The unique sites in SulA protein preferentially cleaved by ATP-dependent Lon protease from Escherichia coli Wataru Nishii 1 , Takafumi Maruyama 1 , Rieko Matsuoka 1 , Tomonari Muramatsu 2 and Kenji Takahashi 1 1 School of Life Science, Tokyo University of Pharmacy and Life Science, Hachioji, Japan; 2 Biophysics Division, National Cancer Center Research Institute, Chuo-ku, Tokyo, Japan SulA protein is known to be one of the physiological substrates of Lon protease, an ATP-dependent protease from Escherichia coli. In t his study, we i nvestigated the cleavage speci®city of Lon protease toward SulA protein. The enzyme w as shown to cleave  27 peptide bonds in the presence of ATP. Among them, six peptide bonds were cleaved preferentially in the early stage of digestion, which represented an apparently unique cleavage sites with mai nly Leu and Ser r esidues at the P 1 ,andP 1 ¢ positions, r espectively, and one or two 1 Gln r esidues i n positions P 2 ±P 5 . They w ere located in the central region and partly in the C-terminal region, both of which are known to be important for the function of SulA, such as inhibition of cell growth and interaction with Lon prote- ase, respectively. T he other cleavage sites did not represent such consensus s equences, though hydrophobic or non- charged residues appeared to be relatively preferred at the P 1 sites. On the other hand, the cleavage in the absence of ATP was very much slower, especially in the central region, than in the presence of A TP. The central region was predicted to be rich in a he lix and b sheet structures, suggesting t hat the enzyme required ATP for disrupting such structures prior to cleavage. Taken together, SulA is thought to contain such unique cleavage sites in its functionally and structurally important regions whose preferential cleavage accelerates the ATP-dependent degradation of the protein by Lon protease. Keywords: ATP-dependent protease; Lon protease; proteolysis; substrate speci®city; SulA. Lon protease coded by the lon gene of Escherichia coli is an ATP-dependent cytosolic protease [1]. The enzyme degrades two types of substrates in vivo. One type of the substrates includes abnormal proteins such as those with i mproper polypeptide length o r tertiary structure. Their degradation should contribute t o the quality control of i ntracellular proteins. Another involves physiological substrates, such a s SulA, kN, RcsA, CcdA and Pem1, which are short-lived regulatory proteins, whose speci®c and rap id degradation is crucial for normal cell g rowth [2±7]. SulA i s one of the most physiologically important substr ates among the second type. The protein is transcriptionally induced by environ- mental stresses, such as UV irradiation, and prevents premature segregation of damaged DNA into daughter cells during DNA repair processes [8,9]. Induced SulA prevents the self-assembly of FtsZ protein, leading to the inhibition of cell division (®lamentation) [10]. The substrate recogn ition mechanism of Lon protease has not yet been well clari®ed. The cleavage sites by the enzyme in v itro have been reported for kN [5] and C cdA [3] proteins, oxidized in sulin B c hain and g lucagon [5], a nd several ¯uorogenic substrates [11]. In these proteins and peptides, the cleavages occurred mainly a fter hydrophobic residues, in spite that not all such sites were cleaved. So far, however, no more consensus features have been reported in the p rimary or higher-order structures of the substrates. There has been little s tudy on the cleavage sites, particularly for SulA, by Lon p rotease. This is presumably because recombinant SulA was reportedly rather insoluble and/or unstable [12,13]. In the present study, we were able to prepare SulA in a soluble form and investigated its cleavage sites by Lon protease in vitro in the presence and absence of ATP. The results indicated t hat Lon p rotease preferentially cleaves certain unique sites, mainly in the central region of SulA, which are function ally and structurally impor- tant for the protein, thus triggering further rapid and extensive degradation of SulA in an ATP-dependent manner. EXPERIMENTAL PROCEDURES Preparation of E. coli Lon protease Recombinant Lon protease was expressed in E. coli harboring an expression plasmid for the enzyme using T7 promotor (manuscript in preparation). The expressed enzyme was puri®ed by successive steps of column chro- matography on phosphocellulose, DEAE-cellulose and Sephacryl S-300 as described previously [1]. Correspondence to K. Takahashi, School of Life Science, Tokyo University of Pharmacy and L ife Sc ience, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392. Fax: + 81 426 76 7149, Tel.: + 81 426 76 7146, E-mail: kenjitak@ls.toyaku.ac.jp Abbreviations: LC-MS, liquid chromatography-mass spectrometer; MBP, maltose binding protein; 4MbNA, 4-methoxy-b-naphthyla- mide; suc, succinyl; SulA3±169, SulA r esidues 3±169; SulA23±169, SulA residues 23±169. (Received 5 July 2001, revised 8 Novem ber 2001 , accepted 13 November 2001) Eur. J. Biochem. 269, 451±457 (2002) Ó FEBS 2002 Preparation of SulA3±169, SulA23±169 and maltose binding protein (MBP) The expression vector pMAL-p-SulA was a generous gift from S. Sonezaki (Kyushu Institute of Technology, Tobata, Japan). Using the vector, MBP-SulA was expressed in E. coli DH5 cells, puri®ed by amylose-resin chromatogra- phy and treated with factor Xa to generate MBP, SulA3± 169 and SulA23±169 as described previously [12]. After digestion of 500 lg of SulA, generated SulA3±169 and SulA23±169 were separately puri®ed to homogeneity by using a p reparative disc SDS/PAGE apparatus (Nihon Eido Co., Ltd, Tokyo, Japan). The puri®ed SulA3±169 and SulA23±169 solutions (2.7 mL and 4.5 mL, respectively) were then dialyzed against 2 L of 20 m M Tris/HCl, pH 8.0, at 4 °C for 4 days with three changes of the buffer. After concentration of the protein solutions to 300 lLbyan ultrafreeÒ-15 centrifugal ®lter device (Millipore Co.), 80% glycerol was added to t hem to a ®nal concentration of 20%. The ®nal concentrations of SulA3±169 and SulA23±169 were 1.65 mgámL )1 and 0.775 mgámL )1 , respectively. MBP was puri®ed by using AKTA e xplorer 10S with a HiPrep 26/ 60 Sephacryl S-300 HR column (Amersham Pharmacia Biotech, Ltd). SDS/PAGE analysis SulA3±169 and MBP (15 lg each) were separately incubat- ed at 37 °Cwith3lgofLonin25lLof50m M Tris/HCl, pH 8.0, containing 15 m M MgCl 2 , with or without 4 m M ATP. At appropriate intervals, a 3-lL aliquot was with- drawn and the reaction was stopped by adding 3 lLofthe SDS/PAGE sample buffer. These samples were subjected SDS/PAGE (15% gel) and proteins were detected by the Coomassie Brilliant Blue R250 staining. CD spectroscopy CD spectra were measured in a Jasco J-600 spectropola- rimeter. The protein concentrations were determined by amino-acid analysis after acid hydrolysis (6 M HCl, 150 °C, 2 h) u sing an amino-acid analyzer (model 421, PE Applied Biosys te ms Co ., Lt d) . In vitro Lon protease assay The enzymatic act ivity of Lon p rotease toward s uc-Phe- Leu-Phe-4 MbNA (where 4MbNA is 4-methoxy-b-naph- thylamide and suc is succinyl Bachem Ag) was measured as described previously [1]. Brie¯y, 1 lg of Lon protease and 10 nmol of suc-Ph e-Leu-Phe-4MbNA in 100 lLof50m M Tris/HCl, pH 8.0, containing 7.5 m M MgCl 2 ,0.5m M ATP and 0±0.05% SDS, were incubated at 37 °C for 1 h. The reaction was stopped b y addition of 100 lLof1%SDSand 1.2 mL o f 0.1 M sodium borate, pH 9.1 and then the ¯uorescent intensity (excitation, 335 nm; emission, 410 nm) was measured. Identi®cation of the peptide fragments by LC-MS SulA3±169 samples (each 150 lg) were incubated f or appropriate periods with 30 lg of Lon protease in 250 lL of 50 m M Tris/HCl, pH 8.0, containing 15 m M MgCl 2 ,with or without 4 m M ATP. The reaction was stopp ed by addition of 35 lL of 50% trichloroacetic acid to each reaction mixture. The r eaction mixture was then centrifuged and 100 lL of the supernatant was applied to a LCQ TM DUO mass spectrometer (ThermoQuest Co., Ltd), con- nected to an HPLC apparatus (1100 series, Agilent Tech- nologies C o., L td) with a TSKgel-ODS-120T column (150 ´ 2.2 mm, Tosoh C o., Ltd) f or LC-MS analysis. The amino-acid sequences of the product peptides were deter- mined by using a Xcalibur BIOWORKS 1.0 software installed in the apparatus. Sequencing and quantitative analysis of the peptides Part of the reaction mixture described a bove was also applied t o an HPLC apparatus (class LC-10, Shimadzu Co., Ltd) with a TSKgel-ODS-120T column (250 ´ 4.6 mm, Tosoh Co., Ltd) to separate peptides. Each peptide fraction was l yophilized and applied to an amino-acid sequencer (model 477, PE Applied Biosystems Co., L td) and an amino-acid analyzer (model 421, PE Applied Biosystems Co., Ltd) after acid hydrolysis (6 M HCl, 150 °C, 2 h). RESULTS Preparation of SulA3±169 In this study, SulA3±169 was obtained from the MBP±SulA fusion protein by digestion with factor Xa. The protein was then puri®ed to apparent homogeneity by using a prepar- ative SDS/PAGE apparatus, followed by extensive dialysis to remove SDS, which resulted in a soluble form of the protein. The CD spectrum o f SulA is shown in Fig. 1. Using the K 2 D program [14,15], the secondary structure of the protein w as estimated from the spectrum to be 29% in a helix, 15% in b sheet and 56% in random loop structure, which w ere similar to those (34% in a helix, 19% in b sheet Fig. 1. Far-UV CD spectra of SulA3±169 (solid line) and SulA23±169 (broken line). The C D spectrum w ere measured u sing a 0.1- cm cuvette at 37 °C at a protein concentration of 5.4 l M in 20 m M Tris/HCl, pH 8.0, containing 20% glyc erol. 452 W. Nishii et al. (Eur. J. Biochem. 269) Ó FEBS 2002 and 47% in random loop structure) predicted from the primary structure by the pro®le f ed neural network systems from Heiderberg (PHD) [16,17] (see below). SulA23±169 was a lso prepared in the same way and its CD s pectrum w as almost the same as that of SulA3±169 (Fig. 1). The SulA3±169 preparation might possibly contain a small amount of SDS that had not been completely removed b y dialysis. In that case, t he remaining SDS should interfere with the activity of Lon protease. W e therefore investigated the effect of SDS on the activity of Lon protease toward a ¯uorogenic substrate, suc-Phe-Leu- Phe-4MbNA. Table 1 shows that the activity of the enzyme was inhibited by a low concentration of SDS (about 50% inhibition in the presence of 0.0025% SDS). On the other hand, an extensive degradation of SulA3±169 by the enzyme was shown to take place by both SDS/PAGE and HPLC analyses as described below, whereas the puri®ed SulA3± 169 before dialysis, which contained 0.1% SDS, was not degraded at all (data not shown). SDS/PAGE analysis of the degradation of SulA by Lon protease Using SulA3±169 as substrate, its in vitro sensitivity toward Lon protease was investigated by SDS/PAGE analysis. Figure 2 shows that SulA3±169 was degraded b y the enzyme in the presence of ATP with an apparent half-life of 15 min under the conditions used, which is similar to those of MBP-SulA [12] and kN [5] and much shorter than that of CcdA [3], but was scarcely degraded in t he absence ATP during 120 min of incubation. On the other hand, no degradation of MBP was observed either in the presence or in the absence of ATP under the conditions used. These results indicated that the enzyme speci®cally degraded SulA3±169 in an ATP-dependent manner in vitro.Wealso investigated the sensitivity of SulA23±169 toward the enzyme in the same way. The result was esse ntially the same with SulA3±169 (data not shown). Identi®cation of the peptide fragments and determination of cleavage sites After incubation of SulA3±169 with Lon protease for 3 h in the presence of ATP, 32 peptide fragments were se parated by HPLC (Fig. 3) and their amino-acid sequences were determined b y using both an LC-MS apparatus and an amino-acid sequencer. The sizes of the peptides ranged f rom 3 t o 1 6 r esidues (average, 9.4 residues). The peptide fragments obtained after 3 or 30 min of incubation in the presence of ATP and after 3 h of incubation in the absence of ATP w ere also analyze d in the same way. The yields of the fragments were estimated b y amino-acid analyses. The results are shown in Fig. 4. Twenty-seven cleavage sites were identi®ed with the sample incubated for 30 min in the presence of ATP. During incubation for 3 min in t he presence of ATP, preferential cleavages occurred at six peptide bonds: Leu57-Gly58, Leu67-Thr68, Leu73-Ser74, Ala80-Ser81, Leu94-Ser95 and Leu158-Ser159, which were hydrolyzed over 5% (Fig. 4 and Table 2). It was remark- able that these cleavage sites contained mainly Leu and Ser at the P 1 and P 1 ¢ positions, respectively, representing an apparent consensus in the primary structure. The other cleavage sites contained various residues at the P 1 positions (Table 2). The cleavage occurred almost exclusively after nonch arged amino acids, s uch as Ala, Val, Met, Thr, Ser, Leu, Phe, Gln and Gly (20 of the 21 sites), where hydrophobic residues were predominant. However, no apparent consensus residues at other than P 1 positions were found except that Ser appeared to be preferred at the P 1 ¢ position: seven out of 21 Ser residues in SulA3±169 were here and that Gln was abundant in positions P 2 ±P 5 , especially in the ÔfastÕ cleavage sites. On the o ther hand, degradation was very slow in the absence of ATP (Fig . 3 ), indicating that the degradatio n of SulA normally occurs in an ATP-dependent manner, consistent with the r esult o f the SDS/PAG E analysis. However, ®ve peptide b onds: Ser10-Ser11, Phe13-Ser14, Met145-Arg146, Ala150-Ser151 and Leu158-Ser159, were Table 1. Activity of Lon protease toward suc-Phe-Leu-Phe-4MbNA in the presence o f S DS. The activity in the absence o f SDS (0%) was taken as 100%. The assay conditions were described in the Expe rimental procedures section. Concentration of SDS (%, w/v) 4 Relative activity (%) 0 100 0.001 82 0.0025 56 0.005 20 Fig. 2. SDS/PAGE analysis showing the sus- ceptibility of S ulA3±169 and MBP toward Lon protease in the prese nce and abs ence of ATP . Ó FEBS 2002 Unique cleavage sites of SulA by Lon protease (Eur. J. Biochem. 269) 453 cleaved to signi®cant extents in the absence of ATP (over 10% hydrolysis in 3 h, Fig. 4 and Table 2). It was notable that the c leavage mainly occurred, as in t he presence of ATP, between certain hydrophobic residues, such as Ala, Leu, Met and Phe, and Ser and that cleavages occurred at sites other than the major sites of cleavage that occurred in the presence of ATP, except for the cleavage of Leu158- Ser159. DISCUSSION In the present study, SulA3±169 was used exclusively as the substrate protein for Lon protease. P reviously, it w as reported that the pre-MBP-SulA fusion protein was well soluble in a queous solution, but that the free SulA p rotein (SulA3±169) separated from the fusion p rotein b y factor Xa digestion was rather insoluble [12]. In the present study, however, we could prepare a soluble form of SulA3±169, cleaved from the fusion protein, by preparative SDS/PAGE followed by extensive dialysis. SulA3±169 appeared to have been properly refolded during the preparation procedure used. SDS was found to strongly inhibit Lon protease. This is in sharp contrast with the case of the proteasome, another ATP-dependent protease, which is known to be activated by certain concentration ( 0.04%) of SDS [18]. As Lon protease degraded SulA extensively, t he detergent is thought to have been removed suf®ciently from SulA 3±169 by dialysis. The CD spectrum of SulA3±169 showed that the protein had a signi®cant amount of secondary structures, and the secondary structure contents were almost identical with those predicted from the known amino-acid sequence. These results suggested that the SulA3±169 protein had essentially the same s econdary structures with the native Fig. 3. Separation of degradation products of SulA by reverse-phase HPLC. Su lA was incubated with L on p rotease for 3 min, 30 m in and 3hinthepresenceofATPandfor3hintheabsenceofATPas described in the Experimental proc edures section. Reaction m ixtures were applied to the reverse-phase HPLC eluted with a gradient of acetonitrile (0±60% in 60 min). Fig. 4. The yields of the peptides and cleavage sites. The amino-acid sequence of SulA is shown using one-letter code for amino acids. The number for each peptide stands for the peak number corresponding to that in Fig. 3. The numbers in parenthesis indicate the esti- mated percentage yields of each peptid e after 3-min, 30-min, and 3-h incubations in the presence of ATP and 3-h incubation in the absence of ATP in this order. Large, medium and sm all c losed arrowheads indicate the fast, medium and slow cleavage sites in the presence of ATP (see Table 2). Open a rrowheads show the major cleavage sites (over 10% hydrolysis in 3 h ) in the absen ce of ATP. T he seco ndary structures predicted by the PHD [16,17] are shown below the sequence. h, e and blank indicate a helix, b sheet and random loop, respectively. 454 W. Nishii et al. (Eur. J. Biochem. 269) Ó FEBS 2002 SulA, hence presumably possessing the native or native-like structure. SulA23±169 also showed essentially the same CD spectrum and sensitivity toward Lon protease as SulA3± 169, and therefore the segment of residues 3±22 does not seem to be important for secondary structure formation and degradation by Lon protease. In the p resence of A TP, Lon protease hydrolyzed SulA3±169 extensively, whereas MBP, used as a control, was not cleaved at all. This is consistent with the report that, when the pre-MBP-SulA fusion protein was used as the substrate, only the SulA portion was degraded by Lon protease in an ATP-dependent manner [12]. When the digest of SulA3±169 was analyzed by SDS/PAGE, inter- mediate protein bands, with molecular masses at least over 10±12 kDa, were scarcely detected. This may indicate that the initial cleavage at a certain peptide bond is followed by further extensive degradation of the initial cleavage prod- ucts. Indeed, the initial rapid and preferential cleavages were o bserved at a limited number of peptide bonds, including Leu67-Thr68, Leu57-Gly58, Ala80-Ser81, Leu158-Ser159, Leu73-Ser74, and Leu94-Ser95. Interest- ingly, these peptide bonds are all located in the central region of the polypeptide chain except for Leu158-Ser159, which is in the C-terminal region. The central region was reported to be important for t he activity of SulA as a cell- division inhibitor, including essential residues, Arg62, Leu67, Trp77 a nd Lys87, for the inhibitory activity and to presumably constitute a surface for protein±protein interaction [13]. It is tempting to assume that these initial cleavage sites are strategically placed mainly in the central region of SulA so that the cleavage at any of these sites would lead to rapid inactivation of the protein. As for the C-terminal region, it is interesting to note that the C-terminal 20 residues were suggested to be important for the recognition by Lon protease [13] and that the C-terminal eight residues binds speci®cally to the enzyme and prevent the degradation of SulA in vitro [19]. The preferential cleavage in such a region might also contribute somewhat to the rapid degradation of the protein. However, it should be noticed that the cleavage in that region, including one of the major cleavage site, Leu158- Leu159, occurred well with or without ATP. Comparison of the nucleotide s equences of several enterobacterial sulA genes shows that the amino-acid sequences around the sites corresponding to the major sites in SulA are well conserved [20], suggesting that s uch a regulatory mechanism of SulA might a lso e xist in other e nterobacteria. It is a lso an interesting issue to see whether such a mechanism exists in other proteolytic regulatory systems, such as the protea- some±ubiquitin system [21]. In the absence of ATP, degradation of SulA3±169 was extremely slow, but partial hydrolysis w as observed at Table 2. C leavage sites of SulA by Lon protease. Cleavage rat e (% hydrolysis): fast, over 5% in 3 min; medium, below 5% in 3 min, but ov er 14% in 30 min; slow, below 5% in 30 min. Cleavage site: amino-acid residues at P 5 ±P 1 and P 1 ¢±P 5 ¢ sites a re shown. Asterisks indicate major cleavage sites (over 10% hydrolysis) in 3 h in the absence of ATP. Cleavage rate Cleavage site P 5 P 4 P 3 P 2 P 1 ¯ P 1 ¢ P 2 ¢ P 3 ¢ P 4 ¢ P 5 ¢ Fast W Q L W L67 T68 P Q Q K L L Q Q L57 G58 Q Q S R E W V Q A80 S81 G L P L A T R Q L158 * S159 G L K I P Q Q K L73 S74 R E W V Q I S Q L94 S95 P C H T Medium R P V S A150 * S151 S H A T H A E L V132 D133 A A N E M G F I M145 * R146 P V S A S M V R A106 L107 R T G N R A L R T109 G110 N Y S V N Y S V V115 I116 G W L A I G W L A120 D121 D L T E L I S E V36 V37 Y R E D P L T K V88 M89 Q I S Q Y A H R S10 * S11 S F S S V I G W L119 A120 D D L T M T Q L L49 L50 L P L L L T K V M89 Q90 I S Q L R S S S F13 * S14 S A A S A S K I A21 R22 V S T E T T A G L32 I33 S E V V Slow M M T Q L48 L49 L L P L A E L V D113 A114 A N E G T K V M Q90 I91 S Q L S L G Q Q S61 R62 W Q L W R Q L S G160 L161 K I H S Ó FEBS 2002 Unique cleavage sites of SulA by Lon protease (Eur. J. Biochem. 269) 455 various site s. Interestingly, the major cleavage occurred not in the central region, but in the N- and C-terminal r egions of the protein. Thus, the major cleavage sites w ere topologically quite different i n t he presence and absence of ATP. Although the mechanism of ATP-dependent hydrolysis is not clear, ATP appears to somehow activate Lon protease so that the enzyme may disrupt the h igher-order s tructure of SulA, especially in the central region, and preferentially attack certain peptide bonds that are not well cleaved in the absence of ATP. It may be worthy o f note that the central region of SulA is especially rich in secondary structures. In the case of C cdA degradation, it was also suggested that the enzyme might disrupt the secondary structure o f the protein in an ATP-dependent manner [3]. Although the major cleavage sites were topologically different i n the presence and absence of ATP, amino-acid residue speci®city at the cleavage sites w ere e ssentially the same with or without ATP. The ATP-independent cleavage a t both t erminal regions will not impair t he physiological function of SulA 2 as these regions were reported to be dispensable for its activity in viv o [13]. The f act that in the presence of ATP the initial cleavage at a certain peptide bond appeared to be followed by further extensive degradation of the initial cleavage products suggests the possibility that Lon protease may be a kind of processive enzyme, like the 20S proteasome and C lpAP protease [22±24]. Indeed, such a possibility h as been discussed previously [25]. The oligomeric structure of Lon protease [26], somewhat resembling those of 20S protea- some and ClpAP, is consistent with this supposition, although further studies are necessary to draw a de®nite conclusion in this regard. As for the amino-acid residue speci®city of Lon protease toward SulA, it is notable that all the P 1 positions relative to the scissile bonds were occupied by uncharged amino-acid residues except for one case (Asp) and that nonpolar or hydrophobic amino-acid residues, such as Leu, Ala, Val, Met and Phe were predominant among them. On the other hand, no other clear-cut consen sus residues or sequences o f residues were foun d around the cleavage sites. However, it should be noted that as for the six major sites, Leu was predominant at the P 1 position and Ser was at the P 1 ¢ position. In addition, Gln appears to be also predominat at the P 2 ±P 5 positions. There may be certain subsite interac- tions that render these s ix peptide bo nds particularly susceptible to Lon protease, although it is not clear what these are from the amino-acid sequences. In conclusion, the results presented here suggest that the cleavage of the unique sites in its functionally and structur- ally important regions of SulA may accelerate its ATP- dependent degradation by Lon protease, a nd contribute to a rapid and accurate regulation of the SulA function. T o our knowledge, this is the ®rst time that such a correlation has been suggested for the action of Lon protease toward its physiological substrate protein. ACKNOWLEDGEMENTS 3 This study was supported in part by grants-in-aid for scienti®c research from the Ministry of E ducation, Science, Sports and Culture of Japan. We gre atly t hank D r Shuji Sonezaki (Department o f A pplied Chemistry, Faculty of Engineering, Kyushu Institute of T echnology, Tobata) for providing the pMAL-p-SulA plasmid. REFERENCES 1. Goldberg, A.L., Moerschell, R.P., C hung, C.H. & Maurizi, M.R. (1994) ATP-dependent protease La (Lon) from Escherichia coli. Methods Enzymol. 244, 350±375. 2. Mizusawa, S. & Gottesman, S. (1983) Protein degradation in Escherichia coli:thelon gene controls the stability of sulA protein. Proc. Natl Acad. Sci. USA 80, 358±362. 3. Melderen, L.V., Thi, M.H.D., Lecchi, P., Gottesman, S ., Coutu- rier, M. & Maurizi, M.R. (1996) ATP-dependent degradation of CcdA by Lon protease. J. Biol. Chem. 271, 27730±27738. 4. Maurizi, M.R., Trisler, P. & Gottesman, S. (1985) Insertional mutagenesis of the lon gene in Escherichia coli: lon is dispen sab le . J. Bacteriol. 164, 1124±1135. 5. Maurizi, M.R. (1987) Degradation in vitro of Bacteriophage kN protein b y Lon prote ase from Escherichia coli. J. Biol. Chem. 262, 2696±2703. 6. Torres-Cabbasa, A.S. & Gottesman, S. (1987) Capsule synthesis in Escherichia c oli K-12 is regulated by proteolysis. J. Bacteriol. 169, 981±989. 7. Tsuchimoto, S., Nishimura, Y. & Ohtsubo, E. (1992) The stable maintenance syste m pem of plasmid R100: degradation of Pem1 protein may allow PemK p rotein to inhibit cell growth. J. Bacte- riol. 174, 4205±4211. 8. Huisman, O. & D 'Ari, R . (1981) An inducible DNA replica- tion-cell division coupling mechanism of E. coli. Nature 290, 797±799. 9. Huisman, O., D'Ari, R. & Gottesman, S. (1984) Cell-division control in Escherichia coli: speci®c induction of the SOS function S®A protein is sucient t o block septation. Proc. Natl Acad. Sci. USA 81, 4490±4494. 10. Bi, E. & Lutkenhaus, J. (1993) Cell division inhibitors SulA a nd MinCD pre vent formation of the FtsZ ring. J. Bacteriol. 175, 1118±1125. 11. Waxman, L. & Goldberg, A.L. (1985) Protease La, the lon gene product, cleaves speci®c ¯ uorogenic peptides in an ATP-depen- dent reaction. J. Biol. Chem. 260, 12022±12028. 12. Son ezaki, S., Ishii, Y., Okita, K., Sugino, T., Kondo, A. & Kato, Y. (1995) Overproduction and puri®cation of SulA fusion protein in Escherichia coli and i ts degradation by Lon protease in v it ro . Appl. Microbiol. Biotechnol. 43, 304±309. 13. Higashitani, A., Ishii, Y., Kato, Y. & Horiuchi, K. (1997) Func- tional dissection of a cell-division inhibitor, SulA, of Escherichia coli and its negative regulation by Lon. Mol. Gen. Genet. 254,351± 357. 14. Andrade, M.A., Chacon, P., Merelo, J.J. & Moran, F. (1993) Evaluation of secondary s tructure of proteins from UV c ircular dichroism spectra using an unsupervised learning n eural n etwork. Protein Eng. 6, 383±390. 15. Merelo, J.J., Andrade, M.A., Prieto, A. & Moran, F. (1994) Proteinotopic feature maps. Neurocomputing 6, 443±454. 16.Rost,B.&Sander,C.(1993)Predictionofproteinsecond- ary structure at better than 70% accuracy. J. Mol. Biol. 232, 584±599. 17. Rost, B . & Sander, C . (1994) Combining evolutionary information and neural networks to p redict protein secondary structu re. Proteins 19, 55±72. 18. Saitoh , Y., Yokosawa, H. & Ishii, S. (1989) Sodium dodecyl sulfate-induced conformational and enzymatic changes of multicatalytic proteinase. Biochem. Biophys. Res. Commun. 162, 334±339. 19. Ishii, Y ., Sonez aki, S., Iwasaki, Y., Miyata, Y., Akita, K., Kato, Y. & Amano, F. (2000) Regulatory role of C-terminal residues of SulA in its degradation by lon protease in Escherichia coli. J. Biochem. 127, 837±844. 20. Fleudl, R., Braun, G., Honore, N. & Cole, S.T. (1987) Evolution of the enterobacterial sulA gene: a component of the SOS system encoding an inhibitor of cell division. Gene 52, 31±40. 456 W. Nishii et al. (Eur. J. Biochem. 269) Ó FEBS 2002 21. B ochtler, M., Ditzel, L., G roll, M., Hartmann, C. & Huber, R. (1999) The proteasome. Annu. Rev. Biophys. Biomol. Struc. 28, 295±317. 22. T hompson, M.W ., Setyendra, K.S. & Maurizi, M.R. (1994) Pro- cessive degradation of p roteins by ATP-dependent Clp protease from Escherichia coli. J. Biol. Chem. 269, 18209±18215. 23. Akopian, T.N., Kisselev, A.F. & G oldberg, A.L. (1997) Processive degradation of proteins and other catalytic properties of the pro- teasome from Thermoplasma acidophilum. J. Biol. Chem. 272, 1791±1798. 24. Breyer, W.A. & Matthews, B.W. (2001) A structural basis for processivity. Protein Sci. 10, 1699±1711. 25. Maurizi, M .R. (1992) Proteases and protein degradation in Escherichia coli. Ex per imentia 48, 178±201. 26. S tahlberg, H., Kutejova, E., Suda, K., Wolpensinger, B., Lustig, A., Schatz, G., Engel, A. & Suzuki, C.K. (1999) Mito- chondrial Lon of Saccharomyces cerevisiae is a ring-shaped protease with seven ¯exible subunits. Proc.NatlAcad.Sci.USA 96, 6758±6790. Ó FEBS 2002 Unique cleavage sites of SulA by Lon protease (Eur. J. Biochem. 269) 457 . The unique sites in SulA protein preferentially cleaved by ATP-dependent Lon protease from Escherichia coli Wataru Nishii 1 , Takafumi. h). RESULTS Preparation of SulA3 ±169 In this study, SulA3 ±169 was obtained from the MBP SulA fusion protein by digestion with factor Xa. The protein was then puri®ed