A novel site for ubiquitination: the N-terminal residue, and not internal lysines of MyoD, is essential for conjugation and degradation of the protein
1998; Springer Nature; Volume: 17; Issue: 20 Linguagem: Inglês
10.1093/emboj/17.20.5964
ISSN1460-2075
Autores Tópico(s)Autophagy in Disease and Therapy
ResumoArticle15 October 1998free access A novel site for ubiquitination: the N-terminal residue, and not internal lysines of MyoD, is essential for conjugation and degradation of the protein Kristin Breitschopf Kristin Breitschopf Department of Biochemistry and the Rappaport Family Institute for Research in the Medical Sciences, The Bruce Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, PO Box 9649, Haifa, 31096 Israel Search for more papers by this author Eyal Bengal Eyal Bengal Department of Biochemistry and the Rappaport Family Institute for Research in the Medical Sciences, The Bruce Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, PO Box 9649, Haifa, 31096 Israel Search for more papers by this author Tamar Ziv Tamar Ziv Protein Research Center, Faculty of Biology, Technion-Israel Institute of Technology, PO Box 9649, Haifa, 31096 Israel Search for more papers by this author Arie Admon Arie Admon Protein Research Center, Faculty of Biology, Technion-Israel Institute of Technology, PO Box 9649, Haifa, 31096 Israel Search for more papers by this author Aaron Ciechanover Corresponding Author Aaron Ciechanover Department of Biochemistry and the Rappaport Family Institute for Research in the Medical Sciences, The Bruce Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, PO Box 9649, Haifa, 31096 Israel Search for more papers by this author Kristin Breitschopf Kristin Breitschopf Department of Biochemistry and the Rappaport Family Institute for Research in the Medical Sciences, The Bruce Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, PO Box 9649, Haifa, 31096 Israel Search for more papers by this author Eyal Bengal Eyal Bengal Department of Biochemistry and the Rappaport Family Institute for Research in the Medical Sciences, The Bruce Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, PO Box 9649, Haifa, 31096 Israel Search for more papers by this author Tamar Ziv Tamar Ziv Protein Research Center, Faculty of Biology, Technion-Israel Institute of Technology, PO Box 9649, Haifa, 31096 Israel Search for more papers by this author Arie Admon Arie Admon Protein Research Center, Faculty of Biology, Technion-Israel Institute of Technology, PO Box 9649, Haifa, 31096 Israel Search for more papers by this author Aaron Ciechanover Corresponding Author Aaron Ciechanover Department of Biochemistry and the Rappaport Family Institute for Research in the Medical Sciences, The Bruce Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, PO Box 9649, Haifa, 31096 Israel Search for more papers by this author Author Information Kristin Breitschopf1, Eyal Bengal1, Tamar Ziv2, Arie Admon2 and Aaron Ciechanover 1 1Department of Biochemistry and the Rappaport Family Institute for Research in the Medical Sciences, The Bruce Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, PO Box 9649, Haifa, 31096 Israel 2Protein Research Center, Faculty of Biology, Technion-Israel Institute of Technology, PO Box 9649, Haifa, 31096 Israel *Corresponding author. E-mail: [email protected] The EMBO Journal (1998)17:5964-5973https://doi.org/10.1093/emboj/17.20.5964 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The ubiquitin proteolytic pathway is a major system for selective protein degradation in eukaryotic cells. One of the first steps in the degradation of a protein via this pathway involves selective modification of ϵ-NH2 groups of internal lysine residues by ubiquitination. To date, this amino group has been the only known target for ubiquitination. Here we report that the N-terminal residue of MyoD is sufficient and necessary for promotion of conjugation and subsequent degradation of the protein. Substitution of all lysine residues in the protein did not affect significantly its conjugation and degradation either in vivoor in vitro. In cells, degradation of the lysine-less protein is inhibited by the proteasome inhibitors MG132 and lactacystin. Inhibition is accompanied by accumulation of high molecular mass ubiquitinated forms of the modified MyoD. In striking contrast, wild-type MyoD, in which all the internal Lys residues have been retained but the N-terminus has been extended by fusion of a short peptide, is stable both in vivo and in vitro. In a cell-free system, ATP and multiple ubiquitination are essential for degradation of the lysine-less protein. Specific chemical modifications have yielded similar results. Selective blocking of the α-NH2 group of wild-type protein renders it stable, while modification of the internal Lys residues with preservation of the free N-terminal group left the protein susceptible to degradation. Our data suggest that conjugation of MyoD occurs via a novel modification involving attachment of ubiquitin to the N-terminal residue. The polyubiquitin chain is then synthesized on an internal Lys residue of the linearly attached first ubiquitin moiety. Introduction Ubiquitination of many proteins plays important roles in basic cellular processes. In most cases, the modification signals proteins for degradation by the 26S proteasome. Formation of ubiquitin conjugates of a specific protein requires the sequential action of three enzymes: the ubiquitin-activating enzyme, E1, one of several ubiquitin-carrier proteins (or ubiquitin-conjugating enzymes), E2s (or UBCs), and a member of the ubiquitin-protein ligase family, E3. E3s play an essential role in specific substrate recognition (Coux et al., 1996; Hochstrasser, 1996; Hershko and Ciechanover, 1998). The ubiquitin pathway is involved in processing and proteolysis of many cellular proteins including, for example, mitotic and G1 cyclins and their regulators, oncoproteins and tumor suppressors, transcriptional activators, cell surface receptors and ER membrane proteins, and MHC class I-restricted antigens. It also removes in a selective manner abnormal and mutated proteins. Proteins destined for degradation by the 26S proteasome are commonly modified by a multi-ubiquitin chain anchored to an internal ϵ-NH2 group of one or more lysine residues. As for specific recognition, the N-terminal domain may play a role in the targeting of a few proteins: in certain rare cases, the stability of a protein is a function of its N-terminal residue, which serves as a binding site for the E3 (‘N-end-rule’; Bachmair et al., 1986; reviewed in Varshavsky, 1996; Hershko and Ciechanover, 1998). For the Mos protein, it was found that its stability is governed primarily by the penultimate Pro residue and by a phosphorylation/dephosphorylation cycle of Ser3 (‘second codon rule’; Nishizawa et al., 1993). One interesting case involves the artificial fusion protein ubiquitin–Pro-β-galactosidase. In this chimera, the ubiquitin moiety has been fused to the N-terminal Pro residue of the protein. Unlike other ubiquitin–X-β-galactosidase species (where X is any of the remaining 19 amino acid residues), here ubiquitin is not removed by isopeptidases and serves as a degradation signal following generation of a polyubiquitin chain that is anchored to Lys48 of the fused ubiquitin moiety (Johnson et al., 1992). However, in this case the attachment of the ubiquitin moiety to the N-terminal residue is not the result of a natural, cellular enzyme-catalyzed, modification. There is no consensus as to the specificity of the internal Lys residues that are targeted by ubiquitin. In some cases distinct lysines are required, while in others there is little or no specificity. Signal-induced degradation of IκBα involves two particular Lys residues, 21 and 22 (Scherer et al., 1995). In the case of Gcn4, lysine residues in the vicinity of a specific PEST degradation signal serve as ubiquitin attachment sites (Kornitzer et al., 1994). Mapping of ubiquitination sites of the yeast iso-2-cytochrome c has revealed that the polyubiquitin chain is synthesized almost exclusively on a single lysine (Sokolik and Cohen, 1991). In two other examples, the proto-oncogene product Mos (Nishizawa et al., 1993) and the ‘N-end rule’ substrate X-β-gal (where X is a short fused peptide not encoded by the native molecule; Chau et al., 1989), one and two lysines, respectively, that reside in proximity to the degradation signal, are required for ubiquitination. In striking contrast, ubiquitination of the ζ chain of the T cell receptor is independent of any particular Lys residue and proceeds as long as one residue is present in the cytosolic tail of the molecule (Hou et al., 1994). Similarly, no single specific lysine residue is required for ubiquitination of c-Jun (Treier et al., 1994). The basic helix–loop–helix (bHLH) protein MyoD is a tissue-specific transcriptional activator that acts as a master switch for muscle development. It interacts with a consensus binding sequence in the enhancer and promoter regions of many genes in the differentiating muscle cell. Expression of the MyoD protein in a large number of cells is sufficient to initiate the myogenic program and to convert these cells into skeletal myoblasts (Davis et al., 1987; Weintraub et al., 1991). Additional members of the muscle-specific transcription factors include myogenin, Myf5 and MRF4 (Weintraub, 1993; Olson and Klein, 1994). MyoD forms heterodimers with other proteins belonging to the bHLH group, such as the ubiquitinously expressed E2A, E12 and E47 (Murre et al., 1989). These dimers are probably the transcriptionally active forms of the factor. Association of MyoD with HLH proteins of the Id family (inhibitors of differentiation that lack the basic domain) inhibits its DNA binding and biological activities (Benezra et al., 1990; Sun et al., 1991). MyoD is a short-lived protein with a half-life of ∼45 min (Thayer et al., 1989; Abu-Hatoum et al., 1998). Degradation of MyoD is mediated by the ubiquitin system both in vitro and in vivo. Furthermore, the process is regulated by its consensus DNA binding site. Addition of Id1 destabilizes the MyoD–E47–DNA complex and renders the protein susceptible to degradation (Abu-Hatoum et al., 1998). Here we show that degradation of MyoD by the ubiquitin system involves a novel mechanism. The NH2 group of the N-terminal residue of the protein, rather then internal lysine(s), serves as an essential and sufficient conjugation site necessary for subsequent degradation of the protein. Results A lysine-less MyoD is conjugated and degraded in an ATP- and ubiquitin-dependent manner in vitro We have shown that the rapid turnover of MyoD is mediated by the ubiquitin–proteolytic system in vitro and in vivo (Abu-Hatoum et al., 1998). To analyze specific ubiquitination sites, we used site-directed mutagenesis to substitute systematically all the lysine residues with arginines. MyoD contains nine lysine residues, most of them located within the N-terminal domain of the molecule. The nine residues are in positions 58, 99, 102, 104, 112, 124, 133, 146 and 241. The various proteins were generated either by expression in bacteria followed by subsequent purification, or by in vitro translation in a eukaryotic system using rabbit reticulocyte lysate and [35S]methionine. Degradation of the proteins was monitored in a reconstituted cell-free system containing, in most cases, ubiquitin-supplemented Fraction II and ATP. Proteins were detected by Western blot analysis or PhosphorImaging. Figure 1A shows that progressive substitution of lysine residues (lanes 3–6) does not affect the efficiency of degradation of a bacterially expressed protein (compare with lanes 1 and 2). Surprisingly, even a MyoD species that lacks all Lys residues is still degraded efficiently in an ATP-dependent manner (lanes 7 and 8). Similar results were obtained using a lysine-less protein translated in vitro in a eukaryotic system (Figure 1B). To demonstrate involvement of the ubiquitin system in the process, we followed the degradation of wild-type (wt) and lysine-less MyoD in the absence and presence of ubiquitin (Figure 1C and D). Like the degradation of the wt protein (Figure 1C), degradation of the lysine-less MyoD is completely dependent upon the addition of ubiquitin (compare lanes 2 and 5). Furthermore, addition of methylated ubiquitin that cannot form polyubiquitin chains and serves as a chain terminator (Hershko and Heller, 1985), inhibits degradation of lysine-less MyoD. The inhibition can be alleviated by the addition of an excess of free ubiquitin (Figure 1C and 1D, compare lanes 3 and 4). The data strongly suggest that polyubiquitination of lysine-less MyoD is necessary for degradation of the protein. Furthermore, they imply that the modification occurs on internal Lys residues of ubiquitin. To demonstrate directly polyubiquitinated lysine-less MyoD, we used in-vitro-translated 35S-labeled protein in a partially reconstituted system. As can be seen in Figure 2, lysine-less MyoD generates high-molecular-mass ubiquitinated adducts. It should be noted that these conjugates are of somewhat lower molecular mass than those of the wt MyoD. This can be attributed to the role that the internal lysine residues also play in the process (see also below). Figure 1.ATP- and ubiquitin-dependent degradation of wt and mutated MyoD proteins in a cell-free reconstituted system. (A) Western blot analysis of ATP-dependent degradation of bacterially expressed purified MyoD. Lanes 1 and 2, wt MyoD (wt); lanes 3 and 4, a MyoD protein in which the last four C-terminal lysines were left (4K); lanes 5 and 6, a MyoD protein in which only the last C-terminal lysine was left (1K); and lanes 7 and 8, lysine-less MyoD (0K). (B) ATP-dependent degradation of 35S-labeled wt (lanes 1 and 2) and lysine-less (lanes 3 and 4) MyoD. (C and D) Polyubiquitination is required for the degradation of wt (wt; C) and lysine-less (0K; D) MyoD. Ubiquitin and methylated ubiquitin (MeUb) were added at the indicated concentrations. Reactions were carried out in the presence of ubiquitin and Fraction II in the absence or presence of ATP as indicated and as described in Materials and methods. Detection of the proteins was carried out using Western blot analysis (A, C and D) or PhosphorImager analysis (B). Ub denotes ubiquitin and MeUb denotes methylated ubiquitin. 35S-MyoD denotes 35S-labeled MyoD. Download figure Download PowerPoint Figure 2.In vitro conjugation of wt and lysine-less (0K) MyoD. 35S-labeled proteins translated in reticulocyte lysate in vitro were incubated in a cell-free reconstituted system containing ubiquitin, ubiquitin aldehyde and Fraction II as described in Materials and methods. Notes are as described in the legend to Figure 1. The weak band seen above the main protein band of translated MyoD is, most probably, the phosphorylated form of the protein left over from the biosynthetic step. We do not see this band in the bacterially expressed protein, as the appropriate kinase is not expressed in E.coli. This band collapses following treatment with alkaline phosphatase (Abu-Hatoum et al., 1998). Download figure Download PowerPoint Ubiquitin-mediated degradation of MyoD in vivo can proceed efficiently in the absence of internal lysine residues To investigate the physiological relevance of the observations in the cell-free system, we followed the fate of the different MyoD lysine mutated proteins in vivo. Figure 3 shows a series of pulse–chase experiments in COS-7 cells transiently transfected with the different MyoD cDNAs, in which we examined the stability of the different proteins. In agreement with our in vitro data, the lysine-less MyoD protein is degraded efficiently. However, we could observe a progressive increase in the half-life of the proteins of up to ∼2-fold with the gradual substitution of the lysine residues (Figure 3A and B). While the half-life of wt MyoD was ∼50 min, that of lysine-less MyoD was ∼2 h (Figure 4A and B). Interestingly, we found that the stability of MyoD is not affected by any specific lysine residue, and it is the total number of these residues that determines the half-life of the protein (data not shown). To identify the system involved in the proteolysis of lysine-less MyoD in vivo, transfected cells were incubated in the presence of inhibitors of the proteasome and of lysosomal degradation. Chloroquine, a general inhibitor of lysosomal proteolysis, and E-64, a cysteine protease inhibitor that affects lysosomal, and also certain cytosolic, proteases, had no effect on the stability of the lysine-less MyoD (Figure 5A, lanes 8–11, and Figure 5B). In striking contrast, the proteasomal inhibitors clasto-lactacystin β-lactone and MG132 block degradation of the lysine-less protein significantly (Figure 5A, lanes 4–7, and Figure 5B). Figure 3.In vivo degradation of MyoD-mutated proteins in which the lysine residues were progressively substituted with Arg. (A) COS-7 cells, transiently transfected with MyoD cDNA, were pulse labeled (1 h) with [35S]methionine and chased (2 h) as described in Materials and methods. Labeled MyoD was immunoprecipitated from aliquots containing equal amounts of labeled proteins as described in Materials and methods. Lanes 1 and 2, wt MyoD (wt); lanes 3 and 4, a MyoD protein in which the last seven C-terminal lysines were left (7K); lanes 5 and 6, a MyoD protein in which the last four lysines were left (4K); lanes 7 and 8, a MyoD protein in which the last three lysines were left (3K); lanes 9 and 10, same as before, but with only the last C-terminal lysine left (1K); lanes 11 and 12, lysine-less MyoD protein (0K). (B) Quantitative (PhosphorImaging) analysis of the degradation data of the different MyoD proteins following a chase period of 2 h. The number of remaining Lys residues in the different MyoD proteins was plotted against the amount of residual protein following the chase period. Download figure Download PowerPoint Figure 4.Time course of degradation of wt and lysine-less MyoD in vivo. (A) The half-lives of wt (wt) and lysine-less (0K) MyoDs in COS-7 cells were determined in a pulse–chase labeling experiment as described in Materials and methods and in the legend to Figure 3. (B) Quantitative analysis of the data depicted in (A). Filled diamonds denote wt and filled circles denote lysine-less MyoD. Download figure Download PowerPoint Figure 5.Sensitivity of lysine-less MyoD (0K) degradation to different inhibitors. (A) Degradation of lysine-less MyoD in COS-7 cells was monitored in a pulse–chase experiment in the absence (lanes 2 and 3) and presence of the proteasome inhibitors clasto-lactacystin β-lactone (lanes 4 and 5) and MG132 (lanes 6 and 7), the general inhibitor of lysosomal proteolysis chloroquine (lanes 8 and 9) and the lysosomal cysteine protease inhibitor E-64 (lanes 10 and 11). Cell lysis and immunoprecipitation were carried out as described in Materials and methods and in the legends to Figures 3 and 4. Lane 1 presents mock-transfected COS-7 cells. 35S-MyoD denotes immunoprecipitated and 35S-labeled MyoD protein. (B) Quantitative analysis of the data depicted in (A) after a chase period of 2 h in the absence and presence of the different inhibitors. Quantities are relative to the amount of protein at time 0. Download figure Download PowerPoint To demonstrate the intermediacy of ubiquitin conjugates in the degradation of lysine-less MyoD, we incubated COS-7 cells, transiently transfected with either wt or lysine-less MyoD cDNAs, with MG132, and followed generation of ubiquitin–MyoD adducts. As can be seen in Figure 6A and B, immunoprecipitation with anti-MyoD antibody followed by Western blot analysis with anti-ubiquitin antibody reveals accumulation of high-molecular-mass compounds in cells transfected with either wt (A) or lysine-less (B) MyoD. Re-probing of the stripped nitrocellulose membrane with anti-MyoD antibody reveals a similar pattern of conjugates of the lysine-less protein (Figure 6C, lane 3). Similar analyses of mock-transfected cells (Figure 6, lanes 1 in all panels) clearly demonstrate the specificity of both the anti-MyoD and anti-ubiquitin antibodies. Figure 6.Detection of ubiquitin–MyoD conjugates in COS-7 cells. COS-7 cells were transiently transfected with 10 μg of expression vector containing either wt (A) or lysine-less MyoD (B and C) cDNA. Following 48 h of transfection, cells were incubated for additional 2 h in the presence or absence of MG132. Following lysis, equal amounts of protein, as determined by the Bradford (1976) method, were subjected to immunoprecipitation with anti-MyoD antibody and ubiquitin conjugates were identified using Western blot analysis and anti-ubiquitin antibody (A and B) or anti-MyoD antibody (C) as described in Materials and methods. (A) wt MyoD was precipitated with a specific antibody and conjugates were identified on the blotted nitrocellulose membrane using anti-ubiquitin antibody. Lane 1: mock-transfected cells. The experiment was performed in the presence of MG132. Lane 2: cells transfected with wt MyoD; the protein was detected in the absence of MG132. Lane 3: same for lane 2, except that the experiment was performed in the presence of MG132. (B) Conjugates of lysine-less MyoD. The experiment was performed, reaction mixtures resolved and data presented in an identical manner as in (A). (C) Conjugates of lysine-less MyoD as detected with anti-MyoD antibody. Following stripping of the nitrocellulose membrane (B), proteins were re-detected with anti-MyoD antibody. Details are as described for (B). Conj. denotes conjugates and Ig indicates the heavy chain of the Ig molecule. The band detected at 57 kDa (A and B) has not been identified. Download figure Download PowerPoint Is a free NH2 terminus of MyoD targeted by ubiquitin and essential for degradation of the protein? Based on the results described above, it was clear that polyubiquitination is essential for targeting MyoD for degradation. The lack of internal Lys residues, the only known targets for ubiquitin modification, made it important to identify the functional group that can serve as an attachment site for ubiquitin. Chemically, several groups can generate covalent bonds with ubiquitin. Ser and Thr can participate in ester bond formation, while Cys can generate a thiol ester bond. However, these bonds are unstable. The stability of the adducts makes it highly unlikely that any of these modifications is the one we observe. A likely candidate is the free amino group of the N-terminal residue of the protein which can generate a stable peptide bond with the C-terminal Gly residue of ubiquitin. Edman degradation of the N-terminal residue has demonstrated that the N-terminal residue of bacterially expressed MyoD as well as that of the in-vitro-translated and the cellularly expressed proteins is the free, unmodified initiator Met. The proteins are not modified and the N-terminal residue is not acetylated (see also Materials and methods). As can be seen in Figure 7A (lanes 7–9), blocking of the α-NH2group, which is the only free amino group left in the molecule, stabilizes the protein completely (compare with lanes 4–6). Figure 7.Selective modification of the α-amino group of the initiator methionine in lysine-less and wt MyoD results in a stable protein that is not conjugated and degraded in vitro. (A) Ubiquitin-and ATP-dependent degradation of the various MyoD derivatives was monitored in a reconstituted cell-free system via Western blot analysis as described in Materials and methods. Lanes 1–6: ATP- and ubiquitin-dependent degradation of bacterially expressed wt (wt) and lysine-less (0K) MyoD. Lanes 7–9: the α-amino group of lysine-less MyoD was methylated (0K-CH3). Lanes 10–12: the α-amino group of wt MyoD was selectively modified by carbamylation (NH2CO-wt). Lanes 13–15: the ϵ-NH2 groups of internal Lys residues of wt MyoD were selectively modified by guanidination (wt-Gd). Lanes 16–18: a lysine-less MyoD protein containing CGC (see Discussion) as a codon for Arg (0K*). (B) In vitro conjugation of wt (wt), lysine-less (0K*) and carbamylated (wt-NH2CO) MyoDs. Equal amounts of bacterially expressed MyoD proteins were incubated in a cell-free reconstituted system containing ubiquitin, ubiquitin aldehyde and Fraction II as described in Materials and methods. MyoD proteins were immunoprecipitated with anti-MyoD antibody and ubiquitin conjugates were identified using Western blot analysis as described in Materials and methods. Lanes 1 and 2: wt MyoD (wt). Lanes 3 and 4: bacterially expressed, lysine-less MyoD protein containing CGC as codon for Arg (0K*). Lanes 5 and 6: carbamylated wt MyoD (wt-NH2CO). Conj. denotes conjugates. (C) Sensitivity of degradation of wt and lysine-less MyoDs to RNase A and ‘N-end rule’ inhibitory peptides. Degradation of wt (wt) and lysine-less (0K*) MyoD was monitored in ubiquitin-supplemented Fraction II as described in Materials and methods. Ribonclease A (lanes 1–8) and one of the dipeptides Arg-Ala (+A; lanes 9, 10, 13 and 14) or Phe-Ala (+B; lanes 11, 12, 15 and 16) was added to the reaction mixtures as indicated and as described in Materials and methods. Download figure Download PowerPoint Whereas in the lysine-less MyoD, a free α-NH2 group appears to be sufficient for ubiquitination and subsequent degradation, it is not clear whether it also plays a physiological role in targeting the wt molecule, which has nine available lysine residues. In order to investigate the role and biological relevance of the free α-NH2 group in the targeting of wt MyoD, we selectively blocked it by carbamylation with potassium cyanate at low pH. This procedure does not modify ϵ-NH2 groups of internal lysine residues (Hershko et al., 1984). Automated Edman degradation along with fluorescamine determination of the extent of remaining free NH2 groups confirmed accurately that the modification affected only the N-terminal group (see Materials and methods). The modified protein was subjected to in vitro degradation and conjugation assays in fractionated reticulocyte lysate. In contrast to lysine-less MyoD, the N-terminally carbamylated protein is stable (compare Figure 7A, lanes 10–12, with lanes 1–6 and 16–18), and cannot be ubiquitinated (compare Figure 7B, lanes 5–6 with lanes 1–4). Thus, a free and exposed NH2 terminus of MyoD appears to be an essential site for degradation, most probably because it serves as an attachment site for the first ubiquitin moiety. As an additional control, we selectively modified the internal Lys residues of wt MyoD by guanidination with O-methylisourea. The modification, which does not affect the N-terminal group (see Materials and methods), generates a protein that is essentially the chemically modified counterpart of the lysine-less MyoD that was generated by site-directed mutagenesis. As can be seen in Figure 7A (lanes 13–15), and similar to the lysine-less protein (Figure 7A, lanes 4–6 and 16–18), this MyoD protein is degraded efficiently in the cell-free system in a ubiquitin- and an ATP-dependent mode. Proteins with acidic NH2-terminal residues are degraded by the ubiquitin system only following conversion of the acidic residue to a basic residue by the addition of an arginine moiety. The reaction utilizes charged tRNAArg for arginylation, and destruction of the tRNA by ribonuclease treatment inhibits degradation of acidic N-termini proteins (Ferber and Ciechanover, 1987). While the N-terminal residue of MyoD is Met and not Asp, Glu, Asn or Gln, and an N-terminal Lys transferase (unlike Arg transferase) has not been described, a formal possibility still existed that the N-terminal residue is modified by lysine, that is then targeted via modification of its ϵ-NH2 group. While such ‘lysinylation’ would have been interesting in itself, we ruled it out by showing that RNase A does not affect the degradation of either the wt or the lysine-less MyoD (Figure 7C, lanes 1–8). As mentioned above, sequencing of the N-terminal residue of the bacterially expressed, the in-vitro translated, and the metabolically labeled, immunoprecipitated, eukaryotic-cell-expressed MyoDs revealed that in all three cases the N-terminal initiator is Met. According to the N-end rule, methionine is a ‘stabilizing’ residue. Furthermore, while according to the ‘N-end rule’, recognition of the protein is mediated via binding of its N-terminal residue to the E3 enzyme while ubiquitination occurs on internal Lys residues, here we demonstrated clearly that the N-terminal group of MyoD is targeted by ubiquitin. Thus, for these two reasons it is clear that targeting of MyoD cannot possibly traverse the ‘N-end rule’ pathway. To rule out the even more remote possibility that the initiator Met is removed and a ‘destabilizing’ basic or acidic residue is exposed that targets the protein for ‘N-end rule’-mediated conjugation, we tested the effect of two ‘N-end rule’ inhibitory peptides, Arg-Ala and Phe-Ala (Reiss et al., 1988) on the degradation of MyoD in vitro. These peptides had no effect on the degradation of the wt and lysine-less proteins (Figure 7C, lanes 9–16). As a preliminary analysis the role of the free N-terminal residue of MyoD in targeting of the protein in vivo, we added a myc tag to the N-terminal residue of the wt protein. As can be seen in Figure 8, the myc-tagged protein is stable both in vitro and in vivo. It should be noted that the two first N-terminal residues of the myc tag, Met and Glu, are identical to the first two N-terminal residues in MyoD. In addition, the myc tag contains also a Lys residue. Nevertheless, the myc tag cannot promote degradat
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