269, 451-457 2002 © FEBS 2002 The unique sites in SulA protein preferentially cleaved by ATP-dependent Lon protease from Escherichia colt Wataru Nishii', Takafumi Maruyama’, Rieko Mats
Trang 1Eur J Biochem 269, 451-457 (2002) © FEBS 2002
The unique sites in SulA protein preferentially cleaved
by ATP-dependent Lon protease from Escherichia colt
Wataru Nishii', Takafumi Maruyama’, Rieko Matsuoka’, Tomonari Muramatsu
and Kenji Takahashi’
‘School of Life Science, Tokyo University of Pharmacy and Life Science, Hachioji, Japan; * 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 this study, we investigated the
cleavage specificity of Lon protease toward SulA protein
The enzyme was 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 mainly Leu and Ser residues at the P,, and P,’
positions, respectively, and one or two Gln residues in
positions P,—-P; They were 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 The other cleavage sites did not represent
such consensus sequences, though hydrophobic or non- charged residues appeared to be relatively preferred at the
P, 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 ATP The central region was predicted to be rich in zhelix and sheet structures, suggesting that 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;
proteolysis; substrate specificity; SulA
Lon _ protease;
Lon protease coded by the /on 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 improper
polypeptide length or tertiary structure Their degradation
should contribute to the quality control of intracellular
proteins Another involves physiological substrates, such as
SulA, AN, RcesA, CcedA and Peml, which are short-lived
regulatory proteins, whose specific and rapid degradation is
crucial for normal cell growth [2—7] SulA is one of the most
physiologically important substrates 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 (filamentation) [10]
The substrate recognition mechanism of Lon protease
has not yet been well clarified The cleavage sites by the
Correspondence to K Takahashi, School of Life Science, Tokyo
University of Pharmacy and Life Science, 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 residues 3-169; SulA23-169,
SulA residues 23-169
(Received 5 July 2001, revised 8 November 2001, accepted 13
November 2001)
enzyme in vitro have been reported for AN [5] and CcdA [3]
proteins, oxidized insulin B chain and glucagon [5], and
several fluorogenic substrates [11] In these proteins and peptides, the cleavages occurred mainly after hydrophobic residues, in spite that not all such sites were cleaved So far, however, no more consensus features have been reported in the primary or higher-order structures of the substrates There has been little study on the cleavage sites, particularly for SulA, by Lon protease 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 that Lon protease preferentially cleaves certain unique sites, mainly in the central region
of SulA, which are functionally 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 £ co/i 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 purified by successive steps of column chro- matography on phosphocellulose, DEAE-cellulose and Sephacryl S-300 as described previously [1]
Trang 2Preparation 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 DHS cells, purified 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 ng of SulA, generated SulA3—169 and
SulA23-169 were separately purified to homogeneity by
using a preparative disc SDS/PAGE apparatus (Nihon Eido
Co., Ltd, Tokyo, Japan) The purified SulA3—169 and
SulA23-169 solutions (2.7 mL and 4.5 mL, respectively)
were then dialyzed against 2 L of 20 mmo 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 uL by an
ultrafree®-15 centrifugal filter device (Millipore Co.), 80%
glycerol was added to them to a final concentration of 20%
The final concentrations of SulA3—-169 and SulA23-169
were 1.65 mgmL"! and 0.775 mgmL™", respectively MBP
was purified by using AKTA explorer 10S with a HiPrep 26/
60 Sephacryl S-300 HR column (Amersham Pharmacia
Biotech, Ltd)
SDS/PAGE analysis
SulA3—169 and MBP (15 pg each) were separately incubat-
ed at 37 °C with 3 ug of Lon in 25 wL of 50 mm Tris/HCl,
pH 8.0, containing 15 mm MgCl, with or without 4 mm
ATP At appropriate intervals, a 3-uL aliquot was with-
drawn and the reaction was stopped by adding 3 uL of the
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) using an amino-acid analyzer (model 421, PE Applied
Biosystems Co., Ltd)
In vitro Lon protease assay
The enzymatic activity of Lon protease toward suc-Phe-
Leu-Phe-4MBNA (where 4MBNA is 4-methoxy-[-naph-
thylamide and suc is succinyl Bachem Ag) was measured as
described previously [1] Briefly, 1 tug of Lon protease and
10 nmol of suc-Phe-Leu-Phe-4MBNA in 100 pL of 50 mm
Tris/HCl, pH 8.0, containing 7.5 mm MgCh, 0.5 mm ATP
and 0-0.05% SDS, were incubated at 37 °C for 1 h The
reaction was stopped by addition of 100 pL of 1% SDS and
1.2 mL of 0.1 m sodium borate, pH 9.1 and then the
fluorescent intensity (excitation, 335 nm; emission, 410 nm)
was measured
Identification of the peptide fragments by LC-MS
SulA3-169 samples (each 150 pg) were incubated for
appropriate periods with 30 ug of Lon protease in 250 pL
of 50 mm Tris/HCl, pH 8.0, containing 15 mm MgCl, with
or without 4mm ATP The reaction was stopped by addition of 35 uL of 50% trichloroacetic acid to each reaction mixture The reaction mixture was then centrifuged and 100 wL of the supernatant was applied to a LCQ™ DUO mass spectrometer (ThermoQuest Co., Ltd), con- nected to an HPLC apparatus (1100 series, Agilent Tech- nologies Co., Ltd) with a TSKgel-ODS-120T column (150 x 2.2 mm, Tosoh Co., Ltd) for LC-MS analysis The amino-acid sequences of the product peptides were deter- mined by using a Xcalibur Bloworks 1.0 software installed
in the apparatus
Sequencing and quantitative analysis of the peptides Part of the reaction mixture described above was also applied to an HPLC apparatus (class LC-10, Shimadzu Co., Ltd) with a TSKgel-ODS-120T column (250 x 4.6 mm, Tosoh Co., Ltd) to separate peptides Each peptide fraction was lyophilized and applied to an amino-acid sequencer (model 477, PE Applied Biosystems Co., Ltd) and an amino-acid analyzer (model 421, PE Applied Biosystems Co., Ltd) after acid hydrolysis (6 mM 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 purified 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 of SulA is shown in Fig 1 Using the K2p program [14,15], the secondary structure of the protein was estimated from the spectrum to be 29% in ahelix, 15% in Bsheet and 56% in random loop structure, which were similar to those (34% in o helix, 19% in B sheet
Wavelength (nm)
Fig 1 Far-UV CD spectra of SulA3-169 (solid line) and SulA23-169 (broken line) The CD spectrum were measured using a 0.1-cm cuvette
at 37 °C at a protein concentration of 5.4 um in 20 mm Tris/HCl,
pH 8.0, containing 20% glycerol.
Trang 3© FEBS 2002
and 47% in random loop structure) predicted from the
primary structure by the profile fed neural network systems
from Heiderberg (PHD) [16,17] (see below) SulA23—169
was also prepared in the same way and its CD spectrum was
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 by dialysis In that case, the remaining SDS
should interfere with the activity of Lon protease We
therefore investigated the effect of SDS on the activity of
Lon protease toward a fluorogenic substrate, suc-Phe-Leu-
Phe-4MBNA Table | 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 purified 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 by 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 AN [5] and much shorter than
that of CcdA [3], but was scarcely degraded in the absence
ATP during 120 min of incubation On the other hand, no
degradation of MBP was observed either in the presence or
Table 1 Activity of Lon protease toward suc-Phe-Leu-Phe-4MBNA in
the presence of SDS The activity in the absence of SDS (0%) was taken
as 100% The assay conditions were described in the Experimental
procedures section
Concentration of SDS (%, w/v) Relative activity (%)
Unique cleavage sites of SulA by Lon protease (Eur J Biochem 269) 453
in the absence of ATP under the conditions used These results indicated that the enzyme specifically degraded SulA3—169 in an ATP-dependent manner in vitro We also investigated the sensitivity of SulA23-169 toward the enzyme in the same way The result was essentially the same with SulA3—169 (data not shown)
Identification 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 separated
by HPLC (Fig 3) and their amino-acid sequences were determined by using both an LC-MS apparatus and an amino-acid sequencer The sizes of the peptides ranged from
3 to 16 residues (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 were also analyzed in the same way The yields of the fragments were estimated by amino-acid analyses The results are shown in Fig 4 Twenty-seven cleavage sites were identified with the sample incubated for 30 min in the presence of ATP During incubation for 3 min in the 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, and P;’ positions, respectively, representing an
apparent consensus in the primary structure
The other cleavage sites contained various residues at the
P, positions (Table 2) The cleavage occurred almost
exclusively after noncharged amino acids, such 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, positions were found except that Ser appeared to be preferred at the P,’ position: seven out of 21 Ser residues
in SulA3—169 were here and that Gln was abundant in positions P,—Ps, especially in the ‘fast’ cleavage sites
On the other hand, degradation was very slow in the absence of ATP (Fig 3), indicating that the degradation of
0.002 5 56 consistent with the result of the SDS/PAGE analysis
Met145-Argl46, Alal50-Ser151 and Leu158-Ser159, were
! +ATP -ATP Ì Ï +ATP = —ATP |
0 510153060120 0 60120 0 60120 0 60120 (min)
66K — ‘3°Te eT: - Ce ee - 4
31K —
21K —
Fig 2 SDS/PAGE analysis showing the sus- _ — —_— —SulA3-169 ceptibility of SulA3-169 and MBP toward Lon 14K —
protease in the presence and absence of ATP.
Trang 4
24 26 2325
Hi
+ATP
Retention time (min) Fig 3 Separation of degradation products of SulA by reverse-phase
HPLC SulA was incubated with Lon protease for 3 min, 30 min and
3 h in the presence of ATP and for 3 h in the absence of ATP as
described in the Experimental procedures section Reaction mixtures
were applied to the reverse-phase HPLC eluted with a gradient of
acetonitrile (0-60% in 60 min)
cleaved to significant extents in the absence of ATP (over
10% hydrolysis in 3 h, Fig 4 and Table 2) It was notable
that the cleavage mainly occurred, as in the 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- Ser 159
DISCUSSION
In the present study, SulA3—169 was used exclusively as the substrate protein for Lon protease Previously, it was reported that the pre-MBP-SulA fusion protein was well soluble in aqueous solution, but that the free SulA protein (SulA3—169) separated from the fusion protein by 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, the detergent is thought
to have been removed sufficiently from SulA 3-169 by dialysis
The CD spectrum of SulA3—169 showed that the protein had a significant 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 secondary structures with the native
voy
TSGYAHRSSSFSSAASKIARVSTENTTAGLISEVVYREDQPMMTOLLL
7 (4,21,15; 14) - 5 (1,6,13; 2) ` 8 (2.15,20; 1) 19 (0,0,10; 0) :
-—3i01525.1)
LPLLQOOLGQOSRWQLWLT PQOQKLSREWVQASGLPLTKVMQISQLSPCHTV
Fig 4 The yields of the peptides and cleavage
26 (1,42,31; 1) 29(12,67,39; 2) 4(3,29,34; 1)9 (7,72,82; 4) 28 (5, 16,10; 0) 13 (4.10,24; 3)
sites The amino-acid sequence of SulA is
32 (0,2,6; 0) 16 (3,14,34; 3) 15 (1,4,8; 1) shown using one-letter code for amino acids
18 (0,0,7; 2) The number for each peptide stands for the 3023160) peak number corresponding to that in Fig 3
YY ự vY vy ¥ 7 The numbers in parenthesis indicate the esti-
150 mated percentage yields of each peptide after
3-min, 30-min, and 3-h incubations in the presence of ATP and 3-h incubation in the
"2 (3,35,40; 13)
3 (0,28,38; 7) 31 (1,7,6; 0) 12 (1,19,23; 8) 22 (2,16, 18; 1)
30 (1,8,5; 1) 27 (2.15.16: 1) 23 (0.0,1; 1)
21(0,9,15;1) 14(0,19,30; 1)
I
Ụ 25 (0,10,22: 0)
: vy,
10 (1,5,5; 1)
absence of ATP in this order Large, medium and small closed arrowheads indicate the fast, medium and slow cleavage sites in the presence
of ATP (see Table 2) Open arrowheads show the major cleavage sites (over 10% hydrolysis
in 3 h) in the absence of ATP The secondary structures predicted by the PHD [16,17] are shown below the sequence h, e and blank indicate «helix, B sheet and random loop, respectively.
Trang 5© FEBS 2002 Unique cleavage sites of SulA by Lon protease (Eur J Biochem 269) 455
Table 2 Cleavage sites of SulA by Lon protease Cleavage rate (% hydrolysis): fast, over 5% in 3 min; medium, below 5% in 3 min, but over 14%
in 30 min; slow, below 5% in 30 min Cleavage site: amino-acid residues at P;—P, and P,’—P;’ sites are shown Asterisks indicate major cleavage sites (over 10% hydrolysis) in 3 h in the absence of ATP
Cleavage
Medium R P Vv 5 A150 * S151 5 H A T
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 presence of ATP, 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 observed at a limited number of peptide bonds,
including Leu67-Thr68, Leu57-Gly58, Ala80-Ser81,
Leul58-Ser159, Leu73-Ser74, and Leu94-Ser95 Interest-
ingly, these peptide bonds are all located in the central
region of the polypeptide chain except for Leul58-Ser159,
which is in the C-terminal region The central region was
reported to be important for the activity of SulA as a cell-
division inhibitor, including essential residues, Arg62,
Leu67, Trp77 and 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 specifically to the enzyme and prevent the degradation of SulA im 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, LeulS8- Leul59, occurred well with or without ATP Comparison
of the nucleotide sequences of several enterobacterial su/A genes shows that the amino-acid sequences around the sites corresponding to the major sites in SulA are well conserved [20], suggesting that such a regulatory mechanism of SulA might also exist in other enterobacteria It is also 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 was observed at
Trang 6various sites Interestingly, the major cleavage occurred not
in the central region, but in the N- and C-terminal regions of
the protein Thus, the major cleavage sites were topologically
quite different in the 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 higher-order structure 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 of note that the central
region of SulA is especially rich in secondary structures In
the case of CcdA degradation, it was also suggested that the
enzyme might disrupt the secondary structure of the protein
in an ATP-dependent manner [3] Although the major
cleavage sites were topologically different in the presence and
absence of ATP, amino-acid residue specificity at the
cleavage sites were essentially the same with or without
ATP The ATP-independent cleavage at both terminal
regions will not impair the physiological function of SulA as
these regions were reported to be dispensable for its activity
in vivo [13]
The fact 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 ClpAP
protease [22-24] Indeed, such a possibility has 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 definite
conclusion in this regard
As for the amino-acid residue specificity of Lon protease
toward SulA, it is notable that all the P, 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 consensus residues or sequences of
residues were found around the cleavage sites However, it
should be noted that as for the six major sites, Leu was
predominant at the P,; position and Ser was at the P,’
position In addition, Gln appears to be also predominat at
the PP; positions There may be certain subsite interac-
tions that render these six peptide bonds 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, and contribute to a
rapid and accurate regulation of the SulA function To our
knowledge, this is the first time that such a correlation has
been suggested for the action of Lon protease toward its
physiological substrate protein
ACKNOWLEDGEMENTS
This study was supported in part by grants-in-aid for scientific research
from the Ministry of Education, Science, Sports and Culture of Japan
We greatly thank Dr Shuji Sonezaki (Department of Applied
Chemistry, Faculty of Engineering, Kyushu Institute of Technology,
Tobata) for providing the pMAL-p-SulA plasmid
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