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Plant Science Letters, 30 (1983) 303--320 303 Elsevier Scientific Publishers Ireland Ltd.
GROUP FRACTIONATION OF WHEAT GERM RIBOSOMAL PROTEINS
MICHAEL M. SIKORSKI a, DANUTA PRZYBYL a, ANDRZEJ B. LEGOCKI a and KNUD H. NIERHAUS b
alnstitute of Biochemistry, Agriculture, University, Wolyhska 35, 60-637 Poznati (Poland) and b Max-Planck-lnstitut ffir Molekulare Genetik, Abt. Wittmann, lhnestrasse 63-73, lOOO Berlin 33 (F.R.G.)
(Received September 22nd, 1982) (Revision received November 25th, 1982) (Accepted December 14th, 1982)
SUMMARY
Proteins from wheat germ ribosomes were isolated by CM-ceUulose chromatography. The 35 proteins of the small subunit (40S) were fraction- ated into 10 main groups by a linear KC1 gradient at pH 7.6, and the 44 ones o.f the large subunit (60S) into six groups by stepwise elution with increasing concentration of potassium acetate at pH 5.5. The proteins of two fractions were further purified using a linear gradient at pH 7.6. Most of the fractions contained only 2--5 proteins. Five proteins from the small subunit and four proteins from the large one could he isolated .to near homogeneity.
Key words: Wheat germ ribosomal proteins -- Protein isolation
INTRODUCTION
Abou t 50% of the mass of eukaryotic ribosomes consists of a large number of different r ibosomal proteins. Since most of the 70--80 ribosomal proteins have similar isoelectric points and molar masses, the isolation and purification of r ibosomal proteins is a challenging task. Various strategies for the isolation of proteins from eukaryotic ribosomes, such as salt fractiona- tion, ionic exchange chromatography and gel filtration, have been employed [1--6].
In recent years, the wheat germ system has become a reference system for eukaryotic protein biosynthesis in vitro. Therefore, we focused our interest on structure and funct ion of wheat germ ribosomes. A prerequisite of a structural analysis is the isolation and characterisation of the r ibosomal constituents. We have recently shown that cytoplasmic ribosomes derived
0304-4211/83/$03.00 © 1983 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
304
from wheat germ contain at least 79 ribosomal proteins [7]. Here we report a simple method for a group fractionation of these ribosomal proteins from both subunits, including the purification of 9 proteins to near homogeneity.
MATERIALS AND METHODS
Preparation of ribosomes, ribosomal subunits and ribosomal proteins Ribosomes were isolated from wheat germ (General Mills, Vallejo, CA)
as described previously [7] with minor modifications. The ribosomal pellet was dissolved in a dissociation buffer: 20 mM Tris--HC1 (pH 7.6), 400 mM KCI, 3 mM MgCI2, 5 mM 2-mercaptoethanol and 5% sucrose (w/v) containing 1 mM puromycin and 1 mM GTP. Ribosomes were then incubated for 30 min at 37°C and the subunits were separated by zonal centrifugation in a Ti 15 Beckman rotor using a 10--38% (w/v) linear sucrose gradient in dissociation buffer. Fractions corresponding to each subunit were pooled and recovered by centrifugation at 35 000 rev./min for 12 h at 4°C in a 42.1 Beckman rotor. The pelleted subunits were suspended in 20 mM Tris-- HOAc (pH 7.6), 50 mM KC1, 5 mM Mg(OAc)2, 5 mM 2-mercaptoethanol containing 10% (v/v) glycerol. Ribosomal proteins were extracted from 40S and 60S subunits by 67% acetic acid according to Hardy et al. [8].
Carboxymethylcellulose Chromatography The proteins extracted from each subunit were applied onto a CH-ceUulose
column at room temperature (CM 52 preswollen, Whatman Inc.; 1.2 × 12 cm). Routinely, 60 A280 units of the total 40S subunit proteins suspended in 15 mM Tris--HOAc (pH 7.6) and 6 M urea were separated on the column which was equilibrated with the same buffer.
The column was washed with the buffer to remove unadsorbed proteins and then developed with 1 1 of a 0--400 mM KC1 linear gradient in the above buffer. Fractions (3-ml) were collected at a flow rate of 15--20 ml/h and were measured for protein content by adsorption at 280 nm.
Total proteins (90 A2s0 units) from the 60S subunit (TP60) in 5 mM KOAe (pH 5.5) and 6 M urea were applied to a CM-cellulose column (1.4 × 15 cm) equilibrated with the same buffer. Adsorbed proteins were then eluted from the column by increasing stepwise the concentration of KOAc (pH 5.5) (5 raM, 250 raM, 400 mM and 600 mM KOAc) in the presence of 6 M urea. Two groups of 60S subunit proteins obtained by this stepwise fractionation were resolved further by chromatography on CM- cellulose at pH 7.6 using a linear gradient of 0--400 mM KC1 in 15 mM Tris--HOAc (pH 7.6) with 6 M urea.
Polyacrylamide gel electrophoresis and identification of proteins The ribosomal proteins were identified by two-dimensional gel electro-
phoresis according to Kaltschmidt and Wittmann [9], except that the second dimension gel contained 16% (w/v) acry!Amide as described earlier [7]. The
305
A
O
- r (3.
e
B
O
"1" a.
e
$7
L8 * ..!:" Lm
Li:l'" ~-':
S26
L2 L&
S13
L6
L18
L39 L40 1 L42 ~ ~ L 4 1
I
pH 8.2 -~ o
$1 E" S2
:.I"~.,$3 Sl. S8 "LI: SS:~lm~.~6
S 1 6 ~ I "" SI m
$25 ~ i ~ C s29
$32 :~i~: $ 3 3
r :
C; S 34
L•43 ~ 4
pH 8.2 o
L1 L3
Q
L7 L9 LIO L12 L13 "L11,
2 ~ ~ L 1 5 L19 L22 L 2 0 ~ l l ~ ' ~1~'
s L z ? ~ ~ z e .,L3z
L35 L36
L39
L42 C:.
z, OS
60S
L37
L38
L40 q~.41
$35
e
Fig. 1. Two4iimensional polyacrylamide gel electrophoresis of proteins from the 40S (A) and 60S (B) ribosomal subunits from wheat germ. Dotted circles indicate the proteins weakly stained. The inset in B shows the fastest migrating proteins L43 and LA4 which run out of the gel under standard conditions (12 h in second dimension), whereas here the run lasted 8 h in the second dimension.
306
purity of the separated proteins was assessed by slab SDS-gel electrophoresis according to Laemmli [10]. The proteins isolated by CM-cellulose chromato- graphy were extensively dialyzed against 0.5% acetic acid, lyophylized, and dissolved in sample buffer. Identification of ribosomal proteins by two- dimensional gel electrophoresis was facilitated by the comparison of the sample pattern with that of a mixture containing the sample and a small amount of corresponding total proteins.
Determination of protein concentration The concentration of protein was measured by the Bradford method [11]
using bovine serum albumin as a standard.
RESULTS AND DISCUSSION
Figure 1 shows two<iimensional patterns of 40S and 60S ribosomal proteins. The total number of ribosomal proteins revealed from both isolated subunits is 79.
Thirty-five proteins were found to be constituents of the small subunit in reasonable agreement with a recently published number of 32 [6], 44 proteins to be constituents of the large subunit. In order to facilitate separation and further purification of individual proteins a group fractiona- tion procedure was developed for both the 40S and 60S subunits.
A general strategy used for group fractionation of ribosomal proteins from various sources is often based on successive washing of the subunits with buffers of increasing ionic strength, followed by chromatography according to charge or by gel filtration according to molecular weight. However, preliminary fractionation of wheat germ ribosomal proteins by the NH4C1/EtOH or KC1/EtOH wash procedure or LiC1 splitting appeared to be insufficient, since we observed that the majority of proteins appeared in almost all the fractions obtained (unpublished data).
Fractionation of 40S sub unit proteins The proteins of the small ribosomal subunit were separated into 10 main
groups by gradient fractionation on CM-cellulose (Fig. 2). The proteins of each fraction were then identified by both SDS one<limensional and urea two<iimensional gel electrophoresis (Fig. 3).
All three acidic proteins of the 40S subunit: $7, $13 and $26 together with four basic proteins, $8, $15, $17 and $32, were present in the first fraction (flow through; Fig. 3 (la,b)). Several 40S proteins were recovered in a quite pure state. These were $8 in fraction 2, $21 in fraction 3, $30 in fraction 8 and $9 in fraction 10. In two other fractions two proteins were detected with only minor contaminants: $4 and $14 in fraction 6, $18 and $23 in fraction 7. Table I summm~zes the fractionation of the 40S ribosomal proteins by CM~ellulose chromatography.
D
!I
0.1
0.05
B
B
O
~S
1
2 3
4 5
M
6 7
8 9
10
t~
E
60
10
30
6
J .L
1
--
5 80
12
0 16
0 20
0 F
RA
CT
ION
S
Fig
. 2.
Cl~
om
ato
gm
pb
y o
n c
arb
ozy
me
t~yl
ee
llu
lose
of
40S
rib
oso
ma
l su
bu
nit
pro
tein
s. T
he
pro
tein
s w
ere
an
aly
zed
by
two
-dim
e]~
ion
al
po
lya
czT
lam
ide
gel
e]e
etro
pbor
esis
in u
rea
(F
ig.
3) a
nd
by
SD
S-g
el e
Ject
ropb
ores
is (s
ee p
ho
to).
(M
--
mo
lecu
lar
we
igh
t m
arke
rs:
phos
- p
bo
ryla
se B
-94
00
0;
bo
vin
e s
erum
alb
um
in -
- 67
00
0;
ova
lbu
min
--
43
00
0;
carb
on
ate
ar~
ydra
se -
- 3
00
0;
soyb
ea
n tr
ypsi
n i
nh
ibit
or
--
20 1
00; ]
yso
zy
m-
13 9
00).
4a
~ °
~
II
B
t m
4b
O
lb °
1MI5
e
$22
Q
3
T~
!L
- $23
MI
e~
7
~ ~
8 g
st
Qs~7
qB
#
Q
t0
g
$~
Fig
. 3.
Tw
o~ii
men
sion
al p
olya
cryl
amid
e ge
l el
ectr
oph
ores
is o
f 40
S r
ibos
omal
pro
tein
s af
ter
sepa
rati
on i
nto
ten
gro
ups
on a
car
box
y-
met
hyl
-cel
lulo
se c
olu
mn
at
pH 7
.6.
Th
e p
rote
ins
of e
ach
grou
p w
ere
anal
yzed
by
two-
dim
ensi
onal
gel
ele
ctro
ph
ores
is t
o as
sess
th
eir
pu
rity
, and
th
ey w
ere
also
ele
ctro
ph
ores
ed w
ith
a s
mal
l am
oun
t (a
bou
t 50
~g)
of
TP
40S
as
a b
ack
grou
nd
for
th
e id
enti
fica
tion
of
the
prot
eins
.
r~
310
TABLE I
GROUP FRACTIONATION OF THE PROTEINS FROM THE 40S RIBOSOMAL SUBUNITS
The symbols used are: +++, present in large amounts; ++, present in small amounts; +, present in traces. P~roteins S1, $2, $3, $11, $20, $31, $33, $34 and $35 were not found in any group.
Protein Group
1 2 3 4 5 6 7 8 9 10
$4 $5 $6 S7 + $8 + +++ S9 S10 S12 S13 ++ S14 S15 + S16 + S17 + + S18 S19 821 $22 ++ S23 $24 $25 $25a $26 +++ $27 $28 $29 $30 S32 ++
÷ ÷ ÷ ÷ ÷ ÷
÷ ÷ ÷ ÷
÷ ÷ ÷
÷ ÷ ÷ ÷
÷ ÷ ÷ ÷ ÷
÷ ÷ ÷
÷ ÷ ÷ ÷ ÷ ÷ ÷
÷ ÷ ÷ ÷ ÷
÷ ÷ ÷ ÷ ÷ ÷
÷ ÷ ÷ ÷
÷ ÷ ÷ ÷ ÷ ÷ ÷
• ÷
÷ ÷
÷ ÷ ÷ ÷ ÷ ÷ ÷ ÷
÷ ÷ ÷ ÷ ÷
÷ ÷ ÷ ÷ ÷ ÷ ÷ ÷ ÷
÷ ÷
÷ ÷ ÷
÷ ÷ ÷ ÷
÷ ÷ ÷ ÷ ÷
÷ ÷ ÷ ÷ ÷
÷ ÷ ÷
Fractionation o f 60S subunit proteins T h e 60S p ro te ins were f r ac t i ona t ed into six g roups (A, B1, B2, C1, C2
and D) b y s tepwise e lu t ion f r o m CM-cellulose wi th increasing concen t ra - t i ons o f p o t a s s i u m a c e t a t e ( p H 5.5) , in t h e p resence o f 6 M urea {Fig. 4). F r a c t i o n A c o n t a i n e d a g roup o f s t rong ly acidic p ro te ins which d o n o t b ind to CM~cellulose u n d e r these condi t ions . T h r e e o f these p ro t e in s were LS, L l l and L16 , wh ich in te res t ing ly can be s t rong ly p h o s p h o r y l a t e d in v i t ro [7] . Peak B~ consis ts p r e d o m i n a n t l y o f t he acidic p ro t e in L18. T h e f rac t ions B: and C1 con ta in a b o u t 20 p ro t e in s (see T a b l e II) . T h e r e is a lmos t no over lap b e t w e e n these t w o f rac t ions . In con t ras t , t he f rac t ions CI and C2 con ta in v i r tua l ly t he s ame set o f p ro t e in s e x c e p t L30 in C2 (Fig. 5). T h e
U3
C:)
tO
n U
D
1.5
I --
1.0
• "-
94.0
00
"-67
000
• -,,-
43.
000
-'---
30.
000
! 0.
5~
i ....
..
....
. -
| :
B 20
• "--
20.
100
• '--
13.9
30
I 40
C
r'
C2
I 60
FR
AC
TIO
NS
0.8-
T- i
0.4
Fig.
4.
Gro
up f
ract
iona
tion
of
60S
ribo
som
al s
ubun
it p
rote
ins
by c
hrom
atog
raph
y on
car
boxy
met
hylc
ellu
lose
col
umn
(ste
pwis
e el
utio
n at
pH
5.5
). T
he i
nset
pho
togr
aph
show
s SD
S-ge
l el
ectr
opho
resi
s of
pro
tein
s pr
esen
t in
eac
h gr
oup.
The
sta
ndar
d pn
)tei
ns f
or
the
mol
ecul
ar m
ass
(M)
are
give
n in
the
leg
end
to F
ig.
2. F
or d
etai
ls s
ee M
ater
ials
and
Met
hods
. k
~
t.a
| +
""
i I
+L__
t e
° 4
!
r
r--
I
Ii
It.
" 0
+ 0
lip Ill
L'b
Cl L3
70
~ 8 L32
L35 L36 ~ L 37
, ~ L 3 ~
" 0
'Hill
0
o q P
LI
L L~
O
C2
0
tL3,r,
-L37
4 P L~a
Fig. 5. Two~timensional polyacrylamide gel electrophoresis of proteins from the 60S ribosomal subunit. The isolation of these proteins is shown in Fig. 4.
314
TABLE II
GROUP FRACTIONATION OF THE PROTEINS FROM THE 60S RIBOSOMAL SUBUNITS
Symbols +++, ++ and + are the same as in Table I. Proteins L2, L4, L5, L6, L10, L15, L40, L43 and L44 were not found in any group.
Protein Group
A B~ B2 C, C2 D
L1 L3 L7 L8 L9 L l l L12 L12a L12b L13 L14 L14a L16 L17 L18 L19 L20 L21 L21a L22 L23 L24 L25 L25a L25b L25/26 L27 L28 L29 L30 L31 L32 L33 L34 L35 L36 L37 L38 L39 L41 L42
4-4-
Jr Jr
4-jr
+ j r
Jrjr 4-4-jr 4-4-
Jr4- 4-4-
4 - j r÷ J r ÷
÷ ÷ j r 4- j r j r
Jr-I-jr
-I-jr
Jr4-
4-jr Jr
Jrjr4-
4-
4-4-4-
4-4-
-I-4-
4-jr4-
+
4-4-
4-4-
4-4-
÷4-4-
Jr4-
Jr4-
4-4-4- ÷ j r ÷
4-4-4- Jr4-jr
4-4- 4-4-4-
4-4-4-
÷ ÷ ÷ 4-4-4-
4-4-
4-4-4- 4-4-4-
4-4-4-
4-
4-÷4- 4-
4- 4-4-
4-4- 4-4-4-
4- 4-
4-4- 4-4-4-
4- 4-4-4-
4-
4-4- 4-
4-4-4- 4-4-4-
4-4-4- 4-4-4-
4-4-4-
4-4-4-
+4-
+4-
Jr-t-
4-4-
0,08
60S
B2M
3
4 5
6 7
8 9
10
11
12
13
14
15
16
17
18
0.04
o
---
^ o_
._..~
o ~
_~
_~
--~
88
°
v tn
11
12
13
j"X
....
.""~
.
4,7
,a
2 ~
45 6
7
u ,u
"J
,-
"~
~ "
.~
L .L
t
L J
----
-.-
40
80
120
160
FR
AC
TIO
NS
Fig
. 6
. C
arb
ox
ym
eth
ylc
ellu
lose
ch
rom
ato
gra
ph
y o
f 6
08
rib
oso
mal
pro
tein
s in
gro
up
B 2
(li
near
gra
dien
t at
pH
7.6
). T
he
in
set
ph
oto
- gr
aph
show
s SD
S-ge
l ele
ctro
phor
esls
of
prot
eins
pre
sent
in
each
elu
ted
frac
tion
. M
, sta
ndar
d pr
otei
ns f
or
the
mol
ecul
ar m
n~
(se
e Fi
g. 2
). F
or
furt
her
deta
ils
see
Mat
eria
ls a
nd M
etho
ds.
i t/) E
~5
30
15 r~
t.A
C
J1
t
P
411
b
- ql
b
qb
B
Ill
P
0
0
L25/)
b
13
L2~/2~
? L
2¢
~
11.2
5j26
~t
,2~
.... ¢
~i
0
15
16
D
B
t
~J ~
a t2
7
18
Fig
. 7.
Tw
o~ii
men
sion
al p
olya
cryl
amid
e ge
l el
ectr
oph
ores
is o
f p
rote
ins
in g
rou
p B
2 of
60S
rib
osom
al s
ub
un
it.
Th
e is
olat
ion
of
thes
e p
rote
ins
is s
how
n i
n F
ig.
6. T
he
pro
tein
s of
eac
h f
ract
ion
wer
e an
alyz
ed b
y tw
o-d
imen
sion
al g
el e
lect
rop
hor
esis
to
asse
ss t
hei
r p
uri
ty
and
wit
h 5
0 u
g of
TP
60S
as
bac
kgr
oun
d f
or t
he
iden
tifi
cati
on o
f th
e p
rote
ins.
k-4
--3
e.O
i.
I
0.04
0.02
R~k_S
CI
I',~
9 10
I;I
12
13 1
'4 15
16
1'7 1
8 19
~
21 2
2
oo~ j°
.,
A~
~~
~~
/2
.
23
,
I I
65
130
195
FRA
CTI
ON
S
E
60
30
Fig.
8.
Cal
boxy
mat
hylc
ellu
lose
chr
omat
ogra
phy
of
608
ribo
som
al p
rote
ins
in g
roup
C 1
(lin
ear
grad
ient
at
pH 7
.6).
The
ph
oto
gra
ph
sh
ows
8DS-
gel e
lect
roph
ores
is o
f pr
otei
ns i
n ea
ch e
lute
d fr
acti
on. M
, st
anda
rd p
rote
ins
for
the
mol
ecul
ar m
ass
(see
leg
end
to F
ig.
2).
For
fur
ther
det
ails
see
Mat
eria
ls a
nd M
etho
ds.
ii! ~ i~ i ~ ~
if?if! ~ ~ i i ~
!!iii ~ i ~I ~ iii ~ i . . . . ~ ~
Fig. 9. Two~timensional polyacrylamide gel electrophoresis of proteins isolated as shown in Fig. 8.
320
proteins of fractions B2 and C1 were submitted to a second fractionation step on CM~ellulose, applying a linear KC1 gradient at pH 7.6 (see Figs. 6 and 7). From the B2 group proteins L12 and L25 could be purified to near homogenei ty with increasing ionic strength, and from the C1 fraction protein L22 was obtained in a rather pure state. All the other fractions contained two or more proteins (see Figs. 8 and 9).
In summary, the technique described allows an efficient fractionation of the 79 proteins from wheat germ ribosomes. Most of the fractions contain two to five ribosomal proteins. Five proteins from the small and four from the large subunit could be isolated in a rather pure state.
ACKNOWLEDGEMENTS
We thank Drs. H.-G. Wittmann and R. Brimacombe for advice and discussions.
REFERENCES
1 C. Gualerzi, H.G. Janda and G. StSffler, J. Biol. Chem., 249 (1974) 3347. 2 H. Bielka and J. Stahl, Int. Rev. Biochem., 18 (1978) 79. 3 I.G. Wool, Annu. Rev. Biochem., 48 (1979) 719. 4 K. Tsuguri and K. Ogata, Eur. J. Biochem., 101 (1979) 205. 5 T. Itoh, K. Higo and E. Otaka, Biochemistry, 18 (1979) 5787. 6 C.-Y. Ting Shih, J.E. Toivenen and G.R. Craven, Eur. J. Biochem., 97 (1979) 189. 7 M.M. Sikorski, D. Przybyl, A.B. Legocki, W. Kudlicki, E. Gasior, J. Zajac and T.
Borkowski, Plant Sci Lett., 15 (1979) 387. 8 S.J.S. Hardy, C.G. Kurland, P. Voynow and G. Mora, Biochemistry, 8 (1969) 2897. 9 E. Kaltschmdit and H.G. Wittmann, Anal. Biochem., 36 (1970) 401.
10 V.K. Laemmli, Nature, 227 (1970) 680. 11 H.H. Bradford, Anal. Biochem., 72 (1976) 248.