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Purification of Chlamydomonas 28-kDa ubiquitinated protein and its identification as ubiquitinated histone H2B
ARCHIVES
OF BIOCHEMISTRY
AND BIOPHYSICS
Vol. 294, No. 1, April, pp. 193-199, 1992
Purification of Chlamydomonas 28-kDa Ubiquitinated Protein and Its Identification as Ubiquitinated Histone H2B Kousuke Shimogawara’ Institute
and Shoshi Muto
of Applied Microbiology,
University
of Tokyo, Yayoi, Bunkyo-ky
Received September 13, 1991, and in revised form November
12,199l
One of the most predominantly ubiquitinated protein species in Chlamydomonas, of which the apparent molecular mass in SDS-PAGE was 28 kDa, was found to exist abundantly in nuclei. The 2%kDa ubiquitinated protein was purified to homogeneity from the isolated nuclei of Chlam ydomonas, and its partial amino acid sequence was determined. The N-terminal peptide sequence was identical with that of ubiquitin. Sequences homologous to those oat ubiquitin and wheat histone B2B, and paired sequences of both of them were found in arginylendopeptidase-digested or protease VS-digested polypeptide fragments of the 2%kDa ubiquitinated protein. Based on these results, it was concluded that Chlamydomonaa 28-kDa ubiquitinated protein is monoubiquitinated histone H2B. o 1992 AC,AIGO press, IXW.
Ubiquitin is a small polypeptide with a molecular mass of 8.5 kDa and is composed of 76 amino acid residues. As the name implies, it has been detected ubiquitously in all eukaryotes so far examined. Since the amino acid sequence of ubiquitin shows remarkable evolutional conservation, this protein is believed to be involved in essential life processes in all eukaryotes. Ubiquitin is known to attach to other specific proteins by isopeptidyl linkage between the carboxyl terminus of ubiquitin and the tamino groups of internal lysyl residues in the target proteins. The role of ubiquitination in cytosolic protein degradation by ATP-dependent proteinase has been well elucidated (for reviews, see (1, 2)). There are several proteins ubiquitinated independently of their own degradation. The best elucidated example is histone ubiquitination (3). Ubiquitin conjugates with his1 Present address: Laboratory of Chemistry, School of Medicine, Teikyo University, Ohtsuka, Hachioji, Tokyo 192-03, Japan. ’ To whom correspondence should be addressed 0003-9Sfx/92 $3.00 Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
Tokyo 113, Japan
tone H2A and H2B in the nuclei, forming uH2A (previously termed A24) and uH2B semihistone, respectively. The degree of ubiquitination of histone varies from species to species and from tissue to tissue. For example, the degree of ubiquitination of H2A is 1, 7, 8-10, 10, and 1214% of total H2A in Chirorwmus tenatans (4), slime molds (5), cultured hamster cells (6), mouse cells (7, 8), and terminally differentiated rat cerebral cortex neurons (9), respectively. The degree of ubiquitination of H2B is 1-2, l-2, and 6% of total H2B in mouse cells, rat cerebral cortex neurons, and slime molds, respectively (refs. cited above). The degree of ubiquitination of histones is also variable depending on the physiological conditions. The ubiquitination of histone is assumed to cause some changes on the nucleosome structure, resulting in changes of the transcriptional activities of specific genes (10-14). There are some reports suggesting the conjugation of ubiquitin to certain proteins, including cell surface and extracellular proteins (15-18). These ubiquitinations are believed to have certain physiological functions like in other protein modifications such as phosphorylation and adenylation. In our previous study (19), we found two remarkably ubiquitinated protein species in Chlamydamonas, of which apparent molecular masses in SDS-PAGE3 were 28 and 31 kDa. The isoelectric points of the 28- and 31-kDa ubiquitinated proteins were strongly basic, i.e., 8.9 and 10.3, respectively. The amounts of these ubiquitinated proteins were found to change in response to heat and active oxygen stress, suggesting that the ubiquitination of these proteins plays some roles in adaptation of cells to stress condition (19, 20). The amounts of them also changed during cell cycle and gamete induction (21). However, the 3 Abbreviations used: HPLC, high-performance liquid chromatography; PVDF, polyvinylidene difluoride; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; SH, sulfhydryl; TFA, trifluoroacetic acid, Mes, 4-morpholineethanesulfonic acid. 193
194
SHIMOGAWARA AND MUTO
significance of these changes of uhiquitination still remains unclear. For clarification of this, it is beneficial to identify these ubiquitinated proteins. Here, we report identification of the 2%kDa ubiquitinated protein as histone H2B based on its intracellular distribution and ammo acid sequence. MATERIALS
AND METHODS
Isolation of Chlnmydomonas nuclei.
To overcome difficulties in isolating the nuclei from wild-type cells, cells of the wall-less strain Chlumydonwnus reinhurdtii CW-15 (mating type -) were used. The nuclei were isolated according to Keller et al. (22) with partial modifications as described below. Chlumydomonns was cultured mixotrophically with acetate as the carbon source at 30°C with gentle shaking under continuous illumination with fluorescent light as described elsewhere (23). Cells grown up to ca. 0.1% (packed cell volume per volume) were harvested by continuous centrifugation at 23,000g at 4°C. The following procedures were done at ice-cold temperature. The cell pellets were resuspended in a fivefold volume of a medium containing 20 mM HepesKOH (pH 7.6), 10 mM CaClx, 10 mM MgCl,, 0.6 M sucrose, 2.5 mM spermidine, 0.75 mM spermine, 2 mM dithiothreitol, 5 mM diisopropylfluorophosphate, and 0.5 mM phenylmethylsulfonyl fluoride. One part of 20% (v/v) Triton X-100 was added to twenty parts of the cell suspension and the mixture was kept for few minutes to lyse the cell wall. Lysis was monitored with a differential interference microscope. The lysate was diluted with 2 vol of 2 M sucrose and centrifuged at 18,000g for 1 h. The supernatant was discarded and viscous greenish pellets consisting of the nuclei and few chloroplaste were resuspended in the above medium enriched with sucrose up to 1.8 M. The nuclei were then collected by centrifugation at 32,000g for 30 min. Purity of the nuclei was checked by a fluorescence microscope after staining with 1 fig/ml H 33258 fluorescent dye (Hoechst).
Extraction of nuclear protein. Chlumydomonus nuclei were resuspended in an extraction buffer consisting of 25 mM Tris-HCl (pH 8.0), and 0.5 mM phenylmeth5 mM EDTA, 2 M NaCl, 1 mM dithiothreitol, ylsulfonyl fluoride. The resulting viscous suspension was sonicated for a few minutes by a Handy Sonic UR-20P (Tomy Seiko, Tokyo) to shear DNA and reduce viscosity, and centrifuged at 32,000g for 30 min. The supernatant was used as nuclear extract. Purification of the 26kDa ubiquitinuted nuclear protein. Unless otherwise mentioned, all purification procedures were performed at 4’C. After adding NaCl to a final concentration of 4 M, Chlamydomonus nuclear extract was loaded onto a hydrophobic chromatography TSK-gel butyl-Toyopearl650M column (2.0 X 20 cm, Tosoh, Tokyo) preequilibrated with 25 mM Tris-HCl (pH 8.0) containing 4 M NaCl. Elution was performed with a descending linear gradient of NaCl concentration from 4 M to zero and in the total eluant volume of 300 ml. The 28kDa protein-containing fractions determined by dot blot assay (see below) were gathered. This was enriched with NaCl up to 4 M and then rechromatographed on another butyl-Toyopearl column as above with the exceptions of column dimension (1.2 X 11 cm), initial NaCl concentration (3 M), and total eluant volume (150 ml). The resulting 28-kDa enriched fraction determined as above was collected and concentrated on Diaflo YMlO membrane (Amicon) in a Model 8MC microultrafiltration system (Amicon). After replacing the buffer with 20 mM Mes-KOH (pH 6.0) containing 4 M NaCl, the 28-kDa enriched fraction was subjected to a hydrophobic HPLC TSK-gel phenyl-5PW column (7.5 X 75 mm, Tosoh) (pH 6.0) containing 3 M NaCl preequilibrated with 20 mM Mes-KOH and eluted by a descending gradient of NaCl (3 M to zero) at room temperature. The 28-kDa ubiquitinated protein was collected and concentrated by ultrafiltration as above. This was then subjected to gelfiltration HPLC using a TSK-gel G30OOSWxL column (7.8 X 300 mm, Tosoh) preequilibrated with 20 mM Mes-KOH (pH 6.0) containing 1 M NaCl. Elution was performed using the same buffer at a flow rate of 0.5 ml/min at room temperature. The 28-kDa-peak fraction determined as
above was then subjected to reverse-phase HPLC using an Asahipak ODP-50 column (4.6 X 250 mm, Asahi Chemical Industry, Tokyo) preequilibrated with 10% acetonitrile in 0.05% TFA. Elution was done at a flow rate of 0.6 ml/min at room temperature with a two-step linear gradient of acetonitrile in 0.05% TFA: 10 to 30% from start to 5 min and 30 to 70% from 5 to 60 min. The fractions containing the 28-kDa ubiquitinated protein were collected and lyophilized. Putative SH groups in the 28-kDa polypeptide were pyridylethylated as described (24). The pyridylethylated proteins were separated by gel-filtration HPLC under denaturing condition using a TSK-gel G3000SWxL column (7.8 X 300 mm, Tosoh) preequilibrated with 20 mM Mes-KOH (pH 6.0) containing 6 M guanidine-HCl and 1 mM EDTA. Elution was carried out at a flow rate of 0.4 ml/min at room temperature with the same solution. The 28-kDa ubiquitinated protein and a 20-kDa protein, eluted subsequently, were collected separately and further purified by the reverse-phase HPLC as above.
SDS-PAGE and radioimmurwblot anulyses of ubiquitinated proteins. SDS-PAGE was performed as described (19). To detect ubiquitinated proteins on the SDS-polyacrylamide gel, these were labeled by radioimmunoblotting using affinity-purified anti-pea ubiquitin antibody and ‘261-labeled Staphylococcus aureus protein A (230 mCi/mg, ICN) as described (19). Radiolabeled proteins were visualized by autoradiography. To visualize total proteins on the SDS-polyacrylamide gel, silver staining was performed using a staining kit (2D-Silver Stain II, Daiichi Pure Chemicals, Tokyo).
Dot blot assay. PVDF membrane (Durapore GVHP, Milipore), prewetted with methanol, was equilibrated with 30% glycerol solution for a few minutes. The membrane was piled on the same size of filter paper (0.7 mm thick, No. 526, Advantec Toyo, Tokyo), presoaked by the same solution, and surplus solution oozed on the membrane was removed. Five microliters of each of samples was spotted in array on the membrane and left for a few minutes until soaked. Ubiquitinated proteins on the membrane were labeled by radioimmunoblotting as described above. Spots were separated with each other by cutting the membrane and their radioactivities were determined with a y-ray counter (Auto-Gamma 5650, Packard). Endoproteinase digestion and amino acid sequencing of polypeptide fragments. The 28-kDa ubiquitinated protein was digested with either arginylendopeptidase (mouse submandibular protease; EC 3.4.21.40; Takara Shuzo, Kyoto) in 50 mM Tris-HCl (pH 8.5) containing 1 mM EDTA, or protease V8 (Staphylococcus aureus protease; EC 3.4.21.19; ICN ImmunoBiologicals) in 100 mM ammonium acetate (pH 4.0) containing 1 mM EDTA. Substrate concentrations were 0.8 and 0.14 pg/pl, and substrate to enzyme ratios were 8 and 12 for the former and the latter treatments, respectively. Both reactions were performed at 27’C for 3 h and resulting peptides were separated by reverse-phase HPLC as described above. Each peptide was subjected to an automatic peptide sequencer (Model 477A+120A, Applied Biosystems) to determine its N-terminal sequence.
RESULTS AND DISCUSSION Intracellular distribution of Chlamydomonas ubiquitinated proteins. The immunoblot analysis using anti-ubiquitin antibody showed that the 28kDa protein is a unique ubiquitinated protein in the nuclear fraction except for a very faint band around 50 kDa (Fig. 1B). This indicates that the 28kDa protein is distributed in nuclei, and other immunoreactive bands detected in the crude extract, including the 31-kDa protein and free ubiquitin (Fig. 1A) are distributed in extranuclear space. The results suggest that the Sl-kDa protein is not ubiquitinated histones and most of nuclear ubiquitin exists in a conjugated form as the 28-kDa protein.
Chlumydomonus
31kDaD 28kDam
UBIQUITINATED
-28kDa ,“__ .-”
3” -..
HISTONE
195
H2B
peptides suggests that they are associated with each other or they have very similar characteristics under the separation conditions so far employed. Therefore next we tried gel-filtration HPLC under a denatured condition. Prior to this, the sample was alkylated with 4-vinylpyridine to block putative SH groups, which may form disulfide bonds between the two polypeptides and thus prevent their separation. In this way, the 28-kDa ubiquitinated protein was separated from the 20-kDa protein (peaks A and B in Fig. 4). Each of the resulting 28- and 20-kDa fractions gave a single peak in reverse-phase HPLC and a single band after silver staining in SDSPAGE (Fig. 4 inset), indicating that the 28-kDa ubiquitinated protein and the 20-kDa protein are purified to homogeneity. Amino acid sequence of the 28-kDa ubiquitinated protein. The N-terminal amino acid sequences of the 28-
FIG. 1. Immunoblot detection of ubiquitinated proteins of total cell extract and nuclear fractions isolated from Chlamydomonas. Chlumydomonas total cell extract (A) and nuclear fractions (B) were subjected to SDS-PAGE and ubiquitinated proteins were detected by radioimmunoblotting using antiubiquitin antibody. The 28- and 31-kDa ubiquitinated proteins and free ubiquitin are indicated by arrowheads.
Purification ated protein.
of the Chlumydomonas
kDa protein and its protease-digested fragments separated by HPLC are shown in Fig. 5. The initial yields and repetitive yields of these sequencing were 5-30% and 8095%, respectively. The 28-kDa whole protein (Fig. 5A)
28-kDa ubiquitin-
The 28-kDa ubiquitinated protein was solubilized from chromatin by a high concentration of salt (2 M NaCl), and precipitated when desalted by dialysis, suggesting that the protein was reassociated with chromatin at low-salt concentrations. To separate total proteins from the nuclear DNA, hydrophobic interaction chromatography using a TSK-gel butyl-Toyopearl and/ or a phenyl-5PW column was satisfactorily employed. The bulk DNA was eluted from the butyl-Toyopearl column in the pass-through fraction and the 28-kDa protein was eluted between 2.4 and 1.6 M NaCl when monitored by dot blot analysis with anti-ubiquitin antibody. Gel-filtration HPLC separation of the proteins from the butyl-Toyopearl column revealed that the apparent molecular mass of the native 28-kDa protein is 96 kDa (Fig. 2). This suggests that the 28-kDa protein exists as an oligomeric structure under this elution condition. Figure 3 shows a chromatogram of reverse-phase HPLC of the next purification step. It was found that a peak fraction eluted at 25.33 min consisted of two polypeptides with apparent molecular masses of 20 and 28 kDa when analyzed by SDS-PAGE (A in Fig. 3 inset). The latter was identified as the 28-kDa ubiquitinated protein by a radioimmunoblot analysis (B in Fig. 3 inset). Attempts to separate these two polypeptides on the same column by changing elution conditions (i.e., column temperature and gradient condition) were unsuccessful. The coincidence of elution time of these two (20- and 28-kDa) poly-
-8 E .L
18
16
20
-6
.%
Time (min)
4
hv
\
Time (mid FIG, 2. Purification of the 28kDa ubiquitinated protein by gel-filtration HPLC using a TSK-gel G30OOSWxL column (7.8 X 300 mm). Elution was carried out as described under Materials and Methods. Protein concentration was monitored by AZBO(-). The 28kDa ubiquitinated protein was detected by radioimmunoblot (- 0 -). The fractions indicated by hatching were used for the next purification step. (inset) Calibration curve of molecular mass for the gel filtration. Standard proteins (molecular mass) were aldorase (158 kDa), bovine serum albumin (68 kDa), ovalbumin (45 kDa), and chymotrypsinogen A (25 kDa). The elution time and calibrated molecular weight of the 28-kDa ubiquitinated protein were 16.88 min and 94 kDa (indicated by the broken line), respectively.
196
SHIMOGAWABA
lime (min) FIG, 3. Purification of the 2%kDa ubiquitinated protein by revereephase HPLC. The sample from the gel-filtration HPLC was injected into an Aeahipak ODP-50 (4.6 X 250 mm) column and eluted aa described under Materials and Methods. The protein profile was monitored by Azza (-). Acetonitrile concentration was indicated by the dashed line. The 28-kDa peak indicated by hatching was ueed for the next purification step. (inset) SDS-PAGE and radioimmunoblot analysis of the hatched fraction. (A) Silver-stained gel; (B) radioimmunoblot. The positions of the 2%kDa and coeluted 20-kDa proteins are indicated by arrowheads.
AND
MUTO
were blocked (26,28). These reports suggest that the first alternative is most likely. If there was an isopeptidyl linkage in the 28-kDa polypeptide chain, peptide fragments including isopeptidyl linkage might have two free N-termini and thus give a double sequence; i.e., one is from ubiquitin and the other from H2B. Indeed, peaks 5 and 8 of the arginylendopeptidase digest (Fig. 5B) and peak 9 of the protease V8 digest (Fig. 5C) gave such double sequences. Figure 6 shows a proposed sequence of the 28-kDa ubiquitinated protein, which was composed of the determined sequences by referring to those of wheat histone H2B and oat ubiquitin. As far as determined, all the amino acid sequences of Chlamydomonas 28-kDa protein were attributable to some part of either ubiquitin or histone H2B. However, a sequence corresponding to the region from the N-terminus to the 56th residue of wheat H2B could not be found in the protease digests of the 28-kDa protein. This may be due to the possibility that there is no cleavable amino acid (Arg or Glu) by both proteases in the corresponding region of Chlumydomonas histone H2B and thus it not sequenced or that these fragments were not sufficiently recovered from the column used here because of their strongly basic nature. The former explanation is not likely since the corresponding region of wheat H2B has three Arg and six Glu residues (26). The position of isopeptidyl linkage between both peptides was estimated as below. Peak 5 of the arginylen-
AS kDa
gave a sequence coinciding with the N-terminal region of oat ubiquitin (25) (Fig. 6), while protease-digested fragments of the 28kDa protein (Figs. 5B and 5C) gave sequences homologous to either oat ubiquitin or wheat histone H2B (26) (Fig. 6). These results strongly suggest that the 28kDa protein is an ubiquitinated histone H2B. The fact that the 2%kDa whole protein gave only one sequence homologous to ubiquitin could be explained by the following alternatives: (a) the ubiquitin moiety conjugates to the internal lysyl residue of histone by isopeptidy1 linkage but the N-terminal amino acid of the histone moiety is blocked to interrupt Edman degradation, (b) the ubiquitin moiety attaches only to the a-amino group of the N-terminal amino acid of histone, and (c) two or more molecules of ubiquitin conjugate with both internal lysine and the N-terminal residue of histone. Olson et al. (27) reported that since the N-terminus of calf thymus histone H2A was blocked by an acetyl group, its ubiquitin adduct (uH2A) gave only one sequence homologous to ubiquitin by Edman degradation. It was reported for wheat histones that the N-termini of one of three variants of histone H2A and all of three variants of histone H2B
4
I lo
15
20
25
30
Time (min) FIG. 4. Purification of the 28-kDa ubiquitinated protein by gel-filtration HPLC under denatured conditions. The sample purified by reverse-phase. HPLC was pyridylethylated and then injected into a TSKgel G3000SWxL column (7.8 X 300 mm). Elution was carried out as described under Materials and Methods and monitored by Am. A and B are the 28-kDa ubiquitinated protein and 20-kDa protein, respectively. (inset) SDS-PAGE analysis of the purified Chlumydomonus 28-kDa ubiquitinated protein and the copurified 20-kDa protein. (A) Peak A; (B) peak B (20~kDa protein).
Chlamydomonus
UBIQUITINATED
A MWVKlLTGKllTLEVESSDTlENVKAKlQDKEGlPF’DQC+l??FAGKQ
r
LIFAGKOLEffiFlTLADVNIPKE??LHLV
Elution Time (min)
1’5
2b
;5
3b 3’5 Elution Time (min)
40
45
FIG. 5. Partial N-terminal amino acid sequences of the 28-kDa ubiquitinated protein and its endoproteinase digests. (A) Partial N-terminal amino acid sequence of the 28-kDa protein. The 28-kDa ubiquitinated protein digested by arginylendopeptidase (B) or protease V8 (C) was separated by reverse-phase HPLC. Peak X in B and peak Y in C were ascribed to the proteases. Numbered peaks were collected and sequenced. Sequencing was continued as long as possible, but was stopped when the sequence overlapped other peaks. C-termini of such sequences are marked by periods. Undetermined cycles are expressed by “?.” Peaks 5 and 8 in B and peak 9 in C gave partial double sequences which were presented alongside. In these cases, two amino acids were assigned to either the ubiquitin or the histone H2B sequence which had been determined in other peaks.
HISTONE
197
T ? F T S) and whole ubiquitin (M Q I F V K T L T . . .). However, the position of the last lysyl residue in the H2B sequence of arginylendopeptidase-digest peak 5 was not detected in the H2B sequence of VB-digest peak 9. The e-amino group of this lysine residue in the former fragment became free after two cycles of Edman degradation and thus was sequenced at the 12th cycle. While in the latter, this lysine remained to be modified with the long side chain even when Edman sequencing came up to this lysine. Therefore, the position of isopeptidyl linkage was determined to be the last lysine in the former sequence (H A V S E G T K A V T K). The estimated position of isopeptidyl linkage in the Chlamydomonas 28kDa protein (uH2B) coincides to those of calf and porcine uH2B (29). Relationship between the 28-kDa ubiquitinuted protein Since the 28-kDa ubiquiand the 20-kDa polypeptide. tinated polypeptide and the 20-kDa polypeptide were copurified throughout the purification processes until the first reverse-phase HPLC, these proteins were supposed to have some relation to each other. Therefore, determination of the N-terminal sequence of the 20-kDa polypeptide was tried. No significant signal for the amino acid was, however, detected in the whole polypeptide, indicating that the N-terminal amino group of this peptide is blocked. Therefore, protease digestion was applied next. One polypeptide fragment produced by its arginylendopeptidase digestion gave an sequence (L V L P G E L A K H A V S E G T K A V T K F T S) identical to the Cterminal region of the H2B moiety of the 28-kDa protein. This result strongly suggests that the 20-kDa polypeptide is histone H2B and that the 28-kDa polypeptide is a ubiquitinated form of the 20-kDa polypeptide. Indeed, the difference in molecular mass between these polypeptides (8 kDa) is close to the molecular mass of ubiquitin (8.5 kDa). The crude nuclear fraction did not give any positive band at 20 kDa in the immunoblot analysis probed with anti-ubiquitin antibody (Fig. I), indicating that the 20kDa polypeptide does not include a ubiquitin moiety, and thus only one ubiquitin moiety is present in the 28-kDa ubiquitinated protein. CONCLUDING
dopeptidase digest (Fig. 5B) gave a double sequence in the first and second cycles of Edman degradation. One of the double sequence (H A V S E G T K A V T K) was attributable to the C-terminal fragment of histone H2B and the other (G G) to the C-terminus of ubiquitin. Two lysyl residues as the candidate for the position of isopeptidy1 linkage were present in this C-terminal fragment of H2B (H A V S E G T K A V T K ). A sequence overlapping this region was found in another double sequence of the protease V8 digest (Fig. 5C, peak 9). Judging from the sequence homology, this fragment was estimated to be composed of C-terminal fragments of H2B (G T K A V
H2B
REMARKS
The 28-kDa ubiquitinated protein of Chlamydomonas was identified as histone uH2B. The protein is monoubiquitinated but its molecular mass is significantly larger than those of ubiquitinated histones from other organisms. This seems to be attributed to the molecular mass of histone H2B. Morris et al. (30) reported that Chlamydomonus histone H2B had a significantly larger molecular mass than calf thymus histone H2B and thus gave a slower mobility in SDS-PAGE than histone H3. A similar property has been reported with histone H2Bs of many higher plants (31). The physiological importance of the ubiquitination of histone H2B is still unclear. We have found that the
198
SHIMOGAWARA
AND
MUTO
,~-.-‘-..“--.------.~---.-----------.----.----------.---------------------------------------~----------~-------~~------
Oat ubiquitin. 7.0 1.6 j; ;~IM<~T&~~ITLEM~~~TIWVKAKIOOKEGIPPOZ~QRLIFAGK 1 10 ZP ?Q 40 S? 6.0 -YNIoKEslLHL~m L_____________-_________________________-------------------------------------------------------------------------------
Chlamydomonas 28-kDa ubiquitinated protein
~;-~~ii~i~-i~~i~~-i~~-~ . 60
70
. 60
90
100
___---_____________________________ ______--_______-----________________ I-I ULAMMPTITSREIOTSVRLM# . . . 110
120
130
149
149 j
Wheat H2B L-____-_______..____-..--....-----..----..------~-----.--------------------------------------~~-----------------~~---~~~~~~.-------~~-----~~-~ FIG. 6.
Proposed primary polypeptide structure of the Chlumydomonas 28-kDa ubiquitinated protein. Ubiquitin (upper) and histone H2B (lower) moieties of Chlumydomonus were arranged by referring to the sequence of oat ubiquitin (34) and wheat histone H2B (26), respectively. Positions of amino acids not coinciding are indicated by asterisks. The ranges where no corresponding sequence was observed are filled with “?“. Ubiquitin and histone moieties are assumed to join by isopeptidyl linkage between the C-terminus of ubiquitin and the c-amino group of Lys (indicated by an arrow) in histone.
amount of 28-kDa ubiquitinated protein varied depending on various physiological states, i.e., heat and active oxygen stress, progress of the cell cycle, and gamete induction (19-21). It has been assumed that ubiquitination of histones disturbs tight packing of nucleosomes and such disturbance is necessary to keep transcriptional activity of some genes (10). Varshavsky and co-workers showed that nucleosomal uH2A is preferentially localized on transcribed genes in Drosophila (11) and mouse cells (12). Haas et al. (32) reported that in cultured mammalian cells infection of a certain virus, which leads to turning off the expression of the host genome, caused a decrease of ubiquitinated histone H2A. Nickel et al. (14) reported that in calf, chicken, and Tetrahymena nuclei the most selectively ubiquitinated histone species in the region of transcriptionally active chromatin was H2B, not H2A. Davie and Murphy (33) showed that in human breast cancer cells the ubiquitination level of H2B but not of H2A is coupled to ongoing transcription. These observations strongly suggest that the ubiquitination of histones, especially of histone H2B, plays an important role in the regulation of transcription, and our present results also support this. In contrast to the fact that uH2A is the predominantly ubiquitinated histone in animal nuclei, there was essentially no detectable ubiquitinated protein other than the 28-kDa protein (uH2B) in the Chlamydomonas nuclear fraction (Fig. 1). The reason why no ubiquitinated H2A was observed in the Chlamydomonas nuclei is obscure. As reported so far, the degree of histone H2B ubiquitination in other organisms such as Physarum (5) and Tetrahymena (14) is relatively higher than that in animals. Swerdlow et al. (25) reported that ubiquitinated histone uH2A is also absent in Saccharomyces cerevisiae. These observations and our present results suggest that the ubi-
quitination of H2B is more general than that of H2A in all eukaryotes, while the predominant ubiquitination of H2A observed in several kinds of animals is exceptional. REFERENCES 1. Hershko,
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A. (1986) Prog. Nucleic Acid Res.
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Chlamydomonas Franche, U., Fried, V. A., Ullrich, Nature 323,226-232. 18. Zhaung, Z., and McCauley, 14,596.
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UBIQUITINATED
L. T. (1986)
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P. L., Schuster, T., and Finley, D. (1990) Mol. Cell. Biol.
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HISTONE
H2B
199
26. Brandt, W. F., Rodrigues, J. D. A., and von Holt, C. (1988) Eur. J. Biochem. 173,547-554. 27. Olson, M. 0. J., Goldknopf, I. L., Guetzow, K. A., James, G. T., Hawkins, T. C., Mays-Rothberg, C. J., and Busch, H. (1976) J. Biol. Chem. 251,5901-5903. 28. Rodrigues, J. D. A., Brandt, W. F., and von Holt, C. (1988) Eur. J. Biochem. 173,555-560. C. 29. Thorne, A. W., Sautiere, P., Briand, G., and Crane-Robinson, (1987) EMBO J. 6, 1005-1010. 30. Morris, R. L., Keller, L. R., ZweidIer, A., and Rizzo, P. J. (1990) J. Protozool. 37, 117-123. 31. Spiker, S. (1985) Annu. Reu. Plant. Physiol. 36,235-253. 32. Haas, A. L., Ahrens, P., Bright, P. M., and Ankel, H. (1987) J. Biol. Chem. 262,11,315-11,313. 33. Davie, J. R., and Murphy, L. C. (1990) Biochemistry 29,4752-4757. 34. Vierstra, R. D., Langan, S. M., and Schaller, G. E. (1986) Biochemistry 25, 31053108.
OF BIOCHEMISTRY
AND BIOPHYSICS
Vol. 294, No. 1, April, pp. 193-199, 1992
Purification of Chlamydomonas 28-kDa Ubiquitinated Protein and Its Identification as Ubiquitinated Histone H2B Kousuke Shimogawara’ Institute
and Shoshi Muto
of Applied Microbiology,
University
of Tokyo, Yayoi, Bunkyo-ky
Received September 13, 1991, and in revised form November
12,199l
One of the most predominantly ubiquitinated protein species in Chlamydomonas, of which the apparent molecular mass in SDS-PAGE was 28 kDa, was found to exist abundantly in nuclei. The 2%kDa ubiquitinated protein was purified to homogeneity from the isolated nuclei of Chlam ydomonas, and its partial amino acid sequence was determined. The N-terminal peptide sequence was identical with that of ubiquitin. Sequences homologous to those oat ubiquitin and wheat histone B2B, and paired sequences of both of them were found in arginylendopeptidase-digested or protease VS-digested polypeptide fragments of the 2%kDa ubiquitinated protein. Based on these results, it was concluded that Chlamydomonaa 28-kDa ubiquitinated protein is monoubiquitinated histone H2B. o 1992 AC,AIGO press, IXW.
Ubiquitin is a small polypeptide with a molecular mass of 8.5 kDa and is composed of 76 amino acid residues. As the name implies, it has been detected ubiquitously in all eukaryotes so far examined. Since the amino acid sequence of ubiquitin shows remarkable evolutional conservation, this protein is believed to be involved in essential life processes in all eukaryotes. Ubiquitin is known to attach to other specific proteins by isopeptidyl linkage between the carboxyl terminus of ubiquitin and the tamino groups of internal lysyl residues in the target proteins. The role of ubiquitination in cytosolic protein degradation by ATP-dependent proteinase has been well elucidated (for reviews, see (1, 2)). There are several proteins ubiquitinated independently of their own degradation. The best elucidated example is histone ubiquitination (3). Ubiquitin conjugates with his1 Present address: Laboratory of Chemistry, School of Medicine, Teikyo University, Ohtsuka, Hachioji, Tokyo 192-03, Japan. ’ To whom correspondence should be addressed 0003-9Sfx/92 $3.00 Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
Tokyo 113, Japan
tone H2A and H2B in the nuclei, forming uH2A (previously termed A24) and uH2B semihistone, respectively. The degree of ubiquitination of histone varies from species to species and from tissue to tissue. For example, the degree of ubiquitination of H2A is 1, 7, 8-10, 10, and 1214% of total H2A in Chirorwmus tenatans (4), slime molds (5), cultured hamster cells (6), mouse cells (7, 8), and terminally differentiated rat cerebral cortex neurons (9), respectively. The degree of ubiquitination of H2B is 1-2, l-2, and 6% of total H2B in mouse cells, rat cerebral cortex neurons, and slime molds, respectively (refs. cited above). The degree of ubiquitination of histones is also variable depending on the physiological conditions. The ubiquitination of histone is assumed to cause some changes on the nucleosome structure, resulting in changes of the transcriptional activities of specific genes (10-14). There are some reports suggesting the conjugation of ubiquitin to certain proteins, including cell surface and extracellular proteins (15-18). These ubiquitinations are believed to have certain physiological functions like in other protein modifications such as phosphorylation and adenylation. In our previous study (19), we found two remarkably ubiquitinated protein species in Chlamydamonas, of which apparent molecular masses in SDS-PAGE3 were 28 and 31 kDa. The isoelectric points of the 28- and 31-kDa ubiquitinated proteins were strongly basic, i.e., 8.9 and 10.3, respectively. The amounts of these ubiquitinated proteins were found to change in response to heat and active oxygen stress, suggesting that the ubiquitination of these proteins plays some roles in adaptation of cells to stress condition (19, 20). The amounts of them also changed during cell cycle and gamete induction (21). However, the 3 Abbreviations used: HPLC, high-performance liquid chromatography; PVDF, polyvinylidene difluoride; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; SH, sulfhydryl; TFA, trifluoroacetic acid, Mes, 4-morpholineethanesulfonic acid. 193
194
SHIMOGAWARA AND MUTO
significance of these changes of uhiquitination still remains unclear. For clarification of this, it is beneficial to identify these ubiquitinated proteins. Here, we report identification of the 2%kDa ubiquitinated protein as histone H2B based on its intracellular distribution and ammo acid sequence. MATERIALS
AND METHODS
Isolation of Chlnmydomonas nuclei.
To overcome difficulties in isolating the nuclei from wild-type cells, cells of the wall-less strain Chlumydonwnus reinhurdtii CW-15 (mating type -) were used. The nuclei were isolated according to Keller et al. (22) with partial modifications as described below. Chlumydomonns was cultured mixotrophically with acetate as the carbon source at 30°C with gentle shaking under continuous illumination with fluorescent light as described elsewhere (23). Cells grown up to ca. 0.1% (packed cell volume per volume) were harvested by continuous centrifugation at 23,000g at 4°C. The following procedures were done at ice-cold temperature. The cell pellets were resuspended in a fivefold volume of a medium containing 20 mM HepesKOH (pH 7.6), 10 mM CaClx, 10 mM MgCl,, 0.6 M sucrose, 2.5 mM spermidine, 0.75 mM spermine, 2 mM dithiothreitol, 5 mM diisopropylfluorophosphate, and 0.5 mM phenylmethylsulfonyl fluoride. One part of 20% (v/v) Triton X-100 was added to twenty parts of the cell suspension and the mixture was kept for few minutes to lyse the cell wall. Lysis was monitored with a differential interference microscope. The lysate was diluted with 2 vol of 2 M sucrose and centrifuged at 18,000g for 1 h. The supernatant was discarded and viscous greenish pellets consisting of the nuclei and few chloroplaste were resuspended in the above medium enriched with sucrose up to 1.8 M. The nuclei were then collected by centrifugation at 32,000g for 30 min. Purity of the nuclei was checked by a fluorescence microscope after staining with 1 fig/ml H 33258 fluorescent dye (Hoechst).
Extraction of nuclear protein. Chlumydomonus nuclei were resuspended in an extraction buffer consisting of 25 mM Tris-HCl (pH 8.0), and 0.5 mM phenylmeth5 mM EDTA, 2 M NaCl, 1 mM dithiothreitol, ylsulfonyl fluoride. The resulting viscous suspension was sonicated for a few minutes by a Handy Sonic UR-20P (Tomy Seiko, Tokyo) to shear DNA and reduce viscosity, and centrifuged at 32,000g for 30 min. The supernatant was used as nuclear extract. Purification of the 26kDa ubiquitinuted nuclear protein. Unless otherwise mentioned, all purification procedures were performed at 4’C. After adding NaCl to a final concentration of 4 M, Chlamydomonus nuclear extract was loaded onto a hydrophobic chromatography TSK-gel butyl-Toyopearl650M column (2.0 X 20 cm, Tosoh, Tokyo) preequilibrated with 25 mM Tris-HCl (pH 8.0) containing 4 M NaCl. Elution was performed with a descending linear gradient of NaCl concentration from 4 M to zero and in the total eluant volume of 300 ml. The 28kDa protein-containing fractions determined by dot blot assay (see below) were gathered. This was enriched with NaCl up to 4 M and then rechromatographed on another butyl-Toyopearl column as above with the exceptions of column dimension (1.2 X 11 cm), initial NaCl concentration (3 M), and total eluant volume (150 ml). The resulting 28-kDa enriched fraction determined as above was collected and concentrated on Diaflo YMlO membrane (Amicon) in a Model 8MC microultrafiltration system (Amicon). After replacing the buffer with 20 mM Mes-KOH (pH 6.0) containing 4 M NaCl, the 28-kDa enriched fraction was subjected to a hydrophobic HPLC TSK-gel phenyl-5PW column (7.5 X 75 mm, Tosoh) (pH 6.0) containing 3 M NaCl preequilibrated with 20 mM Mes-KOH and eluted by a descending gradient of NaCl (3 M to zero) at room temperature. The 28-kDa ubiquitinated protein was collected and concentrated by ultrafiltration as above. This was then subjected to gelfiltration HPLC using a TSK-gel G30OOSWxL column (7.8 X 300 mm, Tosoh) preequilibrated with 20 mM Mes-KOH (pH 6.0) containing 1 M NaCl. Elution was performed using the same buffer at a flow rate of 0.5 ml/min at room temperature. The 28-kDa-peak fraction determined as
above was then subjected to reverse-phase HPLC using an Asahipak ODP-50 column (4.6 X 250 mm, Asahi Chemical Industry, Tokyo) preequilibrated with 10% acetonitrile in 0.05% TFA. Elution was done at a flow rate of 0.6 ml/min at room temperature with a two-step linear gradient of acetonitrile in 0.05% TFA: 10 to 30% from start to 5 min and 30 to 70% from 5 to 60 min. The fractions containing the 28-kDa ubiquitinated protein were collected and lyophilized. Putative SH groups in the 28-kDa polypeptide were pyridylethylated as described (24). The pyridylethylated proteins were separated by gel-filtration HPLC under denaturing condition using a TSK-gel G3000SWxL column (7.8 X 300 mm, Tosoh) preequilibrated with 20 mM Mes-KOH (pH 6.0) containing 6 M guanidine-HCl and 1 mM EDTA. Elution was carried out at a flow rate of 0.4 ml/min at room temperature with the same solution. The 28-kDa ubiquitinated protein and a 20-kDa protein, eluted subsequently, were collected separately and further purified by the reverse-phase HPLC as above.
SDS-PAGE and radioimmurwblot anulyses of ubiquitinated proteins. SDS-PAGE was performed as described (19). To detect ubiquitinated proteins on the SDS-polyacrylamide gel, these were labeled by radioimmunoblotting using affinity-purified anti-pea ubiquitin antibody and ‘261-labeled Staphylococcus aureus protein A (230 mCi/mg, ICN) as described (19). Radiolabeled proteins were visualized by autoradiography. To visualize total proteins on the SDS-polyacrylamide gel, silver staining was performed using a staining kit (2D-Silver Stain II, Daiichi Pure Chemicals, Tokyo).
Dot blot assay. PVDF membrane (Durapore GVHP, Milipore), prewetted with methanol, was equilibrated with 30% glycerol solution for a few minutes. The membrane was piled on the same size of filter paper (0.7 mm thick, No. 526, Advantec Toyo, Tokyo), presoaked by the same solution, and surplus solution oozed on the membrane was removed. Five microliters of each of samples was spotted in array on the membrane and left for a few minutes until soaked. Ubiquitinated proteins on the membrane were labeled by radioimmunoblotting as described above. Spots were separated with each other by cutting the membrane and their radioactivities were determined with a y-ray counter (Auto-Gamma 5650, Packard). Endoproteinase digestion and amino acid sequencing of polypeptide fragments. The 28-kDa ubiquitinated protein was digested with either arginylendopeptidase (mouse submandibular protease; EC 3.4.21.40; Takara Shuzo, Kyoto) in 50 mM Tris-HCl (pH 8.5) containing 1 mM EDTA, or protease V8 (Staphylococcus aureus protease; EC 3.4.21.19; ICN ImmunoBiologicals) in 100 mM ammonium acetate (pH 4.0) containing 1 mM EDTA. Substrate concentrations were 0.8 and 0.14 pg/pl, and substrate to enzyme ratios were 8 and 12 for the former and the latter treatments, respectively. Both reactions were performed at 27’C for 3 h and resulting peptides were separated by reverse-phase HPLC as described above. Each peptide was subjected to an automatic peptide sequencer (Model 477A+120A, Applied Biosystems) to determine its N-terminal sequence.
RESULTS AND DISCUSSION Intracellular distribution of Chlamydomonas ubiquitinated proteins. The immunoblot analysis using anti-ubiquitin antibody showed that the 28kDa protein is a unique ubiquitinated protein in the nuclear fraction except for a very faint band around 50 kDa (Fig. 1B). This indicates that the 28kDa protein is distributed in nuclei, and other immunoreactive bands detected in the crude extract, including the 31-kDa protein and free ubiquitin (Fig. 1A) are distributed in extranuclear space. The results suggest that the Sl-kDa protein is not ubiquitinated histones and most of nuclear ubiquitin exists in a conjugated form as the 28-kDa protein.
Chlumydomonus
31kDaD 28kDam
UBIQUITINATED
-28kDa ,“__ .-”
3” -..
HISTONE
195
H2B
peptides suggests that they are associated with each other or they have very similar characteristics under the separation conditions so far employed. Therefore next we tried gel-filtration HPLC under a denatured condition. Prior to this, the sample was alkylated with 4-vinylpyridine to block putative SH groups, which may form disulfide bonds between the two polypeptides and thus prevent their separation. In this way, the 28-kDa ubiquitinated protein was separated from the 20-kDa protein (peaks A and B in Fig. 4). Each of the resulting 28- and 20-kDa fractions gave a single peak in reverse-phase HPLC and a single band after silver staining in SDSPAGE (Fig. 4 inset), indicating that the 28-kDa ubiquitinated protein and the 20-kDa protein are purified to homogeneity. Amino acid sequence of the 28-kDa ubiquitinated protein. The N-terminal amino acid sequences of the 28-
FIG. 1. Immunoblot detection of ubiquitinated proteins of total cell extract and nuclear fractions isolated from Chlamydomonas. Chlumydomonas total cell extract (A) and nuclear fractions (B) were subjected to SDS-PAGE and ubiquitinated proteins were detected by radioimmunoblotting using antiubiquitin antibody. The 28- and 31-kDa ubiquitinated proteins and free ubiquitin are indicated by arrowheads.
Purification ated protein.
of the Chlumydomonas
kDa protein and its protease-digested fragments separated by HPLC are shown in Fig. 5. The initial yields and repetitive yields of these sequencing were 5-30% and 8095%, respectively. The 28-kDa whole protein (Fig. 5A)
28-kDa ubiquitin-
The 28-kDa ubiquitinated protein was solubilized from chromatin by a high concentration of salt (2 M NaCl), and precipitated when desalted by dialysis, suggesting that the protein was reassociated with chromatin at low-salt concentrations. To separate total proteins from the nuclear DNA, hydrophobic interaction chromatography using a TSK-gel butyl-Toyopearl and/ or a phenyl-5PW column was satisfactorily employed. The bulk DNA was eluted from the butyl-Toyopearl column in the pass-through fraction and the 28-kDa protein was eluted between 2.4 and 1.6 M NaCl when monitored by dot blot analysis with anti-ubiquitin antibody. Gel-filtration HPLC separation of the proteins from the butyl-Toyopearl column revealed that the apparent molecular mass of the native 28-kDa protein is 96 kDa (Fig. 2). This suggests that the 28-kDa protein exists as an oligomeric structure under this elution condition. Figure 3 shows a chromatogram of reverse-phase HPLC of the next purification step. It was found that a peak fraction eluted at 25.33 min consisted of two polypeptides with apparent molecular masses of 20 and 28 kDa when analyzed by SDS-PAGE (A in Fig. 3 inset). The latter was identified as the 28-kDa ubiquitinated protein by a radioimmunoblot analysis (B in Fig. 3 inset). Attempts to separate these two polypeptides on the same column by changing elution conditions (i.e., column temperature and gradient condition) were unsuccessful. The coincidence of elution time of these two (20- and 28-kDa) poly-
-8 E .L
18
16
20
-6
.%
Time (min)
4
hv
\
Time (mid FIG, 2. Purification of the 28kDa ubiquitinated protein by gel-filtration HPLC using a TSK-gel G30OOSWxL column (7.8 X 300 mm). Elution was carried out as described under Materials and Methods. Protein concentration was monitored by AZBO(-). The 28kDa ubiquitinated protein was detected by radioimmunoblot (- 0 -). The fractions indicated by hatching were used for the next purification step. (inset) Calibration curve of molecular mass for the gel filtration. Standard proteins (molecular mass) were aldorase (158 kDa), bovine serum albumin (68 kDa), ovalbumin (45 kDa), and chymotrypsinogen A (25 kDa). The elution time and calibrated molecular weight of the 28-kDa ubiquitinated protein were 16.88 min and 94 kDa (indicated by the broken line), respectively.
196
SHIMOGAWABA
lime (min) FIG, 3. Purification of the 2%kDa ubiquitinated protein by revereephase HPLC. The sample from the gel-filtration HPLC was injected into an Aeahipak ODP-50 (4.6 X 250 mm) column and eluted aa described under Materials and Methods. The protein profile was monitored by Azza (-). Acetonitrile concentration was indicated by the dashed line. The 28-kDa peak indicated by hatching was ueed for the next purification step. (inset) SDS-PAGE and radioimmunoblot analysis of the hatched fraction. (A) Silver-stained gel; (B) radioimmunoblot. The positions of the 2%kDa and coeluted 20-kDa proteins are indicated by arrowheads.
AND
MUTO
were blocked (26,28). These reports suggest that the first alternative is most likely. If there was an isopeptidyl linkage in the 28-kDa polypeptide chain, peptide fragments including isopeptidyl linkage might have two free N-termini and thus give a double sequence; i.e., one is from ubiquitin and the other from H2B. Indeed, peaks 5 and 8 of the arginylendopeptidase digest (Fig. 5B) and peak 9 of the protease V8 digest (Fig. 5C) gave such double sequences. Figure 6 shows a proposed sequence of the 28-kDa ubiquitinated protein, which was composed of the determined sequences by referring to those of wheat histone H2B and oat ubiquitin. As far as determined, all the amino acid sequences of Chlamydomonas 28-kDa protein were attributable to some part of either ubiquitin or histone H2B. However, a sequence corresponding to the region from the N-terminus to the 56th residue of wheat H2B could not be found in the protease digests of the 28-kDa protein. This may be due to the possibility that there is no cleavable amino acid (Arg or Glu) by both proteases in the corresponding region of Chlumydomonas histone H2B and thus it not sequenced or that these fragments were not sufficiently recovered from the column used here because of their strongly basic nature. The former explanation is not likely since the corresponding region of wheat H2B has three Arg and six Glu residues (26). The position of isopeptidyl linkage between both peptides was estimated as below. Peak 5 of the arginylen-
AS kDa
gave a sequence coinciding with the N-terminal region of oat ubiquitin (25) (Fig. 6), while protease-digested fragments of the 28kDa protein (Figs. 5B and 5C) gave sequences homologous to either oat ubiquitin or wheat histone H2B (26) (Fig. 6). These results strongly suggest that the 28kDa protein is an ubiquitinated histone H2B. The fact that the 2%kDa whole protein gave only one sequence homologous to ubiquitin could be explained by the following alternatives: (a) the ubiquitin moiety conjugates to the internal lysyl residue of histone by isopeptidy1 linkage but the N-terminal amino acid of the histone moiety is blocked to interrupt Edman degradation, (b) the ubiquitin moiety attaches only to the a-amino group of the N-terminal amino acid of histone, and (c) two or more molecules of ubiquitin conjugate with both internal lysine and the N-terminal residue of histone. Olson et al. (27) reported that since the N-terminus of calf thymus histone H2A was blocked by an acetyl group, its ubiquitin adduct (uH2A) gave only one sequence homologous to ubiquitin by Edman degradation. It was reported for wheat histones that the N-termini of one of three variants of histone H2A and all of three variants of histone H2B
4
I lo
15
20
25
30
Time (min) FIG. 4. Purification of the 28-kDa ubiquitinated protein by gel-filtration HPLC under denatured conditions. The sample purified by reverse-phase. HPLC was pyridylethylated and then injected into a TSKgel G3000SWxL column (7.8 X 300 mm). Elution was carried out as described under Materials and Methods and monitored by Am. A and B are the 28-kDa ubiquitinated protein and 20-kDa protein, respectively. (inset) SDS-PAGE analysis of the purified Chlumydomonus 28-kDa ubiquitinated protein and the copurified 20-kDa protein. (A) Peak A; (B) peak B (20~kDa protein).
Chlamydomonus
UBIQUITINATED
A MWVKlLTGKllTLEVESSDTlENVKAKlQDKEGlPF’DQC+l??FAGKQ
r
LIFAGKOLEffiFlTLADVNIPKE??LHLV
Elution Time (min)
1’5
2b
;5
3b 3’5 Elution Time (min)
40
45
FIG. 5. Partial N-terminal amino acid sequences of the 28-kDa ubiquitinated protein and its endoproteinase digests. (A) Partial N-terminal amino acid sequence of the 28-kDa protein. The 28-kDa ubiquitinated protein digested by arginylendopeptidase (B) or protease V8 (C) was separated by reverse-phase HPLC. Peak X in B and peak Y in C were ascribed to the proteases. Numbered peaks were collected and sequenced. Sequencing was continued as long as possible, but was stopped when the sequence overlapped other peaks. C-termini of such sequences are marked by periods. Undetermined cycles are expressed by “?.” Peaks 5 and 8 in B and peak 9 in C gave partial double sequences which were presented alongside. In these cases, two amino acids were assigned to either the ubiquitin or the histone H2B sequence which had been determined in other peaks.
HISTONE
197
T ? F T S) and whole ubiquitin (M Q I F V K T L T . . .). However, the position of the last lysyl residue in the H2B sequence of arginylendopeptidase-digest peak 5 was not detected in the H2B sequence of VB-digest peak 9. The e-amino group of this lysine residue in the former fragment became free after two cycles of Edman degradation and thus was sequenced at the 12th cycle. While in the latter, this lysine remained to be modified with the long side chain even when Edman sequencing came up to this lysine. Therefore, the position of isopeptidyl linkage was determined to be the last lysine in the former sequence (H A V S E G T K A V T K). The estimated position of isopeptidyl linkage in the Chlamydomonas 28kDa protein (uH2B) coincides to those of calf and porcine uH2B (29). Relationship between the 28-kDa ubiquitinuted protein Since the 28-kDa ubiquiand the 20-kDa polypeptide. tinated polypeptide and the 20-kDa polypeptide were copurified throughout the purification processes until the first reverse-phase HPLC, these proteins were supposed to have some relation to each other. Therefore, determination of the N-terminal sequence of the 20-kDa polypeptide was tried. No significant signal for the amino acid was, however, detected in the whole polypeptide, indicating that the N-terminal amino group of this peptide is blocked. Therefore, protease digestion was applied next. One polypeptide fragment produced by its arginylendopeptidase digestion gave an sequence (L V L P G E L A K H A V S E G T K A V T K F T S) identical to the Cterminal region of the H2B moiety of the 28-kDa protein. This result strongly suggests that the 20-kDa polypeptide is histone H2B and that the 28-kDa polypeptide is a ubiquitinated form of the 20-kDa polypeptide. Indeed, the difference in molecular mass between these polypeptides (8 kDa) is close to the molecular mass of ubiquitin (8.5 kDa). The crude nuclear fraction did not give any positive band at 20 kDa in the immunoblot analysis probed with anti-ubiquitin antibody (Fig. I), indicating that the 20kDa polypeptide does not include a ubiquitin moiety, and thus only one ubiquitin moiety is present in the 28-kDa ubiquitinated protein. CONCLUDING
dopeptidase digest (Fig. 5B) gave a double sequence in the first and second cycles of Edman degradation. One of the double sequence (H A V S E G T K A V T K) was attributable to the C-terminal fragment of histone H2B and the other (G G) to the C-terminus of ubiquitin. Two lysyl residues as the candidate for the position of isopeptidy1 linkage were present in this C-terminal fragment of H2B (H A V S E G T K A V T K ). A sequence overlapping this region was found in another double sequence of the protease V8 digest (Fig. 5C, peak 9). Judging from the sequence homology, this fragment was estimated to be composed of C-terminal fragments of H2B (G T K A V
H2B
REMARKS
The 28-kDa ubiquitinated protein of Chlamydomonas was identified as histone uH2B. The protein is monoubiquitinated but its molecular mass is significantly larger than those of ubiquitinated histones from other organisms. This seems to be attributed to the molecular mass of histone H2B. Morris et al. (30) reported that Chlamydomonus histone H2B had a significantly larger molecular mass than calf thymus histone H2B and thus gave a slower mobility in SDS-PAGE than histone H3. A similar property has been reported with histone H2Bs of many higher plants (31). The physiological importance of the ubiquitination of histone H2B is still unclear. We have found that the
198
SHIMOGAWARA
AND
MUTO
,~-.-‘-..“--.------.~---.-----------.----.----------.---------------------------------------~----------~-------~~------
Oat ubiquitin. 7.0 1.6 j; ;~IM<~T&~~ITLEM~~~TIWVKAKIOOKEGIPPOZ~QRLIFAGK 1 10 ZP ?Q 40 S? 6.0 -YNIoKEslLHL~m L_____________-_________________________-------------------------------------------------------------------------------
Chlamydomonas 28-kDa ubiquitinated protein
~;-~~ii~i~-i~~i~~-i~~-~ . 60
70
. 60
90
100
___---_____________________________ ______--_______-----________________ I-I ULAMMPTITSREIOTSVRLM# . . . 110
120
130
149
149 j
Wheat H2B L-____-_______..____-..--....-----..----..------~-----.--------------------------------------~~-----------------~~---~~~~~~.-------~~-----~~-~ FIG. 6.
Proposed primary polypeptide structure of the Chlumydomonas 28-kDa ubiquitinated protein. Ubiquitin (upper) and histone H2B (lower) moieties of Chlumydomonus were arranged by referring to the sequence of oat ubiquitin (34) and wheat histone H2B (26), respectively. Positions of amino acids not coinciding are indicated by asterisks. The ranges where no corresponding sequence was observed are filled with “?“. Ubiquitin and histone moieties are assumed to join by isopeptidyl linkage between the C-terminus of ubiquitin and the c-amino group of Lys (indicated by an arrow) in histone.
amount of 28-kDa ubiquitinated protein varied depending on various physiological states, i.e., heat and active oxygen stress, progress of the cell cycle, and gamete induction (19-21). It has been assumed that ubiquitination of histones disturbs tight packing of nucleosomes and such disturbance is necessary to keep transcriptional activity of some genes (10). Varshavsky and co-workers showed that nucleosomal uH2A is preferentially localized on transcribed genes in Drosophila (11) and mouse cells (12). Haas et al. (32) reported that in cultured mammalian cells infection of a certain virus, which leads to turning off the expression of the host genome, caused a decrease of ubiquitinated histone H2A. Nickel et al. (14) reported that in calf, chicken, and Tetrahymena nuclei the most selectively ubiquitinated histone species in the region of transcriptionally active chromatin was H2B, not H2A. Davie and Murphy (33) showed that in human breast cancer cells the ubiquitination level of H2B but not of H2A is coupled to ongoing transcription. These observations strongly suggest that the ubiquitination of histones, especially of histone H2B, plays an important role in the regulation of transcription, and our present results also support this. In contrast to the fact that uH2A is the predominantly ubiquitinated histone in animal nuclei, there was essentially no detectable ubiquitinated protein other than the 28-kDa protein (uH2B) in the Chlamydomonas nuclear fraction (Fig. 1). The reason why no ubiquitinated H2A was observed in the Chlamydomonas nuclei is obscure. As reported so far, the degree of histone H2B ubiquitination in other organisms such as Physarum (5) and Tetrahymena (14) is relatively higher than that in animals. Swerdlow et al. (25) reported that ubiquitinated histone uH2A is also absent in Saccharomyces cerevisiae. These observations and our present results suggest that the ubi-
quitination of H2B is more general than that of H2A in all eukaryotes, while the predominant ubiquitination of H2A observed in several kinds of animals is exceptional. REFERENCES 1. Hershko,
A., and Ciechanover,
A. (1986) Prog. Nucleic Acid Res.
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958-963. 15. Siegelman, M., Bond, M. W., Gallatin, W. M., St. John, T., Smith, H. T., Fried, A. V., and Weissman, I. L. (1986) Science 231,823829. 16. Meyer, E. M., West, C. M., and Chau, V. (1986) J. Biol. Chem. 261, 14,365-14,368. 17. Yarden, Y., Escobedo, J. A., Kuang, W.-J., Yang-Feng, T. L., Daniel, T. O., Tremble, P. M., Chen, E. Y., Ando, M. E., Harkins, R. N.,
Chlamydomonas Franche, U., Fried, V. A., Ullrich, Nature 323,226-232. 18. Zhaung, Z., and McCauley, 14,596.
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UBIQUITINATED
L. T. (1986)
R. (1989) J. Biol. Chem. 264,
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19. Shimogawara, 16.
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