2018 May 18
NUFIP1 Is a Ribosome Receptor for Starvation-Induced Ribophagy
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NUFIP1 Is a Ribosome Receptor for Starvation-Induced Ribophagy
The lysosome degrades and recycles macromolecules, signals to the master growth regulator mTORC1 [mechanistic target of rapamycin (mTOR) complex 1], and is associated with human disease. We performed quantitative proteomic analyses of rapidly isolated lysosomes and found that nutrient levels and mTOR dynamically modulate the lysosomal proteome. Upon mTORC1 inhibition, NUFIP1 (nuclear fragile X mental retardation-interacting protein 1) redistributes from the nucleus to autophagosomes and lysosomes. Upon these conditions, NUFIP1 interacts with ribosomes and delivers them to autophagosomes by directly binding to microtubule-associated proteins 1A/1B light chain 3B (LC3B). The starvation-induced degradation of ribosomes via autophagy (ribophagy) depends on the capacity of NUFIP1 to bind LC3B and promotes cell survival. We propose that NUFIP1 is a receptor for the selective autophagy of ribosomes.
Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.
Conflict of interest statement
Competing Interests: D.M.S. is a founding member of the scientific advisory board, a paid consultant, and a shareholder of Navitor Pharmaceuticals, which is targeting for therapeutic benefit the amino acid sensing pathway upstream of mTORC1.
Fig. 1. Regulation of the lysosomal proteome in response to nutrient starvation and mTOR inhibition
(A) Schematic depicting the workflow for the LysoIP-proteomics method. HA-Lyso and Control-Lyso cells refer to cells stably expressing 3xHA-tagged TMEM192 or 2xFlag-tagged TMEM192, respectively. (B) Nutrient starvation and mTOR inhibition regulate the lysosomal proteome. The scatter plot shows relative (fold) changes in protein abundances in lysosomes captured from the HA-Lyso cells starved for one hour of nutrients (amino acids and glucose) or treated for one hour with 250 nM Torin1 versus lysosomes from cells cultured in nutrient-replete media (full media). For each condition, three independent isolations were compared. Colors indicate proteins mentioned in the text. The dots denote the 5339 unique proteins detected amongst all the pre-filtered samples. The majority of these are in the immunoprecipitates from both the HA-Lyso and Control-Lyso cells. TFEB was detected only in the Torin1/Full media comparison and assigned an arbitrary log
2 fold change of 0 in the Starved/Full media comparison. (C) Validation of changes observed in the lysosomal abundances of the some of the proteins highlighted in Figure 1B. The immunoblot shows analyses for indicated proteins in whole cell lysates or lysosomes purified from HEK-293T cells subjected to the indicated treatments for 1 hour. ER, endoplasmic reticulum. (D) Venn diagram representation of the number of proteins defined as lysosomal in each of the three conditions. A protein was deemed lysosomal if it had a significant (q ≤0.1) enrichment value of 1.5 fold (> 0.58, log 2). Proteins not detected at all on the control beads were also classified as lysosomal.
Fig. 2. Upon starvation NUFIP1-ZNHIT3 accumulates at lysosomes in an autophagosome-dependent manner
(A) NUFIP1-ZNHIT3 accumulates at lysosomes upon mTORC1 inhibition. Lysates and immunoprecipitates were prepared from HEK-293T cells cultured in full media, or deprived of amino acids or treated with 250 nM Torin1 for 1 hour as described in the supplementary materials. (B) Upon mTORC1 inhibition NUFIP1-ZNHIT3 shifts from the nuclear fraction to the post-nuclear supernatant that contains lysosomes. HEK-293T cells were fractionated after being deprived of amino acids or treated with 250 nM Torin1 for 1 hour and the amounts of endogenous NUFIP1 and ZNHIT3 analyzed by immunoblotting. RagA and LAMP1 are lysosome-associated proteins; histone H3 and SNU13 are nuclear. (C) mTOR inhibition shifts NUFIP1 from the nucleus to LAMP1-positive lysosomes. HEK-293T cells stably expressing FLAG-NUFIP1 were treated with 250 nM Torin1 for 1 hour and analyzed as described in the supplementary materials. Scale bar, 10 μm. (D) Loss of ATG7 greatly decreases the amount of NUFIP1-ZNHIT3 on lysosomes. Wild-type and ATG7-null HEK-293T cells stably expressing the HA-Lyso tag were deprived of amino acids or treated with 250 nM Torin1 for 1 hour and the amounts of NUFIP1 and ZNHIT3 on lysosomes and in total cell lysates were analyzed as in (A). (E) mTOR inhibition shifts NUFIP1 from the nucleus to LC3B-positive puncta. HEK-293T cells stably expressing FLAG-NUFIP1 were treated with 250 nM Torin1 for 1 hour and processed as in (C). (F) Schematic depicting the localization of the four putative LC3B-binding regions (LIRs) in NUFIP1. (G) mTORC1 inhibition increases the interaction between endogenous LC3B and NUFIP1-ZNHIT3. Anti-LC3B immunoprecipitates were prepared from HEK-293T cells deprived of amino acids or treated with 250 nM Torin1 for 1 hour and lysates and immunoprecipitates were analyzed for the indicated proteins. Immunoprecipitates prepared with an antibody to GSKb were used as negative controls. (H) NUFIP1-ZNHIT3 interacts with LC3B in vitro. Purified HA-GST-LC3B immobilized on a glutathione affinity resin was incubated with the purified FLAG-NUFIP1-HA-ZNHIT3 complex. HA-GST-Rap2a and HA-GST-GABARAP were used as negative controls. Proteins captured in the glutathione resin pull-down were analyzed by immunoblotting for the indicated proteins using anti-epitope tag antibodies. GST, glutathione S-transferase. (I) Identification of a NUFIP1 mutant that does not bind LC3B. Wild-type (WT) FLAG-NUFIP1 or a series of point mutants in its putative LIR motifs were co-expressed with HA-ZNHIT3 and HA-LC3B. HA-Rap2A was used as a negative control. FLAG-immunoprecipitates and lysates were prepared and analyzed by immunoblotting.
Fig. 3. NUFIP1-ZNHIT3 interacts with ribosomes in an mTORC1-dependent fashion
(A) mTOR inhibition increases the amount of NUFIP1-ZNHIT3 that co-migrates with ribosomes. HEK-293T cell lysates prepared from cells in full media or treated with 250 nM Torin1 were fractionated over a 50% sucrose cushion. Fractions were collected and the indicated proteins analyzed by immunoblotting. (B) Amino acid deprivation increases the amount of ribosomes that co-immunoprecipitates with NUFIP1. HEK-293T cells stably expressing FLAG-NUFIP1 were deprived of amino acid for 1 hour. Lysates and FLAG immunoprecipitates were prepared and analyzed for the indicated proteins by immunoblotting. FLAG-Rap2A was used as a negative control. (C) In vitro, purified NUFIP1-ZNHIT3 binds to ribosomes and the interaction is not affected by whether or not NUFIP1-ZNHIT3 was obtained from cells with inhibited mTOR. The FLAG-NUFIP1-HA-ZNHIT3 complex was purified from HEK-293T cells in full media or treated with 250 nM Torin1 for 1 hour and immobilized on a FLAG affinity resin. Equal amounts of ribosomes obtained from cells in full media were added to the immobilized FLAG-NUFIP1-HA-ZNHIT3 complex and the proteins captured analyzed by immunoblotting. Ribosomes were purified as described in the supplementary materials. Purified HA-METAP was used as a negative control. (D) In vitro, ribosomes purified from cells with mTOR inhibition bind better to NUFIP1-ZNHIT3 than those from cells in full media. Ribosomes were purified from HEK-293T cells in full media conditions or treated with 250 nM Torin1 for 1 hour. The FLAG-NUFIP1-HA-ZNHIT3 complex was immobilized on FLAG affinity beads and equal amounts of ribosomes were added. Proteins captured by the FLAG affinity beads were analyzed by immunoblotting. HA-METAP2 served as a negative control.
Fig. 4. NUFIP1 is required for ribophagy
(A) ATG7 loss suppresses the degradation of ribosomes caused by amino acid starvation. Wild-type or ATG7-null HEK-293T cells were deprived of amino acids for the indicated time points. Cell lysates were analyzed by immunoblotting for the total levels and phosphorylation states of the indicated proteins. (B) Loss of NUFIP1 inhibits the degradation of ribosomes caused by amino acid starvation. Wild-type or NUFIP1-null HEK-293T cells were deprived of amino acids for the indicated time points. Cell lysates were analyzed by immunoblotting for the total levels and phosphorylation states of the indicated proteins. (C) Loss of NUFIP1 inhibits the loss of 28S and 18S rRNA caused by mTOR inhibition. Wild-type and NUFIP1-null 8988T cells were treated with 250 nM Torin1 for 10 hrs and total RNA was extracted and analyzed on a formaldehyde agarose gel. RNA from equal numbers of cells was loaded in each lane. (D) For amino acid starvation to cause ribosomal degradation, NUFIP1 must be able to interact with LC3B. Wild-type or NUFIP1-null HEK-293T cells stably expressing the indicated proteins were deprived of amino acids for 10 hours and analyzed for the total levels and phosphorylation states of the indicated proteins. (E) For amino acid starvation to cause ribosomal degradation, NUFIP1 must interact with LC3B. Wild-type or NUFIP1-null 8988T cells stably expressing the indicated proteins were treated as in (B). (F) Autophagosomes from HEK-293T cells lacking NUFIP1 or expressing the LC3B-binding W40A mutant contain fewer ribosomes than those from control cells. NUFIP1-null HEK-293T cells expressing the control protein metap2, NUFIP1, or NUFIP1 W40A were treated with Torin1 and ConcanamycinA for 4 hours and analyzed by electron microscopy. Autophagosomes were identified by the presence of a double membrane. Red arrows indicate ribosomes inside an autophagosome. Blue arrows indicate ribosomes present in the cytoplasm. Scale bar, 500 nm
Fig. 5. NUFIP1 is important for cells to survive starvation
(A) Loss of NUFIP1 or just its capacity to interact with LC3B impairs cell survival upon nutrient starvation. Wild-type or NUFIP1-null 8988T cells stably expressing the indicated proteins were deprived of nutrients by culturing them in Hank’s Balanced Salt Solution (HBSS); after the indicated times the surviving cells were stained and imaged. (B) Loss of NUFIP1 or just its capacity to interact with LC3B impairs cell survival upon nutrient starvation. Wild-type or NUFIP1-null HEK-293T cells stably expressing the indicated proteins were deprived of nutrients by HBSS, and after 48 hours the number of surviving cells was quantified using cell counting. Values are normalized relative to cell numbers at the start of the starvation period and are mean +/− SD (*P<0.05; n=3). (C) Loss of NUFIP1 or ATG7 inhibits the increase in nucleosides caused by mTOR inhibition. Data represent relative change in whole-cell concentrations of nucleosides in wild-type, ATG7-null, and NUFIP1-null HEK-293T cells treated with 250 nM Torin1 for 1 hour. Values are mean −/+ SEM (*P<0.05; n=3) (D) Nucleoside supplementation rescues the survival defects of ATG7-null and NUFIP1-null HEK-293T cells. Indicated cells were deprived of nutrients by culturing in HBSS with or without the indicated nucleosides (2 mM each). After 48 hours, the number of surviving cells was quantified. Values were normalized relative to cell numbers at the start of the starvation period and are mean +/− SD (*P<0.05; n=3). (E) Nucleoside supplementation rescues survival defect of ATG5-null, ATG7-null, and NUFIP1-null 8988T cells. Wild-type, ATG5-null, ATG7-null, or NUFIP1-null cells were deprived of nutrients by culturing in HBSS with or without the indicated nucleosides (2 mM each). After 48 hours the surviving cells were stained and imaged. (F) Ribosomes are highly enriched for arginine and lysine. Protein sequences in the UniProt database (including isoforms) were ranked based on their fraction content of arginine and lysine. Ribosomal proteins are shown in red; all other proteins are shown in blue. Mitochondrial ribosomal proteins were not designated as ribosomal in this analysis. (G) Loss of NUFIP1 suppresses the reactivation of mTORC1 that occurs after long-term arginine deprivation. Wild-type or NUFIP1 HEK-293T cells were deprived of arginine for 50 mins (first lanes of each set) or the indicated times and, where indicated, re-stimulated with arginine for 10 mins. Cell lysates were analyzed by immunoblotting for the levels and phosphorylation states of the indicated proteins.
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Amino Acids / deficiency
Autophagosomes / metabolism
Cell Nucleus / metabolism
Microtubule-Associated Proteins / metabolism
Nuclear Proteins / genetics
Nuclear Proteins / metabolism
RNA-Binding Proteins / genetics
RNA-Binding Proteins / metabolism
Receptors, Cytoplasmic and Nuclear / genetics
Receptors, Cytoplasmic and Nuclear / metabolism
TOR Serine-Threonine Kinases / antagonists & inhibitors
TOR Serine-Threonine Kinases / metabolism
Receptors, Cytoplasmic and Nuclear
TOR Serine-Threonine Kinases
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