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• W1978995903 abstract "Gid2 physically interacts with Gid9 by anti tag coimmunoprecipitation (View interaction) Glucose is the most prominent carbon and energy source for many cells, being essential for mammals. Glucose is metabolized by the catabolic glycolysis pathway. When it is not available glucose must be synthesized by the anabolic pathway gluconeogenesis, which is in part a reversal of the glycolytic pathway. Irreversible and main regulatory catalytic steps of glycolysis are circumvented in gluconeogenesis by various enzymes. These steps are phosphorylation of glucose by hexokinase (glycolysis) and dephosphorylation of glucose-6-phosphate by glucose-6-phosphatase (gluconeogenesis), phosphorylation of fructose-6-phosphate by phosphofructokinase (glycolysis) and dephosphorylation of fructose-1,6-bisphosphate by fructose-1,6-bisphosphatase (FBPase) (gluconeogenesis), synthesis of pyruvate and ATP from phosphoenolpyruvate by pyruvate kinase (glycolysis) and synthesis of phosphoenolpyruvate by cytoplasmic malate dehydrogenase (c-MDH) and phosphoenolpyruvate carboxykinase (PEPCK) [1]. Malregulation of these two central antagonistic pathways in humans leads to type 2 diabetes [2]. When yeast cells are grown on a non-fermentable carbon source such as ethanol, gluconeogenesis is turned on and specific gluconeogenic enzymes such as FBPase, c-MDH and PEPCK are synthesized. Transfer of cells to glucose containing medium leads to rapid inactivation of these enzymes. The fate of FBPase has been studied best up to now. In a process called catabolite inactivation the FBP1 gene is repressed, the enzyme is allosterically inhibited by fructose-2,6-bisphosphate and AMP and finally phosphorylated and degraded [3-10]. Two different mechanisms were published for the degradation step [11]. After glucose addition of cells grown and starved for 48 h on acetate as non-fermentable carbon source a vacuolar degradation of FBPase was reported. This process is dependent on the uptake of FBPase into vesicles and requires so-called Vid proteins [12-14]. In contrast, glucose addition to cells grown on ethanol for 16–18 h results in rapid degradation of the enzyme by the ubiquitin proteasome pathway (UPS) called catabolite degradation [7, 11, 15-20]. Likewise, degradation of PEPCK was shown to follow the proteasomal degradation route [20]. In addition, proteasomal degradation of FBPase and PEPCK requires the Hsp70 chaperone Ssa1 as well as the Cdc48-Ufd1-Npl4 motor complex [21-23]. Ubiquitin triggered degradation of a protein is initiated by a polyubiquitin chain linked to a lysine residue or the amino terminus of the respective protein [24]. This requires activation of the 76 amino acid ubiquitin by an ubiquitin activating enzyme (E1) resulting in a thioester bond between the C-terminal glycine of ubiquitin and a cysteine residue of the E1. Transfer of ubiquitin to an ubiquitin conjugating enzyme (E2) follows. Subsequently ubiquitin is transferred to the target protein in a concerted action of the E2 and ubiquitin ligases (E3's). While HECT domain E3 ligases first receive the ubiquitin from the E2 and then transfer it to the protein to be degraded, RING domain E3 ligases bind the E2 as well as the substrate and catalyse the direct transfer of the ubiquitin from the E2 to the substrate [25]. The responsible ubiquitin ligase for FBPase degradation, the Gid (glucose induced degradation deficient) complex, was discovered [18]. This E3 ligase complex is composed of 7 subunits of which Gid2 contains a RING finger domain, which confers ubiquitin ligase activity to the complex in vitro and in vivo [20]. In addition, the two other gluconeogenic enzymes PEPCK and c-MDH are also targets of the E3 complex [20]. So far, no other proteins than the gluconeogenic enzymes have been found to be ubiquitylated by the Gid complex. A similar complex, the so called CTLH complex, of mainly unknown function was found in mammalian cells [20, 26]. Interestingly, some of the Gid proteins were also identified as Vid proteins required for vesicle and vacuole dependent degradation of FBPase under harsh starvation conditions on acetate. However, no specific biochemical function has been assigned to these proteins in this process [27, 28]. Here we show that besides Gid2 a second subunit of the Gid complex, Gid9, contains a degenerated RING finger domain. When this domain is mutated, the three gluconeogenic enzymes FBPase, PEPCK and c-MDH fail to be ubiquitylated and degraded. Gid9 binds to Gid2 in the absence of other Gid proteins. The two subunits seem to form the heterodimeric E3 ligase unit of the Gid complex. Previously described standard methods were used for media preparation and genetic and molecular biological techniques [29]. Yeast strains were grown at 30 °C. Precultures were grown 16 h in YPD or CM medium containing 2% glucose, diluted 1:12.5 into YPD or CM and grown for additional 6–7 h. Thereafter, cells were resuspended in YPethanol or CM (2% ethanol) and grown for 16 h to allow FBPase synthesis (final OD600 1-2). For induction of FBPase degradation cells were shifted to YPD or CM medium containing 2% glucose. For construction of strain YTP12 see [20]. Strain Y31488 (Δgid9) was obtained from the EUROSCARF collection. Strain YRM49b (Δgid, deletion of GID1,2,4,5,7,8,9) was generated using the technique described (R. Menssen, in preparation); [30]. Δgid GID2-V5, Δgid gid2Δ216-229CTLH-V5 and Δgid gid2Δ138-171LisH-V5 strains were generated by integration of Sfo1 digested plasmids pWO1283, pWO1313 and pWO1314 into YRM49b. The plasmids expressing wildtype Gid9 or RING finger mutated Gid9C434S were constructed by insertion of a GID9 PCR-fragment (primers Gid9fwd, Gid9rev) with its endogenous promotor and terminator regions in a Xho1/EcoR1 digested pRS316 plasmid [31]. Point mutation of the preserved Cys residue 434 of Gid9 was performed using the Transformer site-directed mutagenesis kit (Clontech, Mountain View, CA) (primers Gid9∗RING fwd, Swa1-Pme1). The plasmid JD421 expressing poly-histidine-tagged Ub was obtained from Dohmen et al. [32]. PWO1153 [33] was used as template plasmid for construction of CTLH domain deleted pWO1316 (primers ED3, ED4, ED9, ED10) and LisH domain deleted pWO1317 (primers Gid2∗LisHfwd, Gid2∗LisHrev, ED9, ED10). A cloned PCR fragment from strain YWO1850 obtained with primers RM83 and RM84 was inserted into Sal1/Not1 digested pRS303 resulting in plasmid pWO1283. Table 1-Table 3 list the yeast strains, plasmids and primers used. Ubiquitylation assays were performed by growing cells with a plasmid encoding poly-histidine-tagged ubiquitin (pJD421). Expression of His6-Ub was induced by adding CuSO4 to a final concentration 0.2 mM. Cells were grown 16-22 h in CM medium with ethanol as carbon source without uracil and leucine to allow derepression of FBPase. Thereafter, 150 OD600 of yeast cells were harvested before and 30 min after addition of 2% glucose. After washing in 1 ml of water, cells were frozen in liquid N2 and stored at -80 °C. Cells were then further processed as described in [34]. Western blotting was carried out as described in ref. [17] and [20]. Antibodies used were obtained from BAbCO (Richmond, CA) (hemagglutinin [HA], clone 16B12); Sigma Aldrich (Schnelldorf), (V5). FBPase, PEPCK and c-MDH polyclonal antibodies were produced by immunization of rabbits using a purified FBPase-/PEPCK-/c-MDH-glutathione transferase (GST). Gid9 antibody was produced by immunization of rabbits using a Gid9 peptide (amino acid 471 to 486 of the Gid9 sequence; Charles River). For immunoprecipitations (IPs) cells were cultivated as described above and samples were taken at the indicated time points. Cells (50-70 OD600) were harvested, washed with water and resuspended in 600 μl of phosphate-buffered saline (PBS) buffer pH 7.4 (137 mM NaCl, 1.25 g/l Na2HPO4, and 0.35 g/l NaH2PO4) containing protease inhibitors (ProteoBlock PIC, Thermo Scientific, Fermentas Molecular Biology Tools; 1.1 mM phenylmethyl-sulfonyl-fluoride (PMSF), 0.1-0.2% Triton-X-100) and lysed at 8 °C with glass beads for 15 min. After centrifugation, 500 μl of the supernatant were transferred to a new test tube. 0.8 μl V5-antibody was added and the samples were gently agitated end over end for 2 h at 8 °C. Immunoprecipitates were collected by adding 50 μl of 6% (wt/vol) protein A-Sepharose CL-4B (GE Healthcare, Little Chalfont, United Kingdom) and further incubated for 1.5 h. For IP the Sepharose beads were centrifuged and washed five times with ice-cold PBS buffer. Proteins were released from Sepharose by boiling in 50 μl of urea buffer. Bioinformatic sequence analysis was performed as indicated in [20]. Recently, Gid2 was identified as a RING finger protein providing ubiquitin ligase activity to the seven subunit containing Gid complex. In several cases it has been shown that RING finger E3 ligases exert their optimum activity in a dimeric state whereby homodimers and heterodimers have been found [25, 35-42]. We therefore searched whether the Gid-ubiquitin ligase complex harbors a second subunit which besides Gid2 contains a RING-finger motif. When aligning the amino acid sequences of yeast Gid proteins with the sequence of yeast Gid2, the sequences of the MAEA protein family, formerly found to be mammalian orthologues of Gid9 [20], as well as classical RING finger proteins, Gid9 emerges carrying a degenerated RING finger domain with two conserved residues (Fig. 1 ). For analysis of the function of Gid9 in the Gid complex we fused a triple HA tag onto the C-terminus of Gid9. However, the Gid9-HA3 fusion proved to be not functional: the protein failed to trigger degradation of FBPase when cells were transferred from gluconeogenic to glycolytic growth conditions. Obviously HA-tagging of Gid9 results in a biochemically inactive Gid9 mutant (Fig. 2 A). The outcome of the experiment indicated a central function for a structurally intact Gid9 protein in the Gid-complex. We therefore raised antibodies against Gid9 in rabbits to follow its expression and to monitor the interaction of Gid9 with Gid2, which had been shown to exhibit ubiquitin ligase activity [20]. For this monitoring purpose we tagged Gid2 with the V5 tag. The fusion protein is active in vivo (not shown). For monitoring an interaction of Gid9 with Gid2-V5 we expressed both proteins in a strain deleted in all seven Gid proteins, which is fully vaible but fails to degrade the gluconeogenic enzymes. As can be seen in Fig. 3 , when precipitating Gid2 with V5 antibodies, Gid9 co-precipitates. Clearly, Gid2 and Gid9 interact in the Gid complex. In addition to its RING domain Gid2 contains a CTLH and a LisH domain [20]. Deletion of these domains slows down FBPase degradation (not shown). We asked whether any of these domains were involved in the binding to Gid9. When expressing Gid2-V5 deleted in its CTLH domain or in its LisH domain together with Gid9 in the strain devoid of all other Gid proteins, both Gid2 domain mutant proteins co-precipitated with Gid9 upon application of V5 antibodies. This indicates that these domains are not essential for the Gid2-Gid9 interaction even though the interaction was somehow less pronounced when the CTLH domain in Gid2 was deleted (Fig. 3). The fact that tagging of Gid9 results in stabilization of FBPase indicates that Gid9 has a crucial function in the degradation process (Fig. 2A). We therefore introduced a mutation into the degenerated RING finger domain of Gid9 by changing the conserved cysteine residue 434 to serine to inactivate the RING domain[20, 43]. When testing the consequence of the Gid9C434S mutation on the catabolite degradation of FBPase, it was obvious that degradation of the enzyme is blocked (Fig. 2B). Also catabolite degradation of the gluconeogenic enzymes PEPCK and c-MDH is abrogated by the mutation (Fig. 2C). As the Gid-complex acts as the ubiquitin ligase in the proteasome catalyzed degradation process of these three enzymes, we expected that ubiquitylation is disturbed by the C434S mutation in Gid9. This is indeed the case: expression of the mutated Gid9C434S protein in a strain otherwise deleted for GID9 leads to a lack in polyubiquitylation of FBPase (Fig. 4 A), PEPCK (Fig. 4B) and c-MDH (Fig. 4C), when cells are shifted from gluconeogenic to glycolytic conditions, by this preventing subsequent proteasomal degradation (Fig. 2B and C). We were not able to show any Gid9 ligase activity in vitro (not shown). Interestingly the C434S mutation in Gid9 does not disturb binding of the protein to Gid2 (Fig. 3). This excludes a defective complex formation between Gid2 and Gid9 as the cause of failure of the Gid-complex harboring Gid9C434S to polyubiquitylate and degrade FBPase, PEPCK and c-MDH. Clearly, an intact Gid9 protein has an important impact on the polyubiquitylating activity of the Gid-ubiquitin ligase complex. A model proposing the sequence of events of the catabolite degradation process is depicted in Fig. 5 . We thank J. Dohmen, L. Barbin, J. Schweiggert and E. Diler for plasmids, J. Juretschke for FBPase antibodies and M. Ziegler for PEPCK and c-MDH antibodies. This work was supported by the Deutsche Forschungsgemeinschaft, Bonn." @default.
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• W1978995903 date "2011-10-29" @default.
• W1978995903 modified "2023-09-27" @default.
• W1978995903 title "Gid9, a second RING finger protein contributes to the ubiquitin ligase activity of the Gid complex required for catabolite degradation" @default.
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• W1978995903 doi "https://doi.org/10.1016/j.febslet.2011.10.038" @default.
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