. There was a statistically insignificant increase in rRNA turnover increased in the DR tissue [(11.1 ± 1.7) % Day−1] relative to control with no dietary restriction (AL) tissue [(10.1 ± 1.2) % Day−1]. In DR, although fewer ribosomes were actively translating protein (Fig. 3), the total number was not different between AL and DR tissue (via qPCR, Fig. S1B). The results imply that, on average, ribosomes were less active and had a slightly shorter lifetime (6 days) in tissues of DR animals than in those from the AL animals (7 days). The measurements of protein turnover within the assembled ribosome and the observed proteome support these results.
      Turnover rates of the individual ribosomal proteins (r-proteins) were resolved by monitoring incorporation of deuterium into multiple tryptic peptides for each protein along the labeling time-course (Fig. 5A), as previously described (
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      ). Within the set of 71 (out of 80 total) integral r-proteins monitored, a range of 4–28% per day was observed (Supplemental Table). Most r-proteins turn over at rates that are similar to (within two standard deviations of) that of the rRNA (Figs. 5B-5D gray symbols), implying that this large group of r-proteins and the rRNA are replaced together as a unit (i.e. complete degradation of the complex). Indeed, the average rate for the r-proteins (10.2% in AL and 11.3% in DR) matched the ribophagy rate (10.1% in AL and 11.1% in DR) remarkably well. Comparison of resolved turnover rates of the individual r-protein components (Fig. 5D) makes it apparent that the small DR-dependent increase in rRNA turnover (Fig. 4) is significant (p < 0.0005). Overall, ∼80% of r-proteins, had individual turnover rates that match the ribophagy rate (within two standard deviations of the rRNA). This agrees with earlier studies of average rRNA and r-protein turnover (
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      ).
      The increased rate of ribophagy in DR tissues was surprising. In agreement with previous studies (
      • Karunadharma P.P.
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      Subacute calorie restriction and rapamycin discordantly alter mouse liver proteome homeostasis and reverse aging effects.
      ,
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      ,
      • Bruss M.D.
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      The effects of physiological adaptations to calorie restriction on global cell proliferation rates.
      ,
      • Thompson A.C.
      • Bruss M.D.
      • Price J.C.
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      • Colangelo M.
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      Reduced in vivo hepatic proteome replacement rates but not cell proliferation rates predict maximum lifespan extension in mice.
      ), we observed that DR slows cell proliferation (Fig. 2B) and protein synthesis (Fig. 3). Interestingly, the cellular half-life (169 days in AL) is 25 times greater that the ribosome half-life in AL. In DR, the ribosome turnover is accelerated relative to cell proliferations, becoming 37 times greater. The two-dimensional comparison suggests that the rate of ribophagy, specifically, doubles relative to the rate of other processes like mitochondria-specific degradation or cell division. The up-regulation of ribophagy during DR may explain the previous observation that increased autophagy and lower protein synthesis rates work together to improve cellular fitness and whole organism lifespan (
      • Conn C.S.
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      Nutrient signaling in protein homeostasis: An increase in quantity at the expense of quality.
      ,
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      Subacute calorie restriction and rapamycin discordantly alter mouse liver proteome homeostasis and reverse aging effects.
      ,
      • Price J.C.
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      • Bruss M.D.
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      • Dalidd M.
      • Floreani N.A.
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      • Turner S.M.
      • Holmes W.E.
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      The effect of long term calorie restriction on in vivo hepatic proteostatis: A novel combination of dynamic and quantitative proteomics.
      ,
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      • Claret M.
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      • Kozma S.C.
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      Ribosomal protein S6 kinase 1 signaling regulates mammalian life span.
      ,
      • Eisenberg T.
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      • Andryushkova A.
      • Pendl T.
      • Küttner V.
      • Bhukel A.
      • Mariño G.
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      • Harger A.
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      • Ruckenstuhl C.
      • Ring J.
      • Reichelt W.
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      • Leeb T.
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      • Kamolz L.P.
      • Magnes C.
      • Sinner F.
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      ,
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      ).
      Although the turnover rate of r-proteins averaged over the entire set reflects the ribophagy rate (as previously reported), a small but intriguing set of r-proteins have significantly different turnover rates (Fig. 5B5D, black symbols). Importantly, some of the proteins with unusually fast or slow turnover are the same in both AL and DR tissues (Fig. 2D). The parallel behavior of these proteins in both dietary cohorts suggests that the difference in their turnover rates might be intrinsic and functionally relevant. The difference in turnover may be due to the free pool r-protein turnover rate (Fig. 1B), which is independent of the ribophagy rate. The free pool turnover rate is difficult to measure directly since it is very low concentration for each r-protein (
      • Chen S.S.
      • Sperling E.
      • Silverman J.M.
      • Davis J.H.
      • Williamson J.R.
      Measuring the dynamics of E. coli ribosome biogenesis using pulse-labeling and quantitative mass spectrometry.
      ) and homogenizing the tissue is likely to break assembled ribosomes, contaminating the free pool. However, as shown in Equation 5, the exchange rate of each protein can be calculated without direct measurement of the free pool. The proteins that are exchanged rapidly out of the assembled structure would have turnover rates defined by the free pool. Therefore, fast exchange between the assembled and free pool could explain outliers at both the fast and slow end of the turnover range (Fig. 5).
      When we compared the calculated exchange rates (Fig. 6A) against the ribosome structure, we saw that the fast exchange and fast turnover r-proteins are predominately located at the interface between the 60S and 40S subunits (Fig. 6B). This region is known to undergo significant movement during the catalytic activity of the ribosome (
      • Khatter H.
      • Myasnikov A.G.
      • Natchiar S.K.
      • Klaholz B.P.
      Structure of the human 80S ribosome.
      ). There are at least two possible hypotheses to explain why ribosomal maintenance would include fast exchange of these proteins. First, proteins at this location may be more prone to damage and therefore exchange more rapidly. Second, damage of these proteins dramatically reduces formation of the 80S ribosome and ensures that there is a longer exchange period. Three of these proteins (L19, L24, and L34) are structurally important, acting like long fingers to secure the 40S to the 60S subunit (Fig. 6). The other members of this subgroup (L10, L36A, and L38) are also localized in the interface, either on the beak or directly across from it. Breaking the 80S down to the 40S and 60S could facilitate exchange of these proteins due to lost surface interactions and greater access to the cytosol.
      Cytosolic RPL36a and L10 are critical to formation of the P-site during assembly and guide the structural rotation necessary to form each peptide bond (
      • Sulima S.O.
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      • Anjos M.
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      ). Dysfunction of these and other fast exchange proteins is associated with disease (
      • Xue S.
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      Specialized ribosomes: A new frontier in gene regulation and organismal biology.
      ,
      • Khatter H.
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      • Natchiar S.K.
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      Structure of the human 80S ribosome.
      ); therefore, exchange may represent an important method to maintain ribosomal quality. Greater exchange of these proteins in AL tissue relative to the ribophagy rate may indicate that damage to these specific proteins occurs prior to ribophagy.
      One interpretation of these results is that ribophagy and r-protein exchange are both used to maintain the active pool of ribosomes (Fig. 7). Simplistically, the high synthetic demand and longer lifespan for individual ribosomes observed in AL tissue might result in accumulation of damaged ribosomes, as only a few selected proteins can be repaired. Slower cell proliferation in DR suggests that, because there is less dilution into new cells, protein degradation is up-regulated to match synthesis (
      • Price J.C.
      • Khambatta C.F.
      • Li K.W.
      • Bruss M.D.
      • Shankaran M.
      • Dalidd M.
      • Floreani N.A.
      • Roberts L.S.
      • Turner S.M.
      • Holmes W.E.
      • Hellerstein M.K.
      The effect of long term calorie restriction on in vivo hepatic proteostatis: A novel combination of dynamic and quantitative proteomics.
      ,
      • Miller B.F.
      • Drake J.C.
      • Naylor B.
      • Price J.C.
      • Hamilton K.L.
      The measurement of protein synthesis for assessing proteostasis in studies of slowed aging.
      ). Lower synthetic demand with an accompanying increase in ribophagy, would allow for more extensive turnover of the assembled ribosome pool. Better maintenance of the ribosome might lead to higher quality nascent peptides and improved accuracy (
      • Conn C.S.
      • Qian S.B.
      Nutrient signaling in protein homeostasis: An increase in quantity at the expense of quality.
      ) and efficiency (
      • Borkowski O.
      • Goelzer A.
      • Schaffer M.
      • Calabre M.
      • Mäder U.
      • Aymerich S.
      • Jules M.
      • Fromion V.
      Translation elicits a growth rate-dependent, genome-wide, differential protein production in Bacillus subtilis.
      ) relative to AL tissue. This provides an attractive interpretation for the frequently observed connection between lower rates of protein synthesis, increased autophagy, and improved protein homeostasis and longevity (
      • Karunadharma P.P.
      • Basisty N.
      • Dai D.F.
      • Chiao Y.A.
      • Quarles E.K.
      • Hsieh E.J.
      • Crispin D.
      • Bielas J.H.
      • Ericson N.G.
      • Beyer R.P.
      • MacKay V.L.
      • MacCoss M.J.
      • Rabinovitch P.S.
      Subacute calorie restriction and rapamycin discordantly alter mouse liver proteome homeostasis and reverse aging effects.
      ,
      • Price J.C.
      • Khambatta C.F.
      • Li K.W.
      • Bruss M.D.
      • Shankaran M.
      • Dalidd M.
      • Floreani N.A.
      • Roberts L.S.
      • Turner S.M.
      • Holmes W.E.
      • Hellerstein M.K.
      The effect of long term calorie restriction on in vivo hepatic proteostatis: A novel combination of dynamic and quantitative proteomics.
      ,
      • Houtkooper R.H.
      • Mouchiroud L.
      • Ryu D.
      • Moullan N.
      • Katsyuba E.
      • Knott G.
      • Williams R.W.
      • Auwerx J.
      Mitonuclear protein imbalance as a conserved longevity mechanism.
      ,
      • Selman C.
      • Tullet J.M.
      • Wieser D.
      • Irvine E.
      • Lingard S.J.
      • Choudhury A.I.
      • Claret M.
      • Al-Qassab H.
      • Carmignac D.
      • Ramadani F.
      • Woods A.
      • Robinson I.C.
      • Schuster E.
      • Batterham R.L.
      • Kozma S.C.
      • Thomas G.
      • Carling D.
      • Okkenhaug K.
      • Thornton J.M.
      • Partridge L.
      • Gems D.
      • Withers D.J.
      Ribosomal protein S6 kinase 1 signaling regulates mammalian life span.
      ).
      Figure thumbnail gr7
      Fig. 7In vivo ribosome maintenance requires ribophagy and r-protein exchange. Each day, ∼10% of the ribosomal pool is replaced via assembly of new ribosomes and ribophagy. During the lifetime of the assembled ribosomal structure, ribosome protein exchange occurs primarily when the ribosome disassociates to its individual subunits. This exchange may be a fast, low cost, method to repair and modify ribosomes. Cellular energetics and demand for protein synthesis may modulate the relative contribution of ribophagy versus exchange in response to stalled or damaged ribosomes.
      It has been observed that the RQC complex is required to dissociate stalled 80S ribosomes (
      • Brandman O.
      • Stewart-Ornstein J.
      • Wong D.
      • Larson A.
      • Williams C.C.
      • Li G.W.
      • Zhou S.
      • King D.
      • Shen P.S.
      • Weibezahn J.
      • Dunn J.G.
      • Rouskin S.
      • Inada T.
      • Frost A.
      • Weissman J.S.
      A ribosome-bound quality control complex triggers degradation of nascent peptides and signals translation stress.
      ). Our data suggest that ribosomes exchange components most rapidly when dissociated into subunits, potentially after the RQC has dissociated the complex. These results raise several interesting questions about mechanisms for maintenance. Is there cross talk between RQC activity and ribosome component turnover? What factors control ribosome component exchange? We assume a passive model, but could the RQC coordinate active exchange? Proteins L35a and L7 are fast exchange in AL tissue but are buried within the rRNA. These proteins may require outside assistance to facilitate exchange. Finally, does r-protein exchange improve the quality of nascent peptides? Initial results suggest that reducing global protein synthesis may improve protein quality (
      • Thompson A.C.
      • Bruss M.D.
      • Price J.C.
      • Khambatta C.F.
      • Holmes W.E.
      • Colangelo M.
      • Dalidd M.
      • Roberts L.S.
      • Astle C.M.
      • Harrison D.E.
      • Hellerstein M.K.
      Reduced in vivo hepatic proteome replacement rates but not cell proliferation rates predict maximum lifespan extension in mice.
      ). Further investigation is needed to confirm whether ribosomal maintenance is a mechanistic link explaining how lower protein synthesis burdens are connected to improved protein homeostasis and lifespan.
      In conclusion, this work uses a generally applicable strategy for investigating cellular maintenance of the proteome, including multiprotein and ribonuclear structures. Here, we show that exchange of protein components and degradation of the entire ribosome are important maintenance strategies. Our results suggest a mechanism wherein dissociation of 60S and 40S subunits promotes in vivo r-protein exchange. We find that dietary signals can change both ribophagy and r-protein exchange rates. Future work testing biological models that modulate the RQC activity and/or mechanistic target of rapamycin (mTOR) signaling will test whether this is mechanism is generally applicable. Also, linking changes in longevity to the rates of protein synthesis and autophagy as we have done, may help identify mechanisms of aging.

      Acknowledgments

      We are grateful to J. Martin Bollinger Jr., Alan Buskirk, Barry Willardson, and Natalie Blades for constructive insights and helpful conversations. Earl Albee, Warren Bingham, and the Brigham Young University (BYU) animal care facility for assistance in maintenance of the mice. Nathan Keyes and Ryne Peters for assistance with Supplemental Fig. S1.

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