Tài liệu Báo cáo khoa học: The unique sites in SulA protein preferentially cleaved by ATP-dependent Lon protease from Escherichia coli ppt

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:
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