Isopeptidase: a Novel Eukaryotic Enzyme That Cleaves Isopeptide Bonds (Histone H2A/Ubiquitin/Chromatin/Carbon-Nitrogen Lyase/Cell Cycle) SEI-ICHI MATSUI*, AVERY A
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Proc. NatL Acad. Sci. USA Vol. 79, pp. 1535-1539, March 1982 Cell Biology Isopeptidase: A novel eukaryotic enzyme that cleaves isopeptide bonds (histone H2A/ubiquitin/chromatin/carbon-nitrogen lyase/cell cycle) SEI-ICHI MATSUI*, AVERY A. SANDBERG*t, SHIGERU NEGORO*, BEN K. SEON*, AND GIDEON GOLDSTEIN: *Roswell Park Memorial Institute, Buffalo, New York 14263; and tOrtho Pharmaceutical Corporation, Raritan, New Jersey 08869 Communicated by David Harker, November 20, 1981 ABSTRACT In an attempt to clarify the regulatory mech- MATERIALS AND METHODS anism that accounts for the shift ofprotein A24 in the mitotic cycle, we demonstrated the existence ofan enzyme, provisionally termed Cultured cell lines of a Chinese hamster (DON) and a mouse isopeptidase, that cleaves A24 stoichiometrically into histone H2A (L929) were maintained at 37°C in RPMI 1640 medium/8% fetal and ubiquitin. Properties of this enzyme are (i) most eukaryotes, calf serum (20, 21). E. coli (strain K-12, wild type) and yeast including mammals, amphibia, chicken, and yeast, contain iso- were gifts ofAllen Leonard and Joel Huberman ofour institute. peptidase in the cytoplasm; (ii) a significant increase in enzyme Metaphase chromosomes, nuclei, acid-soluble chromatin pro- binding to chromatin occurs when cells enter mitosis; (iii) Esche- teins, and A24 were prepared as described (13, 21, 22). '25I richia coli does not contain isopeptidase; (iv) isopeptidase has a Labeling ofA24 was carried out by the chloramin T method (23). molecular weight of 38,000; (v) at an ionic strength that induces To protect A24 molecules from radiation-caused disintegration, globular conformation of H2A, isopeptidase activity is repressed; the iodinated sample was immediately adjusted to a concentra- (vi) a SH group is an essential cofactor; and (vii) most divalent cat- tion of 18.7 nmol of A24/ml and A24-free calf thymus histone ions (except Mg2+ and Ca2+) are inhibitory. In view of the stoi- at 1 was chiometric conversion of A24 into H2A and ubiquitin by isopep- mg/ml added as carrier. 125I Stoichiometry, as deter- tidase in vitro, A24 probably contains a Gly-Gly dipeptide in mined by the isopeptidase reaction (see Fig. 1), was 6:1 (H2A/ isopeptide linkage but no other intervening polypeptides. Since ubiquitin). To avoid extreme modification of amino acid side ubiquitin in various eukaryotes binds to proteins other than H2A, chains, labeling of A24 by reductive methylation of a- and e- and is proteolytically released, isopeptidase probably acts on iso- NH2 groups (24), as adapted by others (25), was not used. peptide bonds in general and not uniquely on those of A24. In- For enzyme preparation, 0.5-2.0 g ofcells was washed once asmuch as isopeptidase is present throughout the cell cycle, the with 0.14 M NaCl and disrupted by using a glass/Teflon ho- level of A24 in chromatin appears to be controlled by a balance mogenizer in 2 to 3 vol of50 mM Na2HPO4/NaH2PO4, pH 7.5/ between isopeptidase and an as yet unestablished H2A-ubiquitin 10 mM MgCl2/2 mM EGTA/0.5 mM phenylmethylsulfonyl ligase. fluoride (BzlSO2F). The homogenate was centrifuged for 10 min at 45,000 X g in a Beckman 65 Ti rotor, and the supernatant Despite extensive advances in chromatin biochemistry (1-3), was used as an enzyme source. Intracellular localization of en- the molecular basis of the higher order organization of chro- zyme was determined as follows. The cells were homogenized matin remains unknown. Histone phosphorylation has been im- in buffer/0.25 M sucrose and centrifuged as above. The su- plicated in chromatin condensation in mitosis (4-9); however, pernatant was used as a cytoplasmic enzyme. The pellet was this idea has been questioned recently (10, 11). In attempts to suspended in 2.0 M sucrose and centrifuged over 2.2 M sucrose seek feasible mechanisms, we found another mitosis-specific at 40,000 X g for 60 min in a Beckman SW 41 rotor. This pellet event that involves a reversible stoichiometric shift in one ofthe was washed twice with 0.25 M sucrose, and then the nuclei were structural chromatin proteins, A24 (12-14), a conjugate of his- extracted at 40C with 0.35 M NaCV0.5 mM BzlSO2F. More tone H2A and the nonhistone protein ubiquitin (15-17). A24 than 75% of the nuclear nonhistone chromatin protein re- virtually disappears as a structural component of chromatin in covered in 0.35 M NaCl extracts was dialyzed against phosphate mitosis (12-14, 18) and, at the same time, a 10% increase in the buffer and used as a nuclear enzyme. The size of the enzyme H2A/DNA mass ratio occurs (12, 13). When cells revert to the molecule was estimated by 5-20% linear sucrose gradient cen- G, phase from mitosis and the chromatin becomes decon- trifugation and by Sephadex G-100 chromatography (26, 27). densed, A24 is reformed by conjugation ofthe core histone H2A The and preformed ubiquitin, apparently without new protein syn- standard assay mixture for the isopeptidase reaction con- thesis (13). tained (total vol, 20,ul) 100,umol ofTris-HCl (pH 8.5), 37.3 pmol In view of the reversible stoichiometric shift of A24 in the ofA24, 2 ,umol of2-mercaptoethanol (or dithiothreitol), 1 ,umol mitotic cycle ofchromatin organization, we predicted the pres- of BzlSO2F, and 0.5-10 ,Ag of enzyme. The reaction was ter- ence ofnovel enzymes that catalyze the interaction of H2A and minated by the addition of5 ,ul of0.5% NaDodSOJpolyacryl- ubiquitin (13, 19). In this article, we present evidence that eu- amide gel electrophoresis buffer. Under these conditions, the karyotes, but not prokaryotes such as Escherichia coli, contain reaction was linear for up to 15 min and proportional to the a cytoplasmic enzyme, termed isopeptidase, that cleaves A24 amount of enzyme. The reaction products were analyzed by into H2A and ubiquitin stoichiometrically in vitro. 15% NaDodSO4 or two-dimensional polyacrylamide gel elec- trophoresis (26, 28). The gels were sliced, the proteins were The publication costs ofthis article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertise- Abbreviation: BzlSO2F, phenylmethylsulfonyl fluoride. ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact. t To whom reprint requests should be addressed. 1535 Downloaded by guest on September 26, 2021 1536 Cell Biology: Matsui et al. Proc. Natd Acad. Sci. USA 79 (1982) quantitatively eluted in 50 mM phosphate buffer, pH 7.5/ strength (Fig. 7). (v) Free SH groups are essential cofactors 0.1% NaDodSO4, and the radioactivity was determined. For (Table 2). (vi) Except for Ca2+ and Mg2+, divalent cations are autoradiography, the stained gels were exposed to a Kodak X- potent inhibitors (Table 2). (vii) Core histones and H1 are not Omat AR film for 16-72 hr. cleaved by isopeptidase (Fig. 3). (viii) The optimal pH is 8.0-8.5 (Fig. 7). RESULTS Primary Structure of A24 Reassessed by Isopeptidase Re- action. Polyacrylamide gel electrophoresis of the isopeptidase Existence of Isopeptidase in Cell Extracts. Our prediction reaction products confirmed that the only components of A24 that the interaction ofH2A and ubiquitin is areversible dynamic are H2A and ubiquitin (Figs. 1 and 3). Because no component process requiring involvement of specific enzymes (18, 19, 29) corresponding to [3H]glycine or [3H]Gly-Gly was produced was tested by addressing three major areas-identification of with [3H]glycine-labeled A24 as substrate (Fig. 3 A and B), the enzyme reaction, intracellular localization, and enzyme level question arises as to whether a Gly-Gly dipeptide is an integral during the cell cycle. Initial attempts were focused on an en- element in the isopeptide bond, as initially proposed (30). This zyme(s) that cleaves A24 into H2A and ubiquitin. The results controversial point was studied. A24was purifiedfrom cells that obtained are summarized as follows. (i) A wide variety of eu- incorporated [3H]glycine for 6 generations to uniformly label karyotic cells from mammals, amphibia, birds, and yeast con- the entire molecule and then incubated with interphase cyto- tains an isopeptidase that cleaves A24 into H2A and ubiquitin plasmic proteins (Fig. 3C). Again, there was no radioactivity at in vitro (Fig. 1A). (ii) A prokaryote such as E. coli does not con- the ion front at which glycine and Gly-Gly would be located tain an isopeptidase (Fig. 1B). This was surprising, because (Fig. 3C). However, the H2A/ubiquitin radioactivity ratio was ubiquitin is present in E. coli (17), and its conjugate may well 2.0, in good agreeement with the ratio between the number of require isopeptidase for turnover. (iii) Isopeptidase attacks iso- glycine residues in H2A and ubiquitin terminating in Gly-Gly peptide bonds ofisolated A24, as well as those integrated in vivo (12:6) (13). as structural nucleosomal components (Figs. 2 and 3); therefore, the enzyme appears to have a very specific recognition site for DISCUSSION isopeptide bonds. (iv) Isopeptidase is localized mostly in the In this article, we report that various eukaryotic species contain cytoplasm (>99%) (Table 1) but it is unclear whether the re- an enzyme, tentatively referred to as isopeptidase, that cleaves sidual activity in nuclei reflects a real localization or merely an protein A24 into its components in vitro stoichiometrically. The absorbed portion during subcellular fractionation. (v) Isopep- failure ofA24 cleavage by E. coli extracts may indicate that iso- tidase is present not only in mitosis but also in interphase (Table peptidase is eukaryote specific. This enzyme selectively attacks 1). (vi) Cleavage ofA24 is stoichiometric; that is, neither inter- on isopeptide bonds between the COOH-terminal Gly-Gly di- vening peptides or Gly-Gly dipeptides, initially proposed to be peptides attached to ubiquitin and the e-NH2 residue oflysine- an integral component between H2A and ubiquitin (30), are 119 on A24 (Figs.