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• W2074676459 abstract "Article26 November 2009free access Cullin neddylation and substrate-adaptors counteract SCF inhibition by the CAND1-like protein Lag2 in Saccharomyces cerevisiae Edyta Siergiejuk Edyta Siergiejuk ETH Zürich, Institute of Biochemistry, Schafmattstrasse 18, Zürich, Switzerland Search for more papers by this author Daniel C Scott Daniel C Scott Howard Hughes Medical Institute, St Jude Children's Research Hospital, Memphis, TN, USA Search for more papers by this author Brenda A Schulman Brenda A Schulman Howard Hughes Medical Institute, St Jude Children's Research Hospital, Memphis, TN, USA Search for more papers by this author Kay Hofmann Kay Hofmann Miltenyi Biotec GmbH, Friedrich-Ebert-Strasse 68, Bergisch-Gladbach, Germany Search for more papers by this author Thimo Kurz Corresponding Author Thimo Kurz ETH Zürich, Institute of Biochemistry, Schafmattstrasse 18, Zürich, Switzerland Scottish Institute for Cell Signaling, Ubiquitylation Unit, University of Dundee, Dundee, UK Search for more papers by this author Matthias Peter Corresponding Author Matthias Peter ETH Zürich, Institute of Biochemistry, Schafmattstrasse 18, Zürich, Switzerland Search for more papers by this author Edyta Siergiejuk Edyta Siergiejuk ETH Zürich, Institute of Biochemistry, Schafmattstrasse 18, Zürich, Switzerland Search for more papers by this author Daniel C Scott Daniel C Scott Howard Hughes Medical Institute, St Jude Children's Research Hospital, Memphis, TN, USA Search for more papers by this author Brenda A Schulman Brenda A Schulman Howard Hughes Medical Institute, St Jude Children's Research Hospital, Memphis, TN, USA Search for more papers by this author Kay Hofmann Kay Hofmann Miltenyi Biotec GmbH, Friedrich-Ebert-Strasse 68, Bergisch-Gladbach, Germany Search for more papers by this author Thimo Kurz Corresponding Author Thimo Kurz ETH Zürich, Institute of Biochemistry, Schafmattstrasse 18, Zürich, Switzerland Scottish Institute for Cell Signaling, Ubiquitylation Unit, University of Dundee, Dundee, UK Search for more papers by this author Matthias Peter Corresponding Author Matthias Peter ETH Zürich, Institute of Biochemistry, Schafmattstrasse 18, Zürich, Switzerland Search for more papers by this author Author Information Edyta Siergiejuk1, Daniel C Scott2, Brenda A Schulman2, Kay Hofmann3, Thimo Kurz 1,4 and Matthias Peter 1 1ETH Zürich, Institute of Biochemistry, Schafmattstrasse 18, Zürich, Switzerland 2Howard Hughes Medical Institute, St Jude Children's Research Hospital, Memphis, TN, USA 3Miltenyi Biotec GmbH, Friedrich-Ebert-Strasse 68, Bergisch-Gladbach, Germany 4Scottish Institute for Cell Signaling, Ubiquitylation Unit, University of Dundee, Dundee, UK *Corresponding authors. Swiss Federal Institute of Technology Zurich (ETH), ETH Zurich, Institute of Biochemistry, Schafmattstrasse 18, Zurich 8093, Switzerland. Tel.: +44 1382 388371; Fax: +44 1382 388500; E-mail: [email protected] or Tel.: +41 44 633 6586; Fax: +41 44 632 1298; E-mail: [email protected] The EMBO Journal (2009)28:3845-3856https://doi.org/10.1038/emboj.2009.354 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Cullin-based E3 ubiquitin ligases are activated through covalent modification of the cullin subunit by the ubiquitin-like protein Nedd8. Cullin neddylation dissociates the ligase assembly inhibitor Cand1, and promotes E2 recruitment and ubiquitin transfer by inducing a conformational change. Here, we have identified and characterized Lag2 as a likely Saccharomyces cerevisiae orthologue of mammalian Cand1. Similar to Cand1, Lag2 directly interacts with non-neddylated yeast cullin Cdc53 and prevents its neddylation in vivo and in vitro. Binding occurs through a conserved C-terminal β-hairpin structure that inserts into the Skp1-binding pocket on the cullin, and an N-terminal motif that covers the neddylation lysine. Interestingly, Lag2 is itself neddylated in vivo on a lysine adjacent to this N-terminal-binding site. Overexpression of Lag2 inhibits Cdc53 activity in strains defective for Skp1 or neddylation functions, implying that these activities are important to counteract Lag2 in vivo. Our results favour a model in which binding of substrate-specific adaptors triggers release of Cand1/Lag2, whereas subsequent neddylation of the cullin facilitates the removal and prevents re-association of Lag2/Cand1. Introduction Ubiquitination regulates many cellular processes by targeting proteins for degradation through the 26S-proteasome (Hershko and Ciechanover, 1998). During this process, the small protein ubiquitin is attached to substrate proteins in three steps. First, ubiquitin is activated by an E1 activating enzyme, which results in the formation of a thioester bond between the C-terminus of ubiquitin and the active site cysteine of the E1. From the E1, ubiquitin is transferred to the active site cysteine of E2 ubiquitin-conjugating enzymes, which subsequently interact with E3 ubiquitin ligases. E3 enzymes recognize the substrate and promote ubiquitin transfer from the E2 onto substrate proteins (Pickart, 2001). Substrate ubiquitination is achieved by the formation of an isopeptide linkage between the C-terminus of ubiquitin and a lysine residue of the substrate protein. Multiple rounds of ubiquitination extend a ubiquitin chain on the first ubiquitin, which depending on the ubiquitin lysine used in chain formation can lead to recognition by 26S-proteasomes, which subsequently degrade the substrate. The largest class of E3 ubiquitin ligases is represented by the multi-subunit cullin–RING E3s (CRLs). Cullins function as scaffolds within the CRL complex, which bind through their N-terminus to variable substrate-specific modules and their C-terminus to the small RING-finger protein Rbx1 (Hrt1 in budding yeast). The cullin/Rbx1 heterodimer acts as the catalytic core by recruiting ubiquitin-charged E2 to the complex. The composition of the substrate-specific module varies depending on the cullin and the substrate (Sumara et al, 2008). The best characterized CRL, the Saccharomyces cerevisiae SCF (Skp1–Cdc53/cullin1-F-box) complex, uses Skp1 to interact with one of several F-box proteins that in turn directly bind targets. For example, the F-box protein Cdc4 promotes cell-cycle progression by mediating degradation of the cyclin-dependent kinase inhibitor Sic1 at the G1/S transition (Schwob et al, 1994; Verma et al, 1997). Owing to their critical role in substrate selection, the activity of E3 ubiquitin ligases is highly regulated. One mode of CRL regulation is the modification of the cullin subunit with the ubiquitin-like protein Nedd8 (Rub1 in S. cerevisiae: related to ubiquitin 1; Lammer et al, 1998; Liakopoulos et al, 1999). Nedd8 is similar to ubiquitin in sequence and structure and is also covalently linked to target proteins. Nedd8 substrates are generally mono-neddylated, and though there are countless substrates for ubiquitination, only very few neddylated proteins are known to date. The best characterized Nedd8 substrates are cullin proteins. Mono-neddylation of cullins at a specific C-terminal lysine residue results in the activation of CRL complexes by triggering structural changes that allow for efficient ubiquitin transfer to the substrate (Duda et al, 2008; Fang et al, 2008; Saha and Deshaies, 2008) and by increasing the affinity of ubiquitin-charged E2 enzyme to the ligase (Kawakami et al, 2001). In addition, Nedd8 counteracts the association of a cullin ligase assembly inhibitor called Cand1 (cullin-associated and neddylation-dissociated 1; Liu et al, 2002; Zheng et al, 2002b). Cand1 preferentially associates with unneddylated cullin and prevents binding of substrate-specific factors, thus inhibiting the formation of an active ligase complex. The X-ray crystal structure of the human Cand–Cul1 complex showed that Cand1 interacts with both the cullin C- and N-termini (Goldenberg et al, 2004). At the N-terminus, Cand1 inserts a β-hairpin loop into the Skp1-binding pocket, sterically preventing association of the substrate-specific module with the ligase (Zheng J et al, 2002; Zheng N et al, 2002). At the cullin C-terminus, Cand1 covers the lysine residue that becomes neddylated in the active complex, providing an explanation for why cullin neddylation and Cand1 binding appear mutually exclusive. However, it remains unclear whether neddylation is required to remove Cand1 and as a result allows the association of substrate-specific factors, or whether the presence of substrate-specific modules counteracts Cand1 and neddylation subsequently activates the complex. Using bioinformatic analysis, we have identified budding yeast Cand1 as a gene described earlier to be involved in longevity assurance, called Lag2 (longevity assurance gene 2). As expected, Lag2 directly interacts with non-neddylated yeast cullin Cdc53 (cullin1), and prevents cullin neddylation in vivo and in vitro. Interestingly, Lag2 is only released from Cdc53 in the presence of substrate-specific adaptors, which subsequently allows neddylation of the cullin, thus establishing a plausible order of molecular events required for CRL activation. Finally, we found that Lag2 is itself a substrate for neddylation and thus the first non-cullin neddylation target identified in yeast. Results Identification of Lag2 as the Cand1 orthologue in S. cerevisiae To determine whether S. cerevisiae carries a Cand1-like activity, we performed a bioinformatic analysis of the yeast genome searching for open reading frames that contained two functionally important regions of human Cand1: the C-terminal β-hairpin structure that inserts into the Skp1-binding pocket on cullins, as well as an N-terminal motif that binds to conserved surfaces in the cullin C-terminus (Figure 1A; Supplementary Figure 1; Zheng J et al, 2002; Zheng N et al, 2002). Using this approach (MAFFT program, L-INS-I algorithm; Katoh et al, 2005), we identified Lag2 as the only gene in the budding yeast genome that carries significant homology in both regions (Figure 1A, alignment). Lag2 homologues were also present in other species that did not carry an obvious Cand1 gene, whereas Lag2 was absent from all examined species with an identifiable Cand1 (Supplementary Table I). This observation further correlates with the notion that Lag2 may act as Cand1 orthologue in these organisms. Figure 1.Lag2 interacts with Cdc53 in a neddylation-dependent manner in vivo. (A) Schematic representation of Cul1/Cand1 and Lag2/Cdc53 complexes. Depicted with dashed lines are regions important for Cand1 inhibitory function (C-terminal β-hairpin and N-terminal patch). The sequence conservation in these regions between Lag2 and Cand1 homologues from different species is shown; identical residues are shaded. The asterisks mark residues that have been mutated to alanine to probe for their functional importance. (B) Extracts prepared from wild type or lag2Δ cells were immunoprecipitated with either control IgG or Lag2-specific antibodies, and bound proteins were immunoblotted as indicated with Cdc53, Lag2, Skp1 or Dcn1 antibodies. One percent of total extracts were loaded as input (sup). (C) Extracts from wild type (wt), deneddylation (rri1Δ) and neddylation-deficient (rub1Δ) cells were subjected to immunoprecipitation assay with either control IgG or Lag2-specific antibodies. Bound proteins were visualized by immunoblotting with Cdc53 (upper panel) and Lag2 (lower panel) antibodies. (D) Extracts from lag2Δ cells expressing wild-type Lag2, Lag2GN(551) (upper panel) or Lag2DDYM(17) (lower panel) were immunoprecipitated with control IgG or Lag2-specific antibodies. Bound proteins were visualized by immunoblotting with Cdc53 and Lag2 antibodies. Download figure Download PowerPoint Lag2 interacts in vivo with cullins in a neddylation state-dependent manner To test whether Lag2 is indeed a budding yeast Cand1, we first determined whether Lag2 interacts with Cdc53. An antibody to Lag2 specifically co-immunoprecipitated Cdc53 from yeast extract (Figure 1B), but not Skp1 or Dcn1, and Lag2 and Cdc53 readily interacted in yeast two-hybrid assays (Supplementary Figure 2A). Conversely, Cdc53 but not Lag2 was found in Skp1 immunoprecipitates (Supplementary Figure 2B). Importantly, Lag2 preferentially co-immunoprecipitated non-neddylated Cdc53, even though the majority of Cdc53 in cells is neddylated (Figure 1C). This observation suggests that similar to Cand1, Lag2 may sequester non-neddylated cullin core complexes. Consistent with this hypothesis, deletion of Rub1/Nedd8, which prevents cullin neddylation, resulted in a slightly stronger interaction between Lag2 and Cdc53 (Figure 1C). Cullin neddylation can be reversed by a specific isopeptidase activity of the COP9–signalosome (CSN) complex (Lyapina et al, 2001). Yeast cells lacking CSN activity are viable, but Cdc53 accumulates in its neddylated form. As predicted, Lag2 no longer co-immunoprecipitated Cdc53 from cells deleted for the CSN core component Csn5/Rri1 (Figure 1C), strongly indicating that neddylation of Cdc53 prevents its interaction with Lag2. The interaction between Lag2 and Cdc53 not only depends on the cullin neddylation state, but also requires the sequences conserved between Lag2 and Cand1. Mutational disruption of either the Lag2 β-hairpin (G551A, N552A, hereafter referred to as Lag2GN(551)) or the N-terminal cullin interaction site (D17A, D19A, Y22A, M23A, hereafter referred to as Lag2DDYM(17)) strongly diminished or abolished the interaction of Lag2 and Cdc53 by co-immunoprecipitation (Figure 1D). Together, these results show that Lag2 resembles Cand1 with respect to cullin binding, and suggest that Lag2 may function as an orthologue of Cand1 in budding yeast. Lag2 inhibits the SCF complex in vivo in a neddylation-dependent manner Association of Cand1 with CRL core complexes prevents their activation and association with substrate-specific adaptors, and as such is predicted to act as an inhibitor of ligase function. To determine whether Lag2 indeed inhibits cullin complexes in vivo, we examined the effect of Lag2 deletions and overexpression on the function of the SCF complex. Interestingly, neither deletion nor overexpression of Lag2 had any discernable effect on viability of wild-type cells (data not shown, Figure 2A). However, Lag2 overexpression was lethal in cells deleted for the yeast Nedd8 homologue Rub1 or the yeast Nedd8 E3 ligase Dcn1 (Kurz et al, 2005, 2008; Figure 2A), and this effect was not additive on simultaneous deletion of rub1 and dcn1. In contrast, Lag2 overexpression had no effect on viability of cells lacking the de-neddylating enzyme Rri1/Csn5 (Figure 2A), supporting the notion that cullin neddylation counteracts the interaction and thus function of Lag2. Indeed, overexpression of the Lag2GN(551) and Lag2DDYM(17) mutants was not detrimental to neddylation-deficient cells (Figure 2B), showing that the conserved stretches between Lag2 and Cand1 and the interaction with cullins are important for Lag2 function. Figure 2.Lag2 inhibits SCF function in vivo. (A) Five-fold serial dilution of an equal number of wild type, rub1Δ, rri1Δ, dcn1Δ and rub1Δ dcn1Δ cells transformed with an empty control plasmid (ev) or a plasmid allowing overexpression of Lag2 from the regulatable GAL1,10-promoter, were spotted on media containing glucose (left, GAL-promoter off) or galactose (right, GAL-promoter on). The plates were photographed after 3 days at 30°C. (B) Five-fold serial dilutions of an equal number of wild-type (wt) (upper plates) or rub1Δ (lower plates) cells transformed with an empty control plasmid (ev) or plasmids allowing as indicated overexpression of wild-type Lag2, Lag2GN(551) or Lag2DDYM(17) from the regulatable GAL1,10-promoter were spotted on media containing glucose (GAL-promoter off) or galactose (GAL-promoter on). The plates were photographed after 3 days at 30°C. (C, D) The morphology of wild type (wt) or rub1Δ cells overexpressing Lag2 from the GAL-1,10 promoter was examined by DIC microscopy 20 h after induction with 2% galactose (C, upper pictures). For control, the morphology of temperature-sensitive cdc53-1 cells shifted to the restrictive temperature for 7 h was included below (C, lower picture). The accumulation of wt, rub1Δ, dcn1Δ and rub1Δ dcn1Δ cells cells with elongated buds was quantified (D), and plotted as percentage of the total number of cells with standard deviations (n=500 in each case). (E) Sic1 levels were analysed by immunoblotting of extracts prepared from wild type (wt) or rub1Δ cells transformed with an empty control plasmid (ev) or plasmids allowing overexpression of wild type Lag2 or Lag2GN(551) from the regulatable GAL1,10-promoter. Immunoblotting with antibodies against Lag2 and actin control for equal Lag2 expression and gel loading, respectively. Download figure Download PowerPoint We next examined the phenotype of these cells and determined whether the lethality associated with Lag2 overexpression is indeed a result of SCF inhibition. The cyclin-dependent kinase inhibitor Sic1 is one of the major substrates of the Cdc53Cdc4 complex, and a failure to degrade Sic1 results in a G1/S cell-cycle arrest accompanied by abnormal, elongated growth of the forming bud. Consistent with Lag2 counteracting SCF function, overexpression of Lag2 in neddylation-deficient cells resulted in the formation of elongated buds (Figure 2C), similar to the morphology of cdc53-1 temperature-sensitive mutants (Willems et al, 1996). The fraction of cells displaying this defect was lower in rub1Δ, dcn1Δ or rub1Δ dcn1Δ cells overexpressing Lag2 (approx. 40%) compared with cdc53-1 mutants at the restrictive temperature (∼60%), suggesting that a failure to degrade Sic1 may not be the only cause of Lag2 overexpression-induced lethality in neddylation-deficient cells (Figure 2D). To verify that Sic1 degradation is indeed impaired, we determined total Sic1 protein levels using western blot analysis in wild type and neddylation-deficient cells overexpressing Lag2. Although overexpression of Lag2 in wild-type cells only marginally increased Sic1 levels (Figure 2E), Sic1 protein abundance was approximately three-fold higher in neddylation-deficient cells overexpressing Lag2 (Figure 2E). Consistent with the phenotypic analysis, this increase was not observed on overexpression of the Lag2GN(551) (Figure 2E), implying that Sic1 accumulation requires the ability of Lag2 to interact with Cdc53. Lag2 prevents Cdc53 neddylation in vitro by forming a heterotrimeric complex with Cdc53 and Hrt1 To investigate the molecular mechanism of Lag2 function in vitro, we tested whether purified Cdc53/Hrt1 forms a heterotrimeric complex similar to Cul1/Rbx1/Cand1. We expressed GST-tagged Hrt1, 6xHis-tagged Cdc53 and untagged Lag2 from a poly-cistronic bacterial expression vector in Escherichia coli, and purified complexes using a two-step affinity purification against the 6xHis and GST-tags. Subsequent gel filtration over a Superose 6 column showed the existence of a stable complex with an apparent size of 670 kDa (Figure 3A), suggesting that Cdc53/Hrt1 and Lag2 form a complex approximately three times the expected size of a single heterotrimeric unit. This oligomerization may be facilitated through dimerization of the GST-tag on Hrt1 and/or the previously reported dimerization of cullins. Figure 3.Lag2 prevents Cdc53 neddylation in vitro. (A) Extracts prepared from E. coli expressing 6xHis-Cdc53, GST-Hrt1 and untagged Lag2 were purified by sequential affinity purification against the 6xHis and GST-tags, and the eluate separated on a Superose 6 column. The fractions were analysed for the presence of Cdc53, Lag2 and Hrt1 by immunoblotting. The positions of marker proteins with known molecular weight (kDa) are indicated below. (B) Cdc53/Rbx1/Skp1 and Cdc53/Rbx1/Lag2 complexes expressed in E. coli and purified using the two-step affinity purification protocol were subjected to in vitro neddylation reactions as described in ‘Materials and methods’. The neddylation state of Cdc53 was visualized by immunoblotting with antibody against Cdc53 (upper panel). The presence of Skp1 (middle panel) or Lag2 (lower panel) was controlled with specific antibodies. Note that the presence of Lag2 but not Skp1 prevents Cdc53 neddylation in vitro. (C–E) Purified Cdc53–Hrt1 or Cul1–Rbx1 complexes were subjected to neddylation reactions as described in ‘Materials and methods’ in the presence of [32P]-Rub1, and analysed by autoradiography after the times indicated. Where indicated (+), the complexes were pre-incubated for 30 min with purified Lag2 or CAND1. The neddylation efficiency of Cdc53 (D) or Cul1 (E) was normalized by phosphorimager analysis from at least three experiments and plotted against time (min). Diamonds: no addition; squares: pre-incubation with Lag2; triangles: pre-incubation with CAND1. (F, G): The ability of purified wild-type (wt) Lag2 (squares), Lag2GN(551) (triangles) and Lag2DDYM(17) (circles) to inhibit neddylation of Cdc53–Hrt1 complexes was examined as described above. Pre-incubation with buffer alone (‘−’ in F; diamonds in G) controls for the efficiency of Cdc53 neddylation. Download figure Download PowerPoint To test whether Lag2 prevents cullin neddylation in vitro, we subjected purified 6xHis-Cdc53/GST-Hrt1/Lag2 complexes to neddylation reactions, and monitored Cdc53 modification by immunoblotting. For control, we included 6xHis-Cdc53/GST-Hrt1/Skp1 complexes, which we purified like the Lag2 complex after expression in E. coli from a poly-cistronic vector. Importantly, though the Cdc53/Hrt1/Skp1 complex was readily neddylated in vitro (Figure 3B), the Cdc53/Hrt1/Lag2 complex was refractory to this modification. To corroborate these results, we purified hCul1/hRbx1 and Cdc53/Hrt1 from baculovirus-infected insect cells (Supplementary Figure 3A), and subsequently monitored Cdc53/hCul1 neddylation using radioactive yeast Rub1 to visualize the conjugates by autoradiography. Titration of purified Lag2 to Cdc53/Hrt1 complexes showed that Lag2 directly binds to Cdc53 in nearly stoichiometric amounts (Supplementary Figure 3B). Although both hCul1/hRbx1 and Cdc53/Hrt1 complexes were efficiently modified by Rub1 (Figure 3C), pre-incubation of Cdc53/Hrt1 with recombinant Lag2 inhibited neddylation of Cdc53 in vitro (Figure 3C). Likewise, pre-incubation of hCul1/hRbx1 with hCand1 almost entirely prevented neddylation of hCul1. When quantified, the presence of Lag2 reduced Cdc53 neddylation to ∼ 20% of the amount detected in the absence of Lag2 (Figure 3D), whereas Cand1 almost entirely abolished neddylation of Cul1 (2%, Figure 3E). The molecular basis for this difference remains unclear, but it is possible that Lag2 requires additional not yet identified factors to fully inhibit cullin neddylation. Despite the large difference of Lag2 and Cand1 on primary sequence level, hCand1 was able to partially inhibit neddylation of ScCdc53 (40%). As expected, purified Lag2GN(551) and in particular Lag2DDYM(17) were defective to block Cdc53 neddylation compared to wild-type controls (Figure 3F and G), implying that efficient binding of Lag2 to the Cdc53/Hrt1 complex is necessary to prevent cullin neddylation in vitro. Lag2 is itself a substrate for neddylation The in vivo function of Lag2 requires an intact neddylation machinery, and the responsible neddylated substrate underlying this effect is thought to be the cullin. Interestingly, however, we noticed that a fraction of Lag2 migrated about 10 kDa slower on SDS–PAGE gels (Figure 4A), which is indicative of a covalent modification by ubiquitin or a ubiquitin-like protein (UBL). To determine whether Lag2 is indeed modified by a UBL, we examined Lag2 modification in yeast cells deleted for all non-essential UBLs. Interestingly, deletion of the yeast Nedd8 homologue Rub1 resulted in the loss of modified Lag2 (Figure 4A). Moreover, expression of an N-terminally HA-tagged version of Rub1 (HA-Rub1) in rub1Δ cells restored the appearance of modified Lag2 (Figure 4A), which as expected migrated slightly slower compared to untagged Rub1. Finally, in contrast to vector controls, modified Lag2 readily co-immunoprecipitated with HA-Rub1 (Figure 4B). Taken together, these data show that a fraction of Lag2 is neddylated in vivo. Figure 4.Lag2 bound to Cdc53 is neddylated in vivo. (A) Total cell extract prepared from wild type (wt) or rub1Δ cells transformed with either an empty HA-vector (HA) or a plasmid expressing HA-tagged Rub1 was analysed by immunoblotting with antibodies against Lag2 (upper panel) and Cdc53 (lower panel). The asterisk marks the position of a slower migrating Lag2 species, which is absent in rub1Δ cells and slightly upshifted in rub1Δ cells expressing HA-Rub1. (B) HA-immunoprecipitates (IP) from extracts prepared from rub1Δ cells transformed with either an empty HA-vector (HA) or a plasmid expressing HA-tagged Rub1 were immunoblotted for the presence of Lag2 (upper panel) or Cdc53 (lower panel) with specific antibodies. An aliquot of the extracts was loaded as input (sup). The asterisk marks the position of Lag2 modified by HA-Rub1. (C) Lag2 (upper panel) and Cdc53 (middle panel) neddylation were examined by immunoblotting of cell extracts prepared from cells lacking the indicated components of the neddylation pathway. An antibody specific for actin controls for equal loading (lower panel). The asterisk marks the position of a neddylated Lag2. (D) Extracts prepared from rub1Δ, lag2Δ or lag2Δ cells with integrated wild-type Lag2, Lag2GN(551) or Lag2DDYM(17) were immunoblotted with antibodies against Lag2 (upper panel) or Cdc53 (lower panel). The asterisk marks the position of neddylated Lag2. Download figure Download PowerPoint Similar to cullin neddylation, Lag2 neddylation required all known components of the Nedd8 conjugation machinery. Deletion of either the Nedd8 E2 Ubc12 or its E3 ligase Dcn1 resulted in a loss of both, Lag2 and Cdc53 neddylation (Figure 4C). However, though deletion of the de-neddylase Csn5/Rri1 shifted all of Cdc53 to its neddylated form, it prevented neddylation of Lag2 (Figure 4C). As Lag2 no longer binds to neddylated Cdc53, the opposite outcomes of Csn5/Rri1 deletion on the neddylation state of Cdc53 and Lag2 could be explained if Lag2 required prior binding to Cdc53 for neddylation. Indeed, Lag2 mutants (Lag2GN(551) and Lag2DDYM(17)) that lost the ability to bind to non-neddylated Cdc53 were no longer neddylated (Figure 4D), indicating that the interaction between Lag2 and Cdc53 is a pre-requisite for Lag2 neddylation. Antagonizing Lag2 function in vivo requires neddylation of Cdc53 but not Lag2 To determine the functional significance of Lag2 neddylation in vivo, we next sought to identify the neddylated lysine on Lag2. To achieve this goal, we systematically mutated all lysine residues to arginine, integrated the mutants into the yeast genome and subsequently determined the neddylation state of the mutated Lag2 by western blot analysis. Using this approach, we identified a single lysine, K16, which when mutated to arginine resulted in a loss of Lag2 neddylation (Figure 5A). Interestingly, K16 is located in close proximity to the site where Lag2, in analogy to Cand1, would interact with the cullin C-terminus (Figure 5B). The Lag2K16R mutant immunoprepitated Cdc53 as efficiently as wild-type controls (Figure 5C), excluding the possibility that the observed loss of Lag2 neddylation is simply caused by a defect in Cdc53 binding. Figure 5.Neddylation of Cdc53 but not Lag2 is necessary to overcome the inhibitory effects of Lag2 on SCF activity. (A) Extracts prepared from rub1Δ, lag2Δ or lag2Δ cells with integrated wild-type Lag2 or Lag2 with lysine 16 mutated to arginine (Lag2K16R) were immunoblotted with antibodies against Lag2 (upper panel) or Cdc53 (lower panel). The asterisk marks the position of neddylated Lag2. (B) Schematic representation of the Cdc53/Lag2 complex with the position of the lysine residues modified by Rub1 indicated (K16 in Lag2 and K760 in Cdc53). (C) Extracts from lag2Δ cells expressing wild-type Lag2 or Lag2K16R were immunoprecipitated with control IgG or Lag2-specific antibodies. Bound proteins were visualized by immunoblotting with Cdc53 (upper panel) and Lag2 (lower panel) antibodies. (D) Five-fold serial dilution of an equal number of wild type (wt) (upper plates) or rub1Δ (lower plates) cells transformed with an empty control plasmid (ev) or plasmids allowing overexpression of wild-type Lag2 (wt), Lag2GN(551) or Lag2K16R from the regulatable GAL1,10-promoter was spotted on media containing glucose (left, GAL-promoter off) or galactose (right, GAL-promoter on). The plates were photographed after 3 days at 30°C. (E) Five-fold serial dilution of an equal number of cdc53Δ cells complemented with wild-type Cdc53 or the neddylation-deficient Cdc53-K760R mutant transformed with an empty control plasmid (ev) or plasmids allowing overexpression of wild-type Lag2 or Lag2GN(551) from the regulatable GAL1,10-promoter was spotted on media containing glucose (left, GAL-promoter off) or galactose (right, GAL-promoter on). The plates were photographed after 3 days at 30°C. Note that neddylation of Cdc53 is important for cells to cope with increased levels of Lag2. Download figure Download PowerPoint To distinguish whether Lag2 and/or Cdc53 neddylation is important to regulate Lag2 function, we first overexpressed the non-neddylatable Lag2K16R mutant in wild type and rub1Δ cells. Similar to wild-type Lag2, Lag2K16R overexpression had no effect on wild-type cells, whereas" @default.
• W2074676459 created "2016-06-24" @default.
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• W2074676459 date "2009-11-26" @default.
• W2074676459 modified "2023-09-26" @default.
• W2074676459 title "Cullin neddylation and substrate-adaptors counteract SCF inhibition by the CAND1-like protein Lag2 in Saccharomyces cerevisiae" @default.
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• W2074676459 doi "https://doi.org/10.1038/emboj.2009.354" @default.
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