Ubiquitin — protein conjugates

U b i q u i t i n - protein conjugates Harris Busch and Ira L.Goldknopf Dept. of Pharmacology, Baylor College of Medicine, Houston, TX 77030, U.S.A. Summary The data available at present indicates there are three distinct functions of ubiquitin, two of which are related to protein conjugation. The first of these has been extensively studied by our laboratory and others interested in nucleosomes and changes in chromatin states. The ubiquitin-histone (Ub-2A, Ub-2B) conjugation reaction now appears to be a very dynamic process. In the deconjugation (lyase) reaction, both the histone 2A and the ubiquitin are left intact and in a form which makes possible ready reconjugation. Accordingly, this may be a mechanism for 'moment-to-moment' control of the genome. The second function in which ubiquitin is conjugated involves proteolytic activity. This activity is correlated with protein turnover. In this process, the ubiquitin-protein conjugate apparently serves as a 'signal' for the protease cleavage of the protein. The released ubiquitin is also intact and is probably available for reconjugation. In the third function, ubiquitin was suggested to serve as a 'hormone'. The studies thus far have been carried out primarily on induction of T- and B-lymphocytes, reduction or delay of Coombs' positivity and reduction of spleen weight. The precise physiological role of this reported function is still unclear, particularly because the ubiquitin used was probably not the physiologically active form. Ubiquitin structure The small polypeptide, ubiquitin (Ub), has a molecular weight of 8,565 and contains 76 amino acid residues. It was initially purified by G. Goldstein and his associates in the course of studies on peptides of the thymus. With the aid of a radioimmunoassay for this peptide, it became apparent that it. was widely distributed in plant, animal, yeast and bacterial cells (1). The sequence determined for amino acids 1-74 of thymus ubiquitin (2) was: 1 5 10 Met-Gln-Ile-Phe-Val-Lys-Thr-Leu-Thr-Gly-Lys15 20 Thr-Ile-Thr-Leu-Glu-Val-Glu-Pro-Ser-Asp-Thr- 25 30 Ile-Glu-Asn-Val-Lys-A1a-Lys-Ile-Gln-Asp-Lys35 40 Glu-Gly-Ile-Pro-Pro-Asp-Gln-Gln-Arg-Leu-Ile45 50 55 Phe-Ala-Gly-Lys-Gln-Leu-Glu-Asp-Gly-Arg-Thr60 65 Leu-Ser-Asp-Tyr-Asn-Ile-Gln-Lys-Glu-Ser-Thr70 74 75 76 Leu-His-Leu-Val-Leu-Arg-Leu-Arg-Gly-Gly Their sequence was notable for NH2-terminal methionine (the only one in the molecule) and the arginine at position 74. The sequence of ubiquitin was confirmed by Low and A. Goldstein (3) who independently studied the polypeptides of thymus and referred to this peptide as polypeptide B1. In Molecuiar and Cellular Biochemistry 40, 173-187 (1981). 0300-8177/81/0403 0173/$03.00. © 1981, Martinus Nijhoff/Dr W. Junk Publishers, The Hague. Printed in The Netherlands. 174 'active ubiquitin' the C-terminus is Arg-Gly-Gly76 (4-8). Preliminary analysis of the structure of the amino-terminal octapeptide of ubiquitin from celery showed that six of the eight residues were the same as for thymus ubiquitin. What is most notable about the sequence in relationship to the three-dimensional structure of ubiquitin is that this molecule is extremely resistant to tryptic digestion despite the presence of seven lysine residues and four arginine residues (2). It is likely that this structure is tightly coiled in such a way that the basic amino acids are unavailable to the active sites of trypsin, and other proteolytic enzymes. It was only after maleylation that the molecule was susceptible to the cleavages necessary for structural analysis. Studies with nuclear magnetic resonance accounted for the resistance to cleavage, i.e., ubiquitin has a highly globular, compact, pH and temperature resistant conformation requiring 7 M guanidine hydrochloride to denature it (9). On the basis of the prediction of protein confromation by Chou and Fasman (10), the NH2-terminal 15 residues (12%) were suggested to be a pleated sheet and the COOH-terminal (28%) was suggested to be a helical region (11). Its activity as a hormone is controversial (12) but this may reflect the absence or presence of the C-terminal Gly-Gly dipeptide. Increased interest in ubiquitin has been stimulated by studies indicating that a number of proteins contain it as all or part of their structures (Table 1). Ubiquitin-histone conjugates Ub-2A (protein A24) was first found in the group of 100 0.4 N H~SO4 soluble proteins of nucleoli and nuclei resolved by two-dimensional polyacrylamide gel electrophoresis (13, 14). The presence of ubiquitin in histone was unsuspected and only was found after purification (15) of Ub-2A (protein A24), peptide analysis (16) and amino acid sequencing procedures (17). We were very surprised to find that the protein had a 'Y-shape' with 2 N-terminals and 1 C-terminal amino acid (14). When the NH2-terminal sequence of Ub-2A (protein A24) was published (17), it was apparent (18) that this was the sequence of ubiquitin (Ub). It was established that ubiquitin was covalently linked to the histone 2A, specifically on the e-amine of lysine 119 (4). This remarkable structure has led to a series of investigations on similar structures in other proteins and on the function of such linkages. Two-dimensional polyacrylamide gel electrophoresis (13) (Fig. la) showed that Ub-2A (protein A24) migrated slightly slower than the histones in the urea-acetic acid first dimension and between histone H1 and H2B in the sodium dodecyl sulfatecontaining second dimension (19). Ub-2A (protein A24) was in acid extracts of whole nuclei (Fig. lb); its relatively similar amount in nuclear, nucleolar, and extranucleolar extracts indicated that Ub-2A (protein A24) was present throughout the cell nucleus (14). Table 1. Studies identifying various proteins as free or conjugated ubiquitin. Form and cellular localization Original nomenclature Isolation Structural identification Cytoplasmic free UBIP, ubiquitin Peptide of bovine Pars Intermedia APFI Goldstein, el al., 1975 (1) Seidah et al., 1978 (83) Ciechanover el al., 1978 (61) Ubiquitin H M G20 Nonhistone protein S A24 = H2A + Ub Goldknopf et Walker et al., Marushige & Goldknopfet uH2B = H2B = Ub West & Bonner, 1980 (48) a Schlesinger et al., 1975 (2) a Seidah et al., 1978 (83) b Hershko et al., 1980 (65) a Wilkinson et al., 1980 (62) a Golfknopf et al., 1978 (25) a Walker et al., 1978 (84) a Watson et al., 1978 (26) b Goldknopf & Busch, 1975 (16) b Goldknopf & Busch, 1977 (3) a Olson et al.. 1976 (17) d Hunt & Dayhoff, 1977 (18) a'UWest & Bonner, 1980 (48) Cytoplasmic free and conjugated Nuclear free Nuclear conjugated a Identified as ubiquitin b Identified as conjugated ubiquitin c Determination of amino terminal sequence d Identity to ubiquitin determined by amino acid sequence comparison al., 1978 (25) 1978 (84) Dixon, 1971 (85) aL, 1975 (15) 175 NORMAL RAT LIVER NUCLEOLAR PROTEINS RAT LIVER NUCLEOLAR PROTEINS 18 II 3 OG~,R 1 c a A REGENERATINGRAT LIVER B~3 % q b A2~ I I I NUCLEO[AR PROTE[NS Cb Bt 3 24-25 f BSL B2 b . . . . Fig. 1. Two-dimensional polyacrylamide gel electrophoresis of 0.4 N H2SO4-soluble proteins. Electrophoresis was from right to left in the first dimension in 10% acrylamide-4 M urea-0.9 acetic acid. The second dimension was from top to bottom in 1% SDS-0.1 M sodium phosphate (pH 7.1) 6 M urea. The histones are spots GAR (histone 4), A 1 (histone 2A), A2 (histone 2B), A3, 4 (histone 3), and A l 1, 17-19, (histone 1). Five hund red micrograms of protein were used for each gel. (a) and (d) from Ballal (22), (b) from Yeoman et aL (14); and (c) from Ballal et al. (21). Protein A24 is Ub-2A. Ub-2A was of physiological interest because its nucleolar content was markedly decreased in rat liver during thioacetamide administration and in regenerating liver after partial hepatectomy (20-22) (Fig. lc & d). Since these nucleoli have high levels of rRNA synthesis (23, 24), the decreased Ub-2A (protein A24) content as well as the presence of free ubiquitin in some active fractions (25-27), suggested that cleavage of Ub-2A (protein A24) might be related to increased gene activity. of Ub-2A (protein A24) (15) was calf thymus chromatin preextracted with 0.35 M NaC1 and 0.5 M perchloric acid (Fig. 2a). Ub-2A (protein A24) was in the 0.4 N H2SO4 extract along with other histones (Fig. 2a). Ub-2A (protein A24) was fractionated on Sephadex G-100 (Fig. 2b) and then on slab gels (Figs. 3 & 4); it migrated in two-dimensional polyacrylamide gel electrophoresis as a single component (Fig. 2c). The primary structure of Ub-2A (protein A24) Isolation of electrophoretically homogeneous Ub2A (protein A24) The starting material employed for purification Presence of histone 2A in Ub-2A (protein A24) The amino acid compositions of Ub-2A (protein ---,.I Fig. 2. (a) Proteins soluble in 0.4 N H2SO 4 obtained from rat liver chromatin after three 0.35 M NaCI extractions followed by two 5% perchloric acid extractions. (b) Sephadex G-100 column chromatography of the protein A24-enriehed acid-soluble proteins from calf thymus, The proteins in the fractions containing protein A24 and histones 2B and 3 (indicated by the shaded area of the graph) were used for preparative electrophoresis (Fig. 3). (c) Two-dimension gel electrophoresis of purified calf thymus protein A24 prepared in Fig. 2 and 3. 177 a b | Fig. 3. Purification of protein A24 by preparative electroph0resis. (a) An Amid o black-stained vertical side strip cut from a preparative 10% polyacrylamide slab gel after electrophoresis of the pooled fractions obtained from column chromatography on Sephadex G-100 (Fig. 2b). The position of protein A24 and histones 2B (A2) and 3 (A3-5, 7) are indicated. Such vertical strips from both sides and the center were used as a guide to cut out horizontal sections of the unstained remainder of the slabs which contained the protein A24 band. The protein A24 was then obtained by electrophoresis out of the gel sections into dialysis tubing. (b) Reelectrophoresis of purified protein A24 obtained in (a) on 10% polyacrylamide gels (15). A24) and histone 2A (Table 2) (16) showed their similar contents of lysine, histidine, aspartic acid, leucine, and phenylalanine. Ub-2A (protein A24) contained a lower molar ratio of arginine and alanine than histone 2A. The contents of threonine, glutamic acid, and proline were higher in Ub-2A (protein A24), and it had a higher acidic-basic amino acid molar ratio than histone 2A. Neither protein contained tryptophan. Histone 2A contained no methionine. The methionine residue was one amino terminal amino acid of Ub-2A (protein A24) as shown by dansylation. A comparison of the peptide maps of these proteins revealed striking similarities (16). The tryptic peptides of histone 2A (28) were present in the corresponding pattern of Ub-2A (protein A24). Similar results were obtained with chymotryptic peptides. Inasmuch as a) Ub-2A (protein A24) had a methionine NH2-terminus and histone 2A contains no methionine and b) Ub-2A (protein A24) had a molecular weight greater than histone 2A and had additional peptides not in histone 2A upon both tryptic and chymotryptic digestion, it seemed that Ub-2A (protein A24) contained the amino acid Table 2. Comparison of Ub-2A (protein A24) and histone 2A. Residue Protein A24 a (mole %) Histone 2A b (mole %) Trp Lys His Arg Asx Thr Ser Glx Pro Gly Ala Val Met lieu Leu Try Phe 0.0 11.3 2.4 7.4 7.3 6.5 4.5 12.3 5.6 9.2 9.6 4.9 0.3 5.8 10.9 1.3 0.9 0.0 10.9 3.1 9.3 6.2 3.9 3.1 9.3 3.9 10.9 13.4 6.2 0.0 4.7 12.4 2.3 0.8 Lys + His + Arg Glx + Asx Glx + A s x / L y s + His + Arg Dansylatable NHz-terminal 21.1 19.6 0.93 Methionine 23.3 15.5 0.67 Blocked (acetyl-Ser) Molecular weight 27 000 14 000 a Goldknopf et al. (1975) b Yeoman et al. 178 Calf Thymus NaCI-EDTA 2X Pellet Supt: NaC1-EDTA Soluble Proteins, Discard Pellet Supt: Tris Soluble Proteins, ~ Discard 0.35 M NaCI 3X Pellet Supt: H M G and L M G Proteins, Discard p c A 2x Pellet Pellet: DNA + R e s i d u a l Discard Proteins, Supt: Histone H1, Supt: Discard A24 and H i s t o n e s save Sephadex H2A, H2B, H3 and H4 G-100 A24 + H2B + H3 Preparative PAGE A24 Fig. 4. Flow sheetforpreparation ofprotein A24. sequence of histone 2A along with an additional amino acid sequence (16). The terminal amino acid sequences o f Ub-2A (protein A24) The peptide designated 16 in Ub-2A (protein A24) was not found by the ninhydrin-cadmium or fluorescamine procedures which require a primary amino group (Table 3); peptides 16 (4) f r o m histone 2A and Ub-2A (protein A24) (Table 3) had identical staining characteristics, amino acid composition, and positive tests for the acetyl group consistent with the structure of the blocked amino terminal tryptic peptide of histone 2A: N-acetyl-Ser-GlyArg. The fact that Ub-2A (protein A24) contained a second NH2-terminus , a methionine, suggested that it had an additional (non-histone) sequence. This unblocked sequence was amenable to automatic E d m a n degradation without prior separation of the two polypeptides. The sequence of the first 37 NH2-terminal residues (17) 5 10 Met-Gln-I!e-Phe-Val-Lys-Thr-Leu-Thr-Gly-Lys_ Table 3. Analysis of tryptic peptide 16 of protein A24 and histone 2A. Staining reactions Fluorescamine Ninhydrin Sakaguchi Rydon-Smith Amino acid ratios set (= 1.0) Gly Arg Molar yielda Acetyl group b Protein A24 Histone 2A + + + + 1.00 1.09 1.12 0.42 + 1.00 1.15 1.21 0.46 + a Nmoles recovered from tryptic peptide maps/nmole of protein digested. b Detected as acetyl hydrazide. 179 15 20 Thr-Ile-Thr-Leu-Glu-Val-Glu-Pro-Ser-Asp-Thr25 30 Ile-Glu-Asn-Val-Lys-Ala-Lys-Ile-Gln-Asp-Lys35 Glu-Gly-Ile-Prowas identical to the first 37 amino acids of ubiquitin (2, 18) as noted above. The known COOH-terminal amino acid sequence (29) and rate-limiting steps for carboxypeptidase B and A digestions of histone 2A were: electrophoretic mobility from peptide 17 of histone 2A (Fig. 5). The amino acid composition and carboxyl terminus of 17' of Ub-2A (protein A24) (Table 5) were the same as that of peptide 17 (residues 119-124) of histone 2A except for the presence of two additional glycine residues. The first cycle of Edman degradation of the i! -His-His-Lys-Ala-Lys-Gly-Lys-COOH A B The values in Table 4 obtained by equating the nanomoles oflysine to 1.0 after carboxypeptidase B and to 3.0 after carboxypeptidase A digestions were identical for both proteins (17). Ub-2A (protein A24) had the same COOH-terminal sequence as histone 2A. Quantitative hydrazinolysis indicated that lysine was the sole COOH-terminal amino acid of Ub-2A (protein A24), and its molar yield was identical to that of histone 2A (4). The branched tryptic peptide of Ub-2A (protein A24) The detection of two amino-termini and one carboxyl-terminus (4) led to the conclusions that the Ub-2A (protein A24) molecule was branched and that the ubiquitin was linked to histone 2A in a manner that prevented detection of its carboxylterminus. A search for an altered tryptic peptide of Ub-2A (protein A24) (4) showed tryptic peptide 17' from Ub-2A (protein A24) had a slightly different Fig. 5. Two-dimensional tryptic peptide maps of (a) histone 2A and (b) protein A24. Vertical dimension is chromatography and horizontal dimension is electrophoresis. Spots 1-16 are common to both proteins. Note the positions of peptides 16 which were negative to the ninhydrin-cadmium stain used in the maps. Note also the position of peptides 17 of histone 2A and 17' of protein A24, which differ slightly in electrophoretic mobility. Table 4. Ratios of free amino acids released from protein A24 and histone 2A digestion with carboxypeptidases A and B. Step 1a residues released from Step 2 b residues released from Amino acid Lysine Histidine Alanine Glycine Protein A24 Histone 2A Protein A24 Histone 2A 1.0 0.1 0.1 0.1 1.0 0.1 0.1 0.1 3.0 0.9 0.9 0.9 3.0 1.0 0.7 0.6 a Carboxypeptidase B: 60-min digestion followed by boiling for 5 min. b Carboxypeptidase A: After step 1, 60-min digestion. 180 Table 5. Amino acid ratios and carboxyl-termini of peptides 17 of histone 2A and 17' of protein A24. Gly Lys His Thr Ser (= 1.00) Glu COOH-terminusa Molar yieldc 17 17' 1.67 1.71 0.91 1.00 0.95 Lys (0.57)b 0.67 1.64 1.68 1.61 0.93 1.00 0.94 Lys (0.64)e 0.74 a Data obtained by hydrazinolysis, only lysine was found. b Data in parentheses are molar yield of carboxyl-terminal lysine. c Nmoles of peptide recovered from tryptic peptide maps/nmole of protein digested. Ub-2A (protein A24) peptide was consistent with a branched structure arising f r o m the additional two glycines being attached to the rest of the peptide in an isopeptide linkage to the e-amino group oflysine (cf below). Coupling with phenylisothiocyanate occurred at the c~-NH 2 groups of both lysine and glycine (4). Cyclization produced two thiazolinones, one containing glycine and one containing lysine with a glycine residue attached to its e-NH2 group. HI hydrolysis of these thiazolinones released two glycine residues and one lysine residue. In addition, conversion of the thiazolinones to phenylthiohydantions (PTH) produced sufficient P T H glycine to account for half of the glycine in the thiazolinones. Gas c h r o m a t o g r a p h i c analysis of phenylthiohydantions showed there was 0.9 nmole of PTH-glycine/ nmole of peptide, or approximately one-half of the a m o u n t obtained from hydrolysis of thiazolinones. These data and subsequent cycles of E d m a n degradation (4), which produced essentially identical results for both peptides (Table 6), supported the branched structure of this peptide and provided the first evidence for the Gly-Gly bond (Fig. 6). Trypsin treatment of Ub-2A (protein A24) yielded intact ubiquitin (4) as expected from the k n o w n resistance of ubiquitin to trypsin (2). The Arg-Gly-Gly linkage was cleaved under these conditions (4). Pretreatment of Ub-2A (protein A24) with the arginine blocking reagent 1,2-cyclohexanedione (30), followed by trypsinization produced (5) a trypsin-resistant polypeptide which included the branched peptide 17' linked to arginine 74 of the carboxyl-terminus of ubiquitin and the glycines of peptide 17'. Recently two-dimensional polyacrylamide gel electrophoretic mobility and a m i n o acid compositions of fragments of Ub-2A (protein A24) f r o m cleavage at tyrosines with N-bromosuccinimide accounted for the whole structure of U b - 2 A (protein A24) (6) as shown in Fig. 7. The novel bifunctional structure of Ub-2A (protein A24) immediateley raised questions about other bifunctional proteins. Other proteins contain two functional units; /3-1ipotropin has both A. Histone 2A peptide 17: Table 6. Sequential Edman degradation of peptides 17 and 17a. Lys-Thr-Glu-Ser-(His)2-Lys Edman cycle Amino acid Peptide 17 of histone 2A Glys Lys Thr Glu Ser Peptide 17' of protein A24 Gly Lys Thr Glu Ser 1 2 3 Edman cycles: 4 B. 0.5 1.0 0.9 0.3 1.6 0.4 Residue no. in Histo~e 2A: 0.8 0.3 a Nmoles of amino acids/nmoles of peptide released from thiazolinones after hydrolysis with HI. 2 3 4 comp.hydraz. 119 120 121 122 123-4 125 Protein A24 peptide 17': O H O . 4 II H2N-CH2-C-N-CH2-C-NH (Gly) (Gly) ] 1 Edman c y c l e s : 0.5 0.1 1.1 1 (~H2) 4 P H2N-CH- 6/-Thr-Glu-Ser - (His) 2 -Lys (Lys) 1 2 3 4 comp. h y d r a z . Fig. 6. Structure and outline of proof of structures of peptides (A) 17and (B) 17'. Note the identical sequence Lys-Thr-Glu-SerHis-His-Lys in both peptides and the position number of these amino acid residues in the histone 2A sequence. Comp., amino acid composition, Hydraz., hydrazinolysis. 181 Histone Ubiquitin Portion 2A Portion 1 5 acetylSer-Gly-Arg-Gly-Lys-Gln-Gly-Gly-Lys-Ala-Arg-Ala1 5 H2N-Met--Gln-lle-Phe-Val-Lys-Thr-Leu-Thr-Gly- i0 ii 15 Lys-Thr-Ile-Thr-Leu-Glu-Val-Glu-Pro-Ser- 20 21 25 Asp-Thr-Ile-Glu-Asn-Val-Lys-Ala-Lys-Ile- 30 31 35 Gln-Asp-Lys-Glu-Gly-lle-Pro-Pro-Asp-Gln- 40 41 45 Gln-Arg-Leu-Ile-Phe-Ala-Gly-Lys-Gln-Leu- 50 51 i0 15 20 Lys-Ala-Lys-Thr-Arg-Ser-Ser-Arg-Ala-Gly-Leu-Gln-Phe- 25 30 35 Pro-Val-Gly-Arg-Val-His-Arg-Leu-Leu-Arg-Lys-Gly-Asn40 45 Tyr-Ala-Glu-Arg-Val-Gly-Ala-Gly-gla-Pro-Val-Tyr-Leu 55 50 Ala- 55 60 65 Ala-Val-Leu-G1u-Tyr-Leu-Thr-Ala-Glu-Ile-Leu-Glu-Leu75 70 Ala-Gly-Asn-Ala-Ala-Arg-Asp-Asn-Lys-Lys-Thr-Arg-lle-lle- 60 80 85 Pro-Arg-His-Leu-Gln-Leu-Ala-Ile-Arg-Asn-Asp-Glu-G1u- G1u-Asp-Gly-Arg-Thr-Leu-Ser-Asp-Tyr-Asn- 61 65 Ile-Gln-Lys-Glu-Ser-Thr-Leu-His-Leu-Val- 90 70 75 I00 Leu-Asn-Lys-Leu-Leu-Gly-Lys-Va1-Thr-I1e-A1a-Gln-Oly- 105 0 71 Leu-grg-Leu-Arg-Gly-Gly 11 - C ( ~ . ~ . ~ ii0 115 Gly-Val L e u - P r o - A s n - I l e - G l n - A l a - V a l - L e u - L e u - P r o - L y s N H a 120 125 Lys-Thr -Glu-Ser-His-His-Lys-AI a-Lys-Gl 129 y-Lys-COOH • Fig. 7. The complete amino acid sequence of Ub-2A (protein A24) wherein the carboxyl terminal glycine 76 of ubiquitin is attached to the e-NH 2 of lysine 119 of histone 2A by an isopeptide linkage. melanocyte-stimulating hormone and/3-endorphin (31-34), but both units are within a single polypeptide chain. The linkage of the histone 2A and ubiquitin by an isopeptide suggested a post-translational conjugation. Isopeptide linkages have been found in collagen (35), fibrin (36, 37), peptidoglycans (38), wheat germ (39), bovine colostrum (40), hair medulla protein (41), chorion of rainbow trout (42), and E. coli ribosomal protein S l l (43). However, histones and nonhistone chromosomal proteins had not been shown to contain such a linkage previously and the ubiquitin-histone 2A bond was the first such conjugate demonstrated. In most systems studied, approximately 10% of histone 2A is in the form of protein A24 (15, 44, 45) and this quantitative distribution is the same for all the postsynthetic modification (46, 47) and amino acid sequence (44, 45) variants of histone 2A. Recently, a ubiquitin adduct of histone 2B, Ub-2B, has been described (48) in which 1-1.5% of histone 2B is conjugated in the carboxyl terminal portion with ubiquitin (48). This concentration of Ub-2B in the nucleus is approximately 1/10 that of Ub-2A (protein A24). Both sequence variants of histone 2B are conjugated to the same extent. C h r o m o s o m a l localization - the presence of U b - 2 A (protein A24) in nucleosomes The distribution of Ub-2A (protein A24) in chromatin (15) is illustrated in Figs. 8 and 9. Ub-2A (protein A24) and the histones were not detected in the saline-EDTA (0.075 M NaC1-0.025 M EDTA, pH 8.0) (Fig. 8b) or the Tris (Fig. 8c) washes, but were found in the 0.4 N H2SOn-soluble proteins of chromatin (Fig. 8d). Ub-2A (protein A24) and the histones were not solubilized when the chromatin was treated with 0.35 M NaC1 (Fig. 9a) (15), which extracted the high mobility group (HMG) and low mobility group (LMG) nonhistone chromosomal proteins (49). The 0.4 N H2SOa-soluble proteins of the chromatin residue contained Ub-2A (protein A24) and the histones (Fig. 9b). The difference in the solubility of Ub-2A (protein A24) and histone H1 (All, 17-19) was evident after the extraction from chromatin of histone H 1 with 0.6 M NaCI or with 0.5 M HC104. Under these conditions, Ub-2A (protein A24) and histones 2A, 2B, 3 and 4 were not extracted (Fig. 2a & 9d, respectively). Ub-2A (protein A24) accounted for 1.9% of the total of histones 1, 2A, 2B, 3 and 4. 182 a Fig. 8. Two-dimensionalpolyacrylamidegel electrophoretic analyses of the distribution of rat liver nuclear proteins during chromatin preparation. (a) 0.4 N H2SO4soluble nuclear proteins;(b) proteins solubilized in two washesof 0.075 M NaC1,0.025 M EDTA, pH 8.0, 1 mM PhCH2SO2F;(c) proteins solubilized in two washes of 0.01 M Tris, pH 8.0, I mM PhCH2SO2F;(d) 0.4 N HzSO4-solublechromatin proteins. When the histones and most nonhistone chromosomal proteins were extracted from chromatin with 3 M NaCI-7 M urea, Ub-2A (protein A24) was extracted (Fig. 9c). Ub-2A (protein A24) and the histones reassociated with the D N A by one-step or gradient analysis to low ionic strength. Ub-2A (protein A24) had similar binding characteristics to histones 2A, 2B, 3 and 4, although it was present in much lower amounts than histones. Thus, Ub-2A (protein A24) is solubilized from chromatin along with histones 2A, 2B, 3 and 4 (15). Two each of those histones compose the octameric globular nucleosome protein core particle (50). Ub-2A (protein A24) is found in a subset of nucleosomes where it replaces free histone 2A in the core octamers (51, 52, 44). The interactions between histone 2A and other core histones (50, 53, 54) and histone 1 (55-57) are apparently not disrupted by ubiquitination (44, 52, 55). It has been speculated that the presence of Ub-2A (protein A24) might affect the flexibility of adjacent nucleosomes (58, 59) inhibiting condensation of the chromatin (58-60). Interestingly Ub-2B has also been found in nucleosome cores (48). Apparently, free ubiquitin is not a D N A binding protein (86, 87, 1 l), although it has been found among the high mobility group proteins (84). Intracellular proteases conjugates and ubiquitin protein In early studies on factors involved in intra- 183 CHROMAT~N 3~MN~CI. 7M UREA SOLUBLE PROTEINS C~ATIN 025M NaCI SOLUBLE PROTEINS a POST 0,35M NaCI DNP 0.4N SOLUBLE ~ E I N S b d Fig, 9. Distribution of rat liver chromatin proteins during extraction with various solutions. Electrophoresis conditions were the same as in Figs. 1 and 2. (a) Proteins solubilized from chromatin by three extractions of 0.35 M NaC1, 0.01 M Tris, pH 8.0, 1 mM PhCH2SO2F; (b) 0.4 N H2SO4-soluble proteins of the residual deoxyribonucleoprotein after 0.25 M NaCI extractions. Note the presence of histone I (A 11, 1.7-19). (c) Proteins dissociated from chromatin in 3 M NaC1, 7 M urea, 0.05 M sodium acetate, pH 6.0, I mM PhCH2SO4F after the DNA was pelleted by centrifugation at 214 000 g for 24 h; (d) 0.4 N H2SO4-soluble proteins of the residual DNP after three (10 ml/g of nuclei) extractions of chromatin with 0.6 M NaCI, 0.01 M Tris, pH 8.0, 1 mM PhCH2SO2F. Note the markedly reduced amounts of histone 1 (spots Al 1, 17-19) compared to (b). cellular protease activities, Ciechanover et al. (61) found that degrad~/tion of globin was stimulated by ATP and that two polypeptides were involved, i.e., a protease and APF-I which was later shown to be ubiquitin (62). The reaction was dependent upon the concentration of ubiquitin. Ubiquitin conjugase Later Ciechanover et al. (63 66) found that ubiquitin formed conjugates by incubation with a reticulocyte fraction retained on DEAE-cellulose. The system required ATP and Mg ++ and was inhibited by N-ethylmaleimide suggesting an intermediary-SH linkage existed as with coenzyme A. The conjugates formed were stable on SDS-polyacrylamide gels and on Sephadex G-75. They resisted acid, alkali, heat, and reduction which led to the conclusion that they were covalently-linked structures. The proteolysis model showed that there are multiple ubiquitin conjugates of the protein substrates used, i.e. lysozymes, globin and lactalbumin. Like the ubiquitin linkage described for Ub-2A 184 (protein A24), these linkages are also isopeptides. Unlike these lifikages, the linkage in Ub-2A is very specific, i.e., only one special site is conjugated. The conjugase required ATP; other nucleoside triphosphates were much less effective. The enzyme that carries out the activation reaction exchanges pyrophosphate with ATP. The linkage was acid stable but was labile to alkali, hydroxylamine, borohydride and mercuric salts (66). Accordingly an SH group ofconjugase is essential for formation of the ester. The essence of the reaction (67) is: o II Ub-GlyCOOH+ ATPF"qUb-Gly-C-OAMP + PPi 76 76 O O II (4-8). Presumably, the loss of glycines in calf thymus ubiquitin resulted from partial proteolysis by a protease of uncertain specificity, although this has not yet been demonstrated. Whether this is a physiological phenomenon or specific processing is uncertain. Deconjugation o f ubiquitin The recently discovered lyase (69) which cleaves the Ub-2A (protein A24) to histone 2A and ubiquitin provides a ubiquitin which has two Cterminal glycine residues (7). The activity of this enzyme may be important in the turnover of ubiquitin which has recently become a topic of increased interest (see below). Ub-GIy-C-OAMP÷ HS-Eq ~Ub-GIy-C-S-E+ AMP 76 76 This activated ubiquitin then is transferred with the aid of other factors (68) to multiple e-NH 2 groups of lysines of proteins prior to degradation. C-terminal-Gly-Gly in active ubiquitin Wilkinson et al. (62) confirmed that ubiquitin formed the same conjugates in the reticulocyte system as were noted above. Inasmuch as there was some uncertainty about the precise amino acid of the Arg-Gly-Gly ubiquitin terminus in the formation of the isopeptide linkages, Hershko et al. (67) attempted to identify the ATP-activated terminal amino acid which they found to be glycine. The problem involved emerged from the sequence studies on calf thymus ubiquitin which has been found by Schlesinger et al. (2) and Low and A. Goldstein (3) to have a C-terminal-Arg-, However, in the studies from our laboratory (4-6), the linkage of ubiquitin to histone 2A was Arg-Gly-Gly. The question then was whether the Gly-Gly was added later or was an intermediate. In this connection, the studies of Watson et al. (26) indicated that in trout nuclear ubiquitin, some termini were Gly, and some Arg- as shown by hydrazinolysis. Hershko et al. (67) treated their ubiquitin-conjugase complexes with NaB3H4, hydrolyzed the product with acid and released ethanolamine derived from the active acid residue. Thus, they concluded that the carboxyl terminus of the activated ubiquitin was glycine. The active form of ubiquitin in protein conjugation probably has an Arg-Gly-Gly carboxyl-terminus Functions of ubiquitin conjugates of protein In the proteolysis system, one can visualize a role for the ubiquitin conjugate as a recognition site for the protease (65). Presumably ubiquitin, which is remarkably insensitive to proteases (2) constitutes either an allosteric binding site or a portion of the protein configuration necessary for the reaction site on the enzyme surface. However, there may be other, more complex possibilities. Exactly how cells identify proteins they no longer need or want and the precise chemistry of the conjugation reactions with respect to substrate site and the enzymes involved remain to be elucidated. It is clear that these reactions are different from those involved in the nucleosomes where the ubiquitin of Ub-2A (protein A24) turns over rapidly and at different times from the synthesis of the histones (70-71). Accordingly, it is likely that the eukaryotic cells have adapted a mechanism that may be involved in protein turnover to a use in chromatin packing, structure or function that does not involve proteolysis. The role of ubiquitin in the chromatin is much less clear. One problem that existed from the beginning of our studies was the fact that Ub-2A (protein A24) was decreased in nucleoli of cells stimulated for cell division (regenerating liver) or for hypertrophy of the nucleoli (thioacetamide treatment) (20-22). These results which were clearcut and readily reproducible were initially thought to relate to the phenomenon of increased rDNA 185 transcription. In the original Miller spreads (23), no nucleosomes were present along the rDNA segments which were being transcribed (88). The RNA polymerase I had supplanted the nucleosomal structures. The idea that Ub-2A (protein A24) was decreased on active ribosomal genes has been confirmed recently by Levinger et al. (72) who showed that they are deficient in Ub-2A (protein A24). In this case, the decrease of the ubiquitin-conjugates makes sense in relation to the displacement of nucleosomes from the rDNA. A decrease in Ub-2A (protein A24) was also found during repression of gene activity. Matsui et al. (60) reported that metaphase chromosomes which are essentially collapsed chromatin, contained no Ub-2A (protein A24) conjugates and hence, ubiquitin residues were lo~t. In addition, the relatively inactive mature chicken erythrocytes did not contain Ub-2A (protein A24) in significant amounts but in the related transcriptionally active 'reticulocytes' the Ub-2A (protein A24) adduct was present in higher concentration (73). Accordingly, the dichotomy existed that in the transcriptionally active chromatin, Ub-2A (protein A24) was present but that in the active states of the rDNA genes, this conjugate was absent. The question then was whether Ub-2A (protein A24) was present along active genes. Levinger et al. (72) showed that active gene regions other than rDNA contained Ub-2A (protein A24) and so did regions containing repetitive DNA. Accordingly, the Ub-2A (protein A24) complex would appear to be distributed rather broadly throughout the chromatin and may or may not be uniformly spaced in nucleosomes in extended chromatin. Barring the development of new evidence which would point to a more specific role for the Ub-2A (protein A24) adducts (and presumably for its other Ub-nucleosome adducts), it would seem that this conjugation would serve a structural role, possibly maintaining the chromatin in a state where it would be available for transcriptional reactions (59, 73) rather than the state of marked condensation as in active chromatin. Several lines of evidence indicate that the metabolism of the ubiquitin portion of Ub-2A (protein A24) is a function of nuclear activity. The ubiquitin portion of the molecule turns over throughout the interphase (70, 71). The rates of ubiquitin synthesis and conjugation into Ub-2A (protein A24) are parallel and drop to minimal levels during mitosis (70) when the Ub-2A (protein A24) is absent from metaphase chromosomes (60). Similarly, Ub-2A (protein A24) and free ubiquitin are lost from nuclei during chicken erythropoiesis (73) when transcription stops (74). Since the mechanisms of conjugation and deconjugation are specific and in progress throughout the cell cycle and in nondividing cells, one question is whether Ub-conjugation is a random or specific event and as noted above, it is still too early to draw conclusions about this point. An interesting recent finding (75) is that the cleavage of Ub-2A itself is tied to its formation. In the chicken reticulocyte, the cleavage of Ub-2A, measured as endogenous lyase activity is brisk, but in the mature, inactive erythrocyte, the lyase activity ceases while little Ub-2A (protein A24) is detected (73). Presumably the dynamic equilibrium between free ubiquitin and Ub-2A must be under rigid control. Marunouchi and his associates (76-78) have utilized an interesting mouse ts mutant which is defective in H1 histone phosphorylation. Under nonpremissive conditions (39 °) they found that Ub-2A (protein A24) disappears from the chromatin. 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