1. Volume 59, Issue 1, Pages 22-34 (July 2015)
The Ubiquitin Ligase SCFUcc1 Acts as a Metabolic Switch for the Glyoxylate Cycle 
Kunio Nakatsukasa, Takashi Nishimura, Stuart D. Byrne, Michiyo Okamoto, Azusa Takahashi-Nakaguchi, Hiroji Chibana, Fumihiko Okumura, Takumi Kamura 
Molecular Cell 
Volume 59, Issue 1, Pages (July 2015)
DOI: /j.molcel
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2. Molecular Cell 2015 59, 22-34DOI: (10.1016/j.molcel.2015.04.013)
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3. Figure 1
The F Box Protein Ucc1 Recognizes Citrate Synthase 2 in the Glyoxylate Cycle
(A) Overview of the pathways of glycolysis, gluconeogenesis, the TCA cycle, and the glyoxylate cycle. See Introduction for detail.
(B) The interaction of Cit2 with Ucc1 was examined by two-hybrid assay in L40 cdc34-2 cells.
(C) Cells expressing 4xHA-tagged Cit2 and/or 3xFLAG-tagged Ucc1 were lysed and subjected to immunoprecipitation with anti-FLAG antibody. Coprecipitated proteins were analyzed by western blotting with anti-HA antibody.
(D) Total yeast lysate was prepared from cit2Δucc1Δ cells expressing Cit2-1xHA-SKL. This yeast lysate was incubated with the 3xFLAG-tagged Ucc1-Skp1 complex that had been immobilized to the anti-FLAG affinity gel. Proteins bound to the 3xFLAG-tagged Ucc1-Skp1 complex were detected by western blotting with anti-HA antibody. 3xFLAG-tagged Ucc1 appears as two bands, possibly due to a processing in the host cells. The asterisk indicates anti-FLAG IgG.
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4. Figure 2 Cit2 Is Localized to Both Peroxisomes and the Cytosol
(A) Wild-type and the indicated mutant cells expressing Cit2-GFP-SKL were grown in glucose-containing medium overnight. Subcellular localization of Cit2-GFP-SKL was analyzed by fluorescent microscopy. The corresponding differential interference contrast (DIC) images are shown.
(B) Subcellular localization of Cit2-GFP-SKL and Pex13-2xmCherry were analyzed by fluorescent microscopy.
(C) Lysates prepared from spheroplasted cells expressing Cit2-GFP-SKL and Pex13-2xmCherry were centrifuged at 13,000 × g. The pellet (P13) was saved and the supernatant was further centrifuged at 20,000 × g. The resultant pellet (P20) and the supernatant (S20) as well as P13 were analyzed by western blotting with the indicated antibodies. Tom70 (mitochondria), Pgk1 (cytoplasm), and Sec61 (ER) served as organelle markers.
(D) Lysates prepared from spheroplast cells expressing 3xFLAG-Ucc1 and Pex11-3HA were fractionated as in (C) and analyzed by western blotting with the indicated antibodies. Pex11 is localized to both peroxisomes and ER depending on its phosphorylation state (Knoblach and Rachubinski, 2010).
See also Figure S1.
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5. Figure 3 Cit2 Is Degraded in a SCFUcc1-Dependent Manner
(A–D) The degradation of Cit2-GFP-SKL or Cit2-1xHA-SKL in glucose-grown cells was assessed by cycloheximide chase analysis. Pgk1 served as a loading control. Where indicated, cells were treated with DMSO or 100 μM MG132 for 30 min before the addition of cycloheximide. To inactivate the temperature-sensitive allele, cells were grown at 25°C and shifted to 37°C for 2 hr before the addition of cycloheximide.
(E) Lysates were prepared from the indicated strains and separated on a 7%+8M Urea SDS-PAGE gel (12 cm). Proteins were analyzed by western blotting with anti-Cit1/2 antibodies. The expression of Cit2 was higher in cit1Δ cells than in wild-type cells due to activation of the retrograde response in cit1Δ cells. Cdc48 served as a loading control.
(F–H) The degradation of endogenous Cit2 was analyzed as in (B)–(D), respectively. When degradation of Cit2 was inhibited, it appeared as two bands. This is likely due to unknown post-translational modifications and/or different folding/denaturation states during separation on a urea-containing gel.
See also Figure S1.
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6. Figure 4
SCFUcc1 Regulates Citrate Homeostasis and Carbon Source Utilization
(A) Ten-fold serial dilutions of the indicated strains were spotted on synthetic complete medium containing glucose or propionate as the sole carbon source. Cells were grown at 30°C for 2 days (glucose) or 5 days (propionate).
(B) The indicated strains were spotted as in (A) on synthetic complete medium containing glucose supplemented with or without 1% (v/v) n-butanol. Plates were incubated at 30°C for 2 days (minus n-butanol) or 4 days (plus n-butanol). Where indicated, Ucc1 was overexpressed under the control of the ADH1 promoter from a low-copy plasmid.
(C) Cellular citrate levels were measured in wild-type and ucc1Δ cells grown in glucose-containing medium. Values on the y axis are arbitrary units and the amount of citrate in wild-type cells was set to 1.0. The bar graph shows the average of five independent experiments (error bars, SD).
(D) Ten-fold serial dilutions of the indicated strains were spotted on synthetic complete or minimum medium containing glucose. Cells were grown at 30°C for 2 days (synthetic complete) or 4 days (synthetic minimum). Where indicated, Cit2 was overexpressed under the control of the ADH1 promoter from a low-copy plasmid.
(E) Ten-fold serial dilutions of the indicated strains were spotted on synthetic minimum plates with glucose at 30°C for 3.5 days. Where indicated, Cit2 was overexpressed as in (D).
(F) Cells were grown in YPglucose or YPacetate medium overnight. The levels of CIT2 and UCC1 mRNA were measured by quantitative RT-PCR.
(G) Ten-fold serial dilutions of the indicated strains were spotted on synthetic complete medium containing glucose or acetate as the sole carbon source. Cells were grown at 30°C for 2 days (glucose) or 5 days (acetate). Where indicated, Ucc1 and Cit2 were overexpressed under the control of the ADH1 promoter and CIT2 promoter, respectively, from a low-copy plasmid.
(H) Growth rates of the indicated strains were measured in synthetic complete medium containing acetate as the sole carbon source at 30°C. Where indicated, Ucc1 and Cit2 were overexpressed as in (G).
See also Figure S1.
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7. Figure 5 Oxaloacetate Prevents the Ubiquitination of Cit2 In Vitro
(A) Cit2 is ubiquitinated in vitro in a SCFUcc1-dependent manner. Cit2-1xHA-SKL immunopurified from yeast cells was incubated with purified E1 (Uba1), E2 (Cdc34), E3 (SCFUcc1), ATP, MgCl2, and ubiquitin at 30°C for 90 min. The extent of ubiquitination was analyzed by western blotting with anti-HA antibody.
(B) The Cit2 in vitro ubiquitination assay was performed as in (A) for the indicated amount of time.
(C) In vivo state of ubiquitination of was analyzed for wild-type and K→R mutant Cit2.
(D) The three-dimensional structure of Cit2 was predicted with Modeler program based on the crystal structure of pig citrate synthase (PCS) in the open conformation (1CTS.pdb). Seven lysine residues reported to be potential sites for ubiquitination in the integrated data base (see Supplemental Experimental Procedures) are shown.
(E) The activity of K→R mutant Cit2 was assessed by its ability to rescue the glutamate auxotrophy of cit1Δcit2Δ cells (see also Figure S3 for the detail of this assay).
(F) The degradation of K→R mutant Cit2 was analyzed as in Figure 3.
(G) Cellular citrate levels were measured in cells expressing either wild-type or K→R mutant Cit2 as in Figure 4C. The bar graph shows the average of three independent experiments (error bars, SD).
(H) Model of the reaction cycle of citrate synthase. Citrate synthase exhibits sequential, ordered kinetics: the binding of oxaloacetate converts the open form of citrate synthase into a closed form, resulting in the creation of a binding site for acetyl-CoA. The enzyme then catalyzes the condensation reaction to produce citrate and returns to the initial open conformation.
(I) Oxaloacetate inhibits ubiquitination of Cit2. In vitro ubiquitination assay of Cit2 was performed as in (A) in the absence or the presence (closed circle) of 500 μM of oxaloacetate (OAA) for the indicated duration. The asterisks indicate a nonspecific band.
See also Figures S2 and S3.
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8. Figure 6
SCFUcc1 Preferentially Recognizes the Open Conformation of Cit2
(A) The three-dimensional structure of Cit2 was predicted as in Figure 5D based on the crystal structure of pig citrate synthase (PCS) in the open (1CTS.pdb) and closed (2CTS.pdb) conformation. The conserved catalytic and hinge residues are shown; H293 (a catalytic/hinge residue, H274 in PCS), G294 (a hinge residue, G275 in PCS), H339 (a catalytic residue, H320 in PCS), and D394 (a catalytic residue, D375 in PCS) are colored with green, red, yellow, and magenta, respectively. Historically, the numbering of amino acids in PCS starts from A28.
(B) The in vitro ubiquitination assay of the wild-type or mutant (H293G) form of Cit2 was performed for 5 min in the presence of the indicated concentration of oxaloacetate (OAA).
(C) The interaction of wild-type or mutant Cit2 with the 3xFLAG-tagged Ucc1-Skp1 complex was analyzed by pull-down assay as in Figure 1D in the absence (−OAA) or presence (+OAA) of 1 mM oxaloacetate.
(D) Pull-down assay was performed as in Figure 1D except that purified GST-Cit2 was used instead of Cit2-1xHA-SKL that was supplied from the yeast lysate. Where indicated, GST-Cit2 was preincubated with 1 mM oxaloacetate.
(E) The in vitro ubiquitination assay of wild-type or mutant forms of Cit2 (G294A and G294V) was performed.
(F) The degradation of wild-type or mutant forms of Cit2-1xHA-SKL (G294A and G294V) was assessed as in Figure 3. The asterisks in (B) and (E) indicate nonspecific bands. The asterisks in (C) and (D) indicate anti-FLAG IgG.
See also Figures S3–S5.
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9. Figure 7
SCFUcc1-Mediated Degradation of Cit2 Is Regulated by the Metabolic Status In Vivo
(A) In vivo state of Cit2 ubiquitination in glucose- or acetate-grown cells was analyzed as in Figure 5C.
(B) The degradation of Cit2 in glucose- or acetate-grown cells was analyzed as in Figure 3. CPY∗, a substrate of ER-associated degradation, served as a control. Since Cit2 and CPY∗ were expressed more in acetate-grown cells than in glucose-grown cells, the lysates for lanes 9–16 were diluted before subjected to SDS-PAGE.
(C) Model for the role of SCFUcc1-mediated regulation of Cit2 in the modulation of metabolic flux. In glucose-grown cells, Cit2 is ubiquitinated by SCFUcc1 and degraded by the proteasome, thereby keeping the activity of the glyoxylate cycle at a low level. In acetate-grown cells, Cit2 is transcriptionally upregulated, Ucc1 is downregulated, and oxaloacetate inhibits the ubiquitination of Cit2 by SCFUcc1, thereby activating the glyoxylate cycle to produce glucose through gluconeogenesis.
See also Figures S6 and S7.
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