1. Volume 171, Issue 4, Pages 890-903.e18 (November 2017)
Cytosolic Protein Vms1 Links Ribosome Quality Control to Mitochondrial and Cellular Homeostasis 
Toshiaki Izawa, Sae-Hun Park, Liang Zhao, F. Ulrich Hartl, Walter Neupert 
Cell 
Volume 171, Issue 4, Pages e18 (November 2017)
DOI: /j.cell
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3. Figure 1 Vms1 Associates with 60S Ribosomes and the RQC Complex
(A) Cell lysates of vms1Δ cells expressing Vms1-3HA or Vms1-3Myc from the GPD1 promoter were subjected to anti-Myc immunoprecipitation. Precipitates were analyzed by SDS-PAGE, Coomassie blue staining, and mass spectrometry (MS). Asterisks, immunoglobulin heavy and light chains.
(B) Volcano plot of the proteins co-precipitated with Vms1-3Myc and identified by label-free proteomics. p values and fold enrichment are shown. Proteins of interest are highlighted (see Table S1 and STAR Methods).
(C) Recovery of 60S and 40S ribosomal proteins by MS analysis as in (A). Median values from label-free quantification (LFQ) of the recovered ribosomal proteins are shown.
See also Figure S1.
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4. Figure 2 Deletion of Vms1 and Ltn1 Causes Respiratory Deficiency
(A) Growth of wild-type (WT), ltn1Δ, vms1Δ, and vms1Δltn1Δ cells. Cells were grown in YPD for 8 to 10 hr at 30°C (to log-phase) and spotted in 10-fold dilution steps on YPD (fermentable carbon source) or YPG plates (non-fermentable carbon source). YPD plates were incubated for 3 days (23°C) or for 2 days (30°C and 37°C) and YPG plates for 4 days (23°C) or for 3 days (30°C and 37°C).
(B) Mitochondria were isolated from WT, ltn1Δ, vms1Δ, and vms1Δltn1Δ cells grown in YPD at 30°C. Proteins were analyzed by SDS-PAGE and immunoblotting using the antibodies indicated.
(C) Cell extracts prepared with SDS-containing buffer from WT or vms1Δltn1Δ cells grown in YPGal medium at 23°C, 30°C, or 37°C were analyzed by SDS-PAGE and anti-Rip1 immunoblotting. i-Rip1, intermediate and m-Rip1, mature Rip1. Phosphoglycerate kinase (Pgk1) was analyzed as loading control.
(D) Mitochondria were isolated from WT, ltn1Δ, vms1Δ, and vms1Δltn1Δ cells grown in YPGal at 37°C. Mitochondria were lysed with 3% digitonin and analyzed by blue native PAGE and immunoblotting against cytochrome c1 (Cyt1) (left) and the β subunit of F1FO-ATP synthase (F1β) (right). III and IV, complexes III and IV of the respiratory chain.
See also Figure S2.
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5. Figure 3
Deletion of VMS1 and LTN1 Leads to Rqc2-Dependent Aggregation of Mitochondrial Proteins
(A) RQC2 deletion rescues growth defect of vms1Δltn1Δ cells. Growth of WT, vms1Δltn1Δ, vms1Δltn1Δrqc2Δ, and vms1Δltn1Δrqc1Δ cells was analyzed on YPD and YPG plates as in Figure 2A.
(B) Solubility of Hsp60, Ssc1, and Ssq1 and of Rip1 in WT and mutant cells. Cells were grown in YPGal at 37°C and lysed in buffer containing 0.5% Triton X-100 (see STAR Methods). Total lysates (T) were separated into pellet (P) and supernatant (S) fractions by centrifugation (15 min, 20,000 × g), followed by immunoblotting as indicated. 10% of T and S fractions and 100% of P fractions were analyzed. Aliquots of the same samples were analyzed on different gels.
(C) Processing defect of Rip1 is dependent on CAT-tailing-active Rqc2. Vms1Δltn1Δrqc2Δ cells harboring empty vector (–) or expressing RQC2-FLAG or the rqc2aaa-FLAG mutant from the GAL1 inducible promoter were grown in SCGal medium for 6 hr at 37°C. Cell extracts were analyzed by immunoblotting with anti-Rip1 and anti-FLAG.
(D) Proteomic analysis by SILAC. Ltn1Δ, vms1Δltn1Δ, and vms1Δltn1Δrqc2Δ cells were grown in SCGal medium at 37°C in the presence of light (L), heavy (H), or medium (M) lysine isotope, as indicated. Cell lysates as in (B) were separated into supernatant and pellet fractions, followed by LC-MS/MS analysis (see STAR Methods).
(E) Number of mitochondrial and non-mitochondrial proteins significantly enriched in insoluble fractions of vms1Δltn1Δ and vms1Δltn1Δrqc2Δ cells relative to ltn1Δ cells. Analysis was as described in (D).
(F) Analysis of functional categories of insoluble mitochondrial proteins enriched in vms1Δltn1Δ cells using the Saccharomyces Genome Data Base.
See also Figure S3.
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6. Figure 4
Aggregation of Mitochondrial NS Proteins Is Dependent on Vms1, Ltn1, and Rqc2
(A) Transcripts for expression of cytosolic (NS-cGFP) and mitochondrial (NS-mtGFP) NS proteins.
(B) Aggregation of NS-cGFP and NS-mtGFP in ltn1Δ cells. Total cell lysates of WT and ltn1Δ cells expressing NS-cGFP or NS-mtGFP from the GAL1 promoter for 20 hr at 30°C were subjected to anti-GFP immunoprecipitation (IP). Total lysates (left) and precipitates (right) were analyzed by anti-GFP immunoblotting (IB). Bracket indicates SDS-resistant aggregates.
(C) Deletion of RQC2 in ltn1Δ cells prevents aggregation. Cell lysates of WT, ltn1Δ, rqc1Δ, rqc2Δ, ltn1Δrqc2Δ, and rqc1Δrqc2Δ cells expressing NS-mtGFP from the GAL1 promoter were subjected to anti-GFP IP and analyzed as in (B). Asterisk, uncharacterized protein species.
(D) Growth of WT, ltn1Δ, and ltn1Δrqc2Δ cells expressing NS-mtGFP from the GAL1 promoter on SCD (– induction) or SCGal (+ induction) plates at 37°C. 6-fold serial dilutions of cells were spotted on agar plates, and plates were incubated for 3 days (– induction) or 4 days (+ induction).
(E) Analysis of NS-mtGFP in ltn1Δ and vms1Δltn1Δ cells by IP and IB as in (B). Co-IP of Tom40 and the 60S ribosomal subunit Rpl3 was detected by IB with specific antibodies. Input fractions were analyzed as controls.
(F) SDS-resistant aggregates in vms1Δltn1Δ cells are retained in the filter trap assay. Total cell lysates of WT, ltn1Δ, vms1Δ, and vms1Δltn1Δ cells expressing NS-mtGFP from the GAL1 promoter were analyzed by filter trap assay. NS-mtGFP aggregates were detected with anti-GFP antibody. Lysates were analyzed without or with prior incubation with 2% SDS. Amounts of total protein analyzed are indicated. See STAR Methods.
(G) Constitutive expression of NS-cGFP-3HA and NS-mtGFP-3HA with CYC1 3′ UTR leads to mild formation of SDS-insoluble aggregates in vms1Δ cells. Upper: schematic of transcripts. Lower: NS-cGFP-3HA and NS-mtGFP-3HA were expressed from the GPD1 promoter in WT, vms1Δ, and vms1Δrqc2Δ cells grown in SCD medium at 30°C. NS proteins were immunoprecipitated with anti-HA antibody and analyzed as in (B). Bracket indicates SDS-resistant aggregates. The section of the immunoblot indicated by a dotted rectangle is also shown after longer exposure (arrow).
See also Figure S4.
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7. Figure 5
Aggregates of Mitochondrial NS Proteins Sequester Mitochondrial Homeostasis Machinery
(A) Interactors on NS-mtGFP in ltn1Δ and vms1Δltn1Δ cells. Lysates were subjected to anti-GFP immunoprecipitation followed by in-solution digest and SILAC-MS. WT cells expressing NS-mtGFP served as controls. Venn diagrams showing numbers of proteins identified in pull-down fractions (Tables S3A and S4). Left: ltn1Δ cells; middle: vms1Δltn1Δ cells; right: overlap of mitochondrial interactors (see STAR Methods).
(B) Functional categories of NS-mtGFP interactors. Left: ltn1Δ cells; right: vms1Δltn1Δ cells (see Tables S3A and S4).
(C) Enrichment of mitochondrial and cytosolic protein synthesis and quality-control factors in the NS-mtGFP interactomes of ltn1Δ and vms1Δltn1Δ cells. Means enrichment of mitochondrial tRNA ligases and ribosomal proteins is shown. Proteins identified in SDS-resistant NS-mtGFP aggregates in ltn1Δ cells are indicated by an asterisk (see Tables S3 and S4). (SDS-resistant proteins in aggregates of vms1Δltn1Δ cells could not be determined, because the aggregates were too large to be separated on SDS-PAGE.)
(D) Overlap of mitochondrial NS-mtGFP interactors with insoluble mitochondrial proteins in vms1Δltn1Δ cells (see Figure 3E; Tables S2A and S4).
(E) Enrichment of Rqc2 in the NS-mtGFP interactomes of ltn1Δ and vms1Δltn1Δ cells.
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8. Figure 6
Vms1 Counteracts Rqc2-Dependent Aggregation of NS Proteins in Mitochondria
(A) Vms1 overexpression reduces NS-mtGFP aggregation. Upper: NS-cGFP or NS-mtGFP were expressed from the GAL1 promoter in ltn1Δ cells without or with overexpression of Vms1-3Myc from the GPD1 promoter (see Figure 1A). Cell lysates were analyzed by anti-GFP immunoprecipitation (IP) and immunoblotting (IB) as in Figure 4B. Lower: aliquots of anti-GFP IP and input fractions were analyzed by IB against Ssq1, Hsp60, and the Myc-tag of Vms1-3Myc.
(B) Vms1 overexpression in ltn1Δ cells expressing NS-mtGFP reduces visible inclusions. Ltn1Δ cells expressing NS-mtGFP as in (A) with or without overexpression of Vms1-3Myc were grown at 30°C. Mitochondrial mCherry (mt-mCherry) was co-expressed. The fraction of cells with fluorescent inclusions was quantified (n = 100). Error bars represent SD from three independent experiments. p values from Student’s t test.
(C) Vms1 overexpression reduces the level of Rqc2 bound to 60S ribosomes. Upper: Vms1-3Myc was expressed in vms1Δ cells either from its own promoter (designated WT) or overexpressed from the GPD1 promoter (VMS1 OE). Cells expressed Rqc2-3HA from its own promoter. Fractions from sucrose gradient centrifugation were analyzed by IB for Rqc2-3HA and Vms1-3Myc. 60S and 80S ribosomes were detected using anti-Rpl3 antibody. Lower: the same experiment was performed with WT and vms1Δ cells expressing Rqc2-3HA. T, top and B, bottom of the gradient.
(D) Domain structure of Vms1. ZnF, zinc finger domain; MTD, mitochondrial targeting domain; Ank R, ankyrin repeat domain; CC, coiled-coil domain; VIM, VCP (Cdc48) interacting motif.
(E) Vms1 lacking the VIM domain interacts with 60S ribosomes. Cell lysates of vms1Δ cells expressing Vms1-3HA, Vms1-3Myc, or Vms1(ΔVIM)-3Myc from the GPD1 promoter were subjected to IP using anti-Myc antibody as in Figure 1A. Precipitates were analyzed by SDS-PAGE and Coomassie staining. Asterisks indicate immunoglobulin G (IgG) H- and L-chains.
(F) Volcano plot of proteins co-precipitated with Vms1(ΔVIM)-3Myc and identified by label-free proteomics. p values and fold enrichment are shown. Proteins of interest are highlighted (see Table S1).
See also Figures S5 and S6.
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9. Figure 7 Roles of Vms1 in mitoRQC of Aberrant Mitochondrial Proteins
(A) RQC of cytosolic NS proteins.
(B) RQC of mitochondrial NS proteins. Translocation of nascent NS proteins into mitochondria is arrested by the 60S ribosome. Ltn1 pathway (upper): Ltn1 gains access to C-terminal lysines of the chain in transit, followed by ubiquitylation, Cdc48-mediated extraction and proteasomal degradation. Vms1 pathway (lower): Ltn1 fails to ubiquitylate as the ribosome is pulled in close contact with the outer membrane (OM) by the import machinery (red arrow). Vms1 associates with 60S ribosome at the OM and counteracts CAT tailing by Rqc2. NS protein is imported into the mitochondrial matrix and either folds or is degraded by mitochondrial quality-control machinery. IM, inner mitochondrial membrane.
(C) Toxic effects of mitochondrial NS proteins in the absence of Vms1 and Ltn1 function. CAT-tailed NS proteins are imported and sequester chaperones and other protein quality-control factors into stable aggregates.
See also Figure S7.
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10. Figure S1 Vms1 Associates with 60S Ribosomes, Related to Figure 1
Lysates from cells expressing Vms1-3Myc from its own promoter (grown in SCD medium) were subjected to 10%–30% sucrose density gradient centrifugation. Fractions were analyzed by SDS-PAGE and immunoblotting using antibodies against the Myc-tag of Vms1 and Rpl3 (subunit of the 60S ribosome). Absorbance at 254 nm and Rpl3 indicate the positions of 40S, 60S, and 80S ribosomes. T, top and B, bottom of gradient.
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11. Figure S2
Deletion of Vms1 and Ltn1 Causes Respiratory Deficiency of Mitochondria, Related to Figure 2
(A) Growth of strains with single and double deletions of RQC components as indicated on YPD and YPG plates, analyzed as in Figure 2A.
(B) Levels of RIP1 mRNA. Total RNA of WT and mutant cells grown in YPD, YPGal or YPG medium at 30°C were analyzed by Northern blotting using probes for RIP1 mRNA and SCR1 (loading control; RNA subunit of signal recognition particle).
(C) Electrophoretic mobility upon SDS-PAGE of the three forms of Rip1, p-Rip1 (precursor), i-Rip1 (processing intermediate); m-Rip1 (mature protein). Total cell extracts of WT and vms1Δltn1Δ cells (grown in SCGal medium at 30°C) and WT cells overexpressing Rip1 from the GPD1 promoter (grown in SCD medium at 30°C) were analyzed by SDS-PAGE and immunoblotting using anti-Rip1 antibodies. Rip1 overexpression (OE) leads to increased levels of p-Rip1 and i-Rip1.
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12. Figure S3
Solubility of Non-mitochondrial Proteins in WT and RQC Mutant Strains, Related to Figure 3
Solubility of Hsp70 chaperone proteins in the cytosol (Ssa1) and ER (BiP) as well as of cytosolic phophoglycerate kinase (Pgk1) in WT and mutant cells from Figure 3B. Cells were grown in YPGal medium at 37°C and lysates prepared in buffer containing 0.5% Triton X-100. Total lysates (T) were separated into insoluble pellet (P) and soluble supernatant (S) fraction by centrifugation (15 min at 20,000 x g), followed by immunoblotting using the antibodies indicated. 10% of total and soluble fractions and 100% of pellet fractions were analyzed.
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13. Figure S4
Aggregation of Mitochondrial NS Proteins Is Dependent on Vms1, Ltn1, and Rqc2, Related to Figure 4
(A) WT and ltn1Δ cells expressing NS-mtGFP were lysed under denaturing conditions (see STAR Methods). Total cell lysates were subjected to anti-GFP IP as in Figure 4B after buffer exchange and analyzed by immunoblotting (IB) with anti-GFP and anti-ubiquitin antibodies. Immunoprecipitation of NS-cGFP from the proteasome deficient pre1-1 mutant served as a positive control for detection of ubiquitylated protein. Brackets indicate SDS-resistant high molecular weight aggregates (left panel) and ubiquitylated species (right panel).
(B) Analysis of NS-mtGFP in WT and RQC mutant cells by fluorescence microscopy. Cells expressing NS-mtGFP as in Figure 4 were grown for 6 hr at 30°C. Mitochondrial mCherry (mt-mCherry) was co-expressed to visualize mitochondria. The brightness of NS-mtGFP in ltn1Δ and vms1Δltn1Δ cells was much stronger than in WT cells and was adjusted to a lower level. The brightness and contrast of the mt-mCherry images was adjusted to show the overlap of mt-mCherry and NS-mtGFP signals. DIC, differential interference contrast.
(C) Aggregation of mtGFP-(AT)10 in WT and rqc2Δ cells. Total cell lysates of WT and rqc2Δ cells expressing mtGFP-(AT)10 (containing 10 Ala-Thr repeats) or mtGFP-(GS)10 (containing 10 Gly-Ser repeats) from the GAL1 promoter for 20 hr at 30°C were subjected to IP with anti-GFP antibody and analyzed as in Figure 4B. Asterisk, uncharacterized protein species.
(D) Accumulation of i-Rip1 in ltn1Δ cells. Total cell lysates (T) of WT, ltn1Δ and ltn1Δrqc2Δ cells expressing NS-mtGFP from the GPD1 promoter (grown in SCGal medium at 37°C) were fractionated into supernatant (S) and pellet (P) as in Figure 3B. Fractions were analyzed by SDS-PAGE and IB using anti-Rip1 antibodies.
(E) Aggregation of NS-cGFP and NS-mtGFP in vms1Δltn1Δ cells. Total cell lysates of WT, ltn1Δ and vms1Δltn1Δ cells expressing NS-cGFP or NS-mtGFP from the GAL1 promoter for 20 hr at 30°C were subjected to IP with anti-GFP antibody and analyzed as in Figure 4B. SDS-resistant aggregates of NS-mtGFP are visible at the top of the gel and in the well.
(F) Analysis of SDS-resistant aggregates of NS-cGFP and NS-mtGFP in vms1Δltn1Δ cells by filter trap assay. Lysates from cells in Figure S4E were analyzed as in Figure 4F. A longer exposure is shown (right panel) demonstrating that vms1Δltn1Δ cells generate only very small amounts of NS-cGFP that are large enough to be retained by the filter.
(G) Growth of vms1Δltn1Δ cells expressing NS-cGFP or NS-mtGFP from the GAL1 promoter on SCD (-induction) or SCGal (+induction) plates at 30°C. Six-fold serial dilutions of cells were spotted on agar plates and incubated for 2 days (- induction) or 3 days (+ induction).
(H) NS-Shm1 but not NS-ΔMTS-Shm1, lacking the mitochondrial targeting signal, aggregates in vms1Δ cells. Cells harboring empty vector (-) or expressing NS-Shm1-2Myc or NS-ΔMTS-Shm1-2Myc from the GPD1 promoter were grown in SCD medium at 30°C. Cell extracts were analyzed by SDS-PAGE and IB with anti-Myc antibody. MTS, mitochondrial targeting sequence.
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14. Figure S5
Vms1 Counteracts Rqc2-Dependent Aggregation of NS Proteins in Mitochondria, Related to Figure 6
(A) Overexpression of Vms1 in ltn1Δ cells markedly reduces aggregation of NS-mtGFP as analyzed by filter trap assay. Ltn1Δ cells expressing NS-cGFP or NS-mtGFP from the GAL1 promoter were grown at 30°C with or without co-expression of Vms1-3Myc from the GPD1 promoter. Cell lysates were subjected to filter trap analysis in the presence or absence of SDS as in Figure 4F.
(B) Vms1 overexpression in ltn1Δ cells expressing mitochondrial NS-proteins reduces accumulation of i-Rip1. Ltn1Δ cells harboring empty vector or expressing either NS-mtGFP or NS-Shm1 were grown in SCGal medium at 37°C with or without co-expression of Vms1 from the GPD1 promoter. Cell extracts were analyzed by SDS-PAGE and immunoblotting (IB) with anti-Rip1 antibodies. Levels of i-Rip1 related to total Rip1 (i-Rip1 + m-Rip1) were quantified by densitometry. Error bars represent SD from four (for NS-mtGFP) and three (for NS-Shm1) independent experiments. p values from Student’s t test.
(C) Vms1 overexpression partially rescues the growth defect of ltn1Δ cells expressing mitochondrial NS-proteins. Cells in (B) were grown on SCD plates at 37°C and analyzed as in Figure 2A.
(D) Overexpression of Vms1 reduces Rqc2-dependent CAT-tailing of NS-mtGFP. Ltn1Δ and ltn1Δrqc2Δ cells expressing NS-mtGFP from the GPD1 promoter were grown in SCGal medium at 37°C with or without overexpression of Vms1-3Myc from the GPD1 promoter. Total cell extracts were subjected to SDS-PAGE and IB with anti-GFP antibody. Bracket, SDS-resistant high molecular aggregates. Arrow, monomeric CAT-tailed NS-mtGFP. In order to resolve monomeric CAT-tailed NS-mtGFP, an NS-mtGFP construct with a CYC1 3′ UTR was used.
(E) Rqc2 deletion or overexpression does not alter the relative amount of Vms1 associated with 60S ribosomes. Upper panel: WT or rqc2Δ cells expressing Vms1-3Myc from its own promoter were grown in YPD medium at 30°C. Cells expressed Rqc2-3HA from its own promoter. Cell lysates were subjected to 10%–40% sucrose gradient centrifugation and fractions analyzed by IB for Vms1 using anti-Myc antibody. 60S and 80S ribosomes were detected using antibody against the 60S subunit Rpl3. Lower panel: The same experiment was performed with WT cells expressing Vms1-3Myc either with or without overexpression of Rqc2 (RQC2 OE). T, top and B, bottom of the gradient.
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15. Figure S6
The VIM Domain Is Not Required for Vms1 to Counteract Rqc2-Dependent Aggregation of NS Proteins in Mitochondria, Related to Figure 6
(A) Vms1 lacking the VIM domain does not interact with Cdc48 in WT and ltn1Δ cells. Lysates of vms1Δ cells and vms1Δltn1Δ cells expressing Vms1-3HA, Vms1-3Myc or Vms1(ΔVIM)-3Myc from the GPD1 promoter were subjected to IP using anti-Myc antibody, followed by immunoblotting (IB) with anti-Myc and anti-Cdc48 antibodies.
(B) Vms1(ΔVIM) retains activity to suppress NS-mtGFP aggregation. Ltn1Δ cells expressing NS-mtGFP from the GAL1 promoter with or without co-expression of Vms1-3Myc or Vms1 (ΔVIM)-3Myc from the GPD1 promoter were grown at 30°C. Immunoprecipitates prepared as in Figure 4B were analyzed by IB using anti-GFP antibody.
(C) Vms1(ΔVIM) rescues the processing defect of Rip1. Cell extracts of vms1Δltn1Δ cells expressing either Vms1-3Myc or Vms1(ΔVIM)-3Myc from the VMS1 promoter and grown in SCGal medium at 37°C were analyzed by IB with anti-Rip1 antibodies.
(D) Vms1 (ΔVIM) rescues the growth defect of vms1Δltn1Δ cells under respiratory conditions. Vms1Δltn1Δ cells expressing Vms1-3Myc or Vms1 (ΔVIM)-3Myc from the GPD1 promoter were grown in SCD medium at 30°C, spotted on YPD and YPG plates and grown at 37°C as described in Figure 2A.
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16. Figure S7
Homologs of the RQC2 Gene Are Present in Archaea, Related to Figure 7
Alignment of N-terminal segments of the Rqc2 proteins from various archaea and eukaryotes (source National Center for Biotechnology; Essential residues characteristic of CAT-tailing activity are highlighted in red; conserved residues throughout archaea and eukaryotes in black; structurally conserved residues in gray.
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