BIOCHEMICAL
Vol. 87, No. 1, 1979
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
March 15, 1979
ACIDIC
PROTEINS
Sdnchez-Madrid, lnstituto
de
FROM
F.,
Saccharomyces
Conde,
Bioquimica
de
C.S.I.C.
Received
281-291
Pages
and
P.,
cerevisiae
Va’zquez,
D.
U.A.M.,
and
Centro
Macromoliculas. Canto
Blanco,
RIBOSOMES
Bal lesta, de
J .P.G.
Biologia
Madrid-34,
Molecular,
Spain
December 28,1978 SUMMARY
Two dimensional gel electrophoresis of ribosomal proteins from Saccharomyces cerevisiae reveals the presence of three spots in the region corresponding to proteins of high acidic character. Washing the ribosomes with 0.4 M NH4Cl and 508 ethanol, followed by chromatography of the extracted proteins on DEAE-cellulose, indicated the presence of two fractions of acidic proteins; (A and Ax), having very similar molecular weights (lZ.OOO-13.000), but phosphorylated to different extents. Fractions A and Ax are immunologically distinct and their immunologic properties differ from acidic proteins found in Escherichia rat 1 iver, and --Artemia sal ina ribosomes. fi, Protein A can be resolved into two bands by electrofocusing, and two dimensional gel electrophoresis. The two components correspond to proteins L44 and L45 according to the standard nomenclature. Proteins Ax seems to correspond to the spot that moves above and to the left of L44 and L45 and is at least three times more phosphoryiated than these two proteins. INTRODUCTION The
presence
to
be
an
isolectric
common
15.000 When one
have these
to
all
point been
(7)
polypeptides.
several types
of
below
pH 5.0
proteins only
among
while
the poor
the homology (10).
in
or
phosphorylation data
proteins with Perhaps
and
from the
of
scarce the
most
the
same
chemical (8,3)
few
properties proteins
weights eukaryotic
in
simple
acidic the
and
acidic
ribosomal
molecular
present
are
strong
Thus,
bacterial
are
differ
with
ribosomes.
in
Although
exist
proteins
reported
acidic
they
copy,
acetylation
rather
of
some
or
12.000
ribosomes
(l-6).
ribosome
in such
two
more
ribosomes from
striking
characteristics
than
as
similarity
proteins
and
unmodified
structural
eukaryotic
having
between
modifications one
seems
seem
studied
Escherichia
(lo),
coli of
to
is these
0006-291X/79/050281-11$01.00/0 281
Copyright All rights
@I 1979
by Academic Press, Inc. in any form reserved.
ofreproduction
Vol. 87, No. 1, 1979
proteins
is
might
the
existance
resemble The
in
E.
some
col
role
i ribosomes in
the
ribosome
role
for
the
however
,
to
identification released
ethanol.
Results
presented
they
in
in
proteins
clarify
the
(15)
and S. or
washing the
S.
AC id ic
be
required
for
are
contradictory
some in
have
ribosomes
of
of the
role required.
We have
cerevisiae
presence
studied
ribosomes of
the
Reports
cerevisiae
three
main
that
(11).
been
ribosomes
functional
molecules
has
factors.
is
myosin
imp1 icated
eukaryotic
their
in
interaction
results, stringent
bacterial
cells
(15).
proteins,
analyzed with
of
a similar
Our
a less
acidic
detail
ribosome
suggest (3).
great
the
their acidic
ammonium-
phosphorylated
proteins
report. AND METHODS
ribosome cerevisiae in low were
The-cells described
are
than
purification
this
to
in
as
there
supernatant
MATERIALS
Cells
seem
Saccharomyces
showing
such
proteins
they
acidic
by
regions
although
proteins
and
proteins
these
and
several
that
for
In order
of
synthesis,
acidic
indicate
requirements
alanine-rich
Specifically,
with
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
proteins
(12)
protein
(13,14).
reports
of
contractil
functional
activity
are
BIOCHEMICAL
Y166 was phosphate co1 I&ted,
grown medium broken.anb
to
(16)
late exponential phase when 32~ labeled cells the ribosomes prepared
in YEPD medium were reauired. as previously
(15). proteins
The acidic proteins were extracted by washing the ribosomes with 0.4 M NH@1 in the presence of 50% ethanol at 0°C (according to Hamel et al. (17)) with the modif ications reported (15). The proteins extracted (SPO.4) were applied to DEAE-cellulose column and eluted essentially in the same condit-ions described for Artemia sal ina proteins (3) but using a steeper gradient (10 mM to 1.5 M)ofium acetate. Proteins were labeled with 125lodine in the presence of chloramine T, when required (18). Electrophores
is
Two
dimensional gel electrophoresis was carried out according to standard system of Kaltschmidt and Wittmann (19) with some modifications (20). When SDS was present in the second dimension, the conditions of Martini and Gould (21) were used. Electrophocusing was done using LKB ampholines between pH 3 and pH 5 in tubes of 10 cm x 0.5 cm following pub1 ished conditions (9).
282
the
BIOCHEMICAL
Vol. 87, No. 1, 1979
When required, spots (22). The absorption estimated.
acid were
Immunological
were at
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
eluted
605 nm and
with 67% acetic of the sample
from the gel plates the radioactivity
techniques
Antisera were rised in rabbit by intramuscular injection of the proteins in complete adjuvant followed by two subcutaneous booster injections in incomplete adjuvant every 15 days. Sera were processed as described (15). lmmunodifusion tests were carried out on 1% agar plates, in 10 mM Tris-HCI, 150 mM NaCl pH 7.4.
125
Radioimmunoassay was carried out using l-labeled protein Ax and anti-SPO.4 bound to CNBr activated Sepharose 4B. About 50000 cpm of labeled Ax (specific activity 18500 cpm/pmol) were added to 40 pl of antiSPO.b-Sepharose (1 mg/ml) and after incubation for 1 h at room temperature and 1 h at 0°C the samples were centrifuged at 5000 rpm for 10 min. The pellet was washed twice with water and finally resuspended in 100 ~1 of water. The bound radiactivity was measured in 50~1 of the final suspension. Competition was done incubating in the presence of increasing concentration of unlabeled proteins.
RESULTS
Two of
S.
dimensional
cerevisiae
character of
shows
moving
E.
coli
have
in
the
L44/45
and
~35/36
detect
the
presence
that
does
and
d).
the
acidic
not When
times
washing
and the
higher
acidic them
the
with
0.4
of
(23)
and
that
region
two
Planta spots
in
second
the
region
dimension
gel
is
resolved
to
nomenclature. in
dimension
carried
into
two
of
spots
in
ri’bosomes
strong
acidic
proteins
Zinker
and
L7 and
L12
Warner
(24)
that
they
named
However,
we
frecuently
L44/45
and
lA,
spots
(Fig.
out
80s
the
with
a fourth a,
presence
b,
of
apparently
c,
SDS,
similar
1B).
the
in
the
presence
specific at
spot can
M NH4Cl
of
activity
absorption in
proteins
spots
and
spots
obtained
to
The
in
(Fig.
radioactive
radioactivity
three
several
from
similar
respective
their
proteins
a position
in
are
ribosomes
to
of
second
weights
d are
gels
of
presence
enter
of
presence
Krujswijk
proteins
molecular In
the
of
the
electrophoresis the
ribosomes.
reported
three
gel
c be and
605
calculated nm
(Table
in
the
a,
from
eluted
b,
the
c,
ratio
spots,
is
at
from
the
ribosomes
When
proteins
and of least
1).
selectively 50%
32,,3- 4 , spots
removed ethanol
283
(15).
thus
by
Vol. 87, No. 1, 1979
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
a
284
BIOCHEMICAL
Vol. 87, No. 1, 1979
Table
1.
Distribution
of
radioactivity
dimensional
among
gel
32 P labeled
spot
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
acidic
proteins
in
Mixture
proteins CPM/A605
A605
‘251-
of
labeled CPM
two
electrophoresis
Ax
and
labeled
un-
A
wm
a
496
0.024
20.6 x lo3
291
b
663
0.028
23.6
866
0.018
60.6 x 103
0.024
24.2
1092
C
d
581
extracted
from
ribosomes
DEAE-cellulose
as
phosphorylated
proteins,
ratio
described
32 P/protein
heavily
in
labeled By
than
of
C marker
or
components
several
fraction
whereas b and Ax,
is seem
proteins in
Ax
fraction
different
spot
was
shown).
32P
and
Methods,
Ax,
were
were
chromatographed two
peaks
obtained
indicated
two
is
of
made made
to
that
Ax
in
of
(Fig.
the
origin
in
very
dimensional
of
(21%)
2).
is
The
more
with
the
second
both
are
to made
molecular
spot
to
the
c.
Both d,
dimension
Ax
close
in
the
a of
weight
one (about
indicated
corresponding
spot of
A and
electrophoresis
moving
formation of
similar
polypeptide
spots
of
a position that
gel
one two
a mixture
suggests
or
only
the
of
This
contaminated
in
939
with
detected
identical
However
contribute
remain
lo3
electrophoresis
of
A is
slightly to
clearly
2666
A.
(not
da1 tons).
that
fractions
a single
cytochrome
Materials A and
103
x
vivo
fractions
gel
SDS,
--in
in
the
polyacrylamide
presence
13.000
labeled
x
position fractions,
thus
part in
to
spot
of
a and
c,
A and of
variable
these amounts
electrophoresis.
Figure 1. Two dimensional gel electrophoresis of 805 ribosomal from S. cerevisiae. A) Second dimension in standard conditions. dimension in the presence of sodium dodecylfulphate.
285
proteins B) Second
Vol. 87, No. 1, 1979
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
‘=Z /’
A
9 IOmM-,
I’
4.0
10
20
30
FRACTION
0 g a.
100
n?
Figure 2. Chromatography on DEAE-cellulose of SPO.4 fraction from 80s r i bosomes . Proteins from the ammonium-ethanol treatment (SPO.4) were applied to a column of 2.5 cm x 20 cm and eluted as indicated in Methods. Radioactivity and protein concentrations were tested in the fractions.
These Ax
and
results
were
unlabeled
proteins
electrophoresis. mainly
to The
sample isolectric three
As spot
main
whose occasionally to
their
from
indicated
of
80s in
fraction
Ax
polyacrylamide
gels
point
close
bands
detected
electrophocused
by
resolving
a mixture
ribosomes
Table
by
two
l-labeled
dimensional
radioactivity
1,
125
of
is
associated
c.
purity in
confirmed
in
intensities
the vary
detected dephosphorylated
to
was between
pH 4.0 when
and the
conditions.
with
the below
electrophocusing pH
corresponds
larger
products.
286
SP
of
the
ones,
the
3). most
proteins
the
the
3 (Fig. to
acidic In
preparation the
by
pH 5 and
total
same
just
confirmed
0.4
ribosomes, probably
has
acidic
(SPo samples
Ax
4)
an of
the
are
faint, are corresponding
bands
BIOCHEMICAL
Vol. 87, No. 1, 1979
Figure Fraction
3.
Electrophocusing SPOe4.
Using possible when is
antisera to
in
with
appears
same
conclusion
and fraction
We have cross
reacts
the
fractions
l-labeled
the
a double can
Ax.
Ax
be
As
1,
acidic give
(Fig. from
as
gels.
total
A and
immunoprecipitation
fact
125
polyacrylamide
against
distinguishable
The SPOs4
that
by
clearly
latter
rised
show
tested
in
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
4).
Fraction
proteins
different The
Ax.
(SPU.,,)
it
precipitation
precipitation
precipitation
2,
band
of
A
was bands
of
band
and
the
Ax
band. drawn
shown
from in
a radioimnunoassay 5 fraction
Fig.
using Ax
does
not
anticompete
A. previously with
antisera
shown rised
that
neither against
(15).
287
of proteins
the
two
fractions
L7 and
L12
A and from
E.
Ax coli
BIOCHEMICAL
Vol. 87, No. 1, 1979
Figure 4 and
4. Double 6, fraction
immunodifusion SPD.4. Well
3,
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
test. Central fraction Ax.
L
10’2
well, Well
anti-SP 5 fracti%4A.We’1s
I
10”
I
100
A@ Df PROTEIN UNLIBELED
288
10’ -O-Q-O-CC.-
A A,
29
Vol. 87, No. 1, 1979
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
DISCUSSION Our three a,
results
main must
L45
and
part
of
of both to
proteins
strong
henceforth
we will
60s
subunit
are
similar
in
different
two
degrees and
Al
and
A2,
are
chemical
fore
possible
that,
if
the
This eL12, the
extent
previously
L44/45
pro&in,
in
yeast,
and
Warner
Protein
Ax
and
variants
of
at Ax
is
in
least
clearly
to
although
it
by
forms fraction might
three
of
Ax,
is
there-
the
site
factor. two
have
to
two
that
process,
not
from
of
It
proteins, been
a single
has
correspond
eL7
shown
to
and be
polypeptide
been spot
(9).
reported PI’ detected s
by
(24).
weight
very
specific times
L45
data
polypeptide.
where
be
represent
suggest
differentiating
and
they
L44/45,
the
that
must
composition
is
autoradiography
high
acid
modifying
L44
find
However,
the
form
dissociation we
that
is
salina,
unphosphorylated c or
its
A.
of
to
and
32 P incorporated
protein.
a single
modification
L44
phosphorylation
amino
equivalent
a molecular
therefore
phosphorylated However,
has
the
b and
They
upon
unlikely
same
probably
spot
only
of
seems
on
as
(24),
ratio
of
spots
nomenclature.
reports
the
the
8)
this
other
cerevisiae
them,
released
extension
of
of
S.
previously
partially
it
of
Two
using
from
similar and
third
the
different
ribosome
named
with
ref.
characteristics
The
Zinker
the is
phosphorylated
be
phosphorylation
of
situation
with
to
Hence
in
proteins
not
them
modification
both
and
to
and
spots,
80s
proteins
agreement
(reported
proteins,
seem
molecules. of
Osawa
refer
phosphorylated the
the
properties.
to
In
of
the
in
acidic
and
(25).
intensity
yeast
presence
respectively
proteins
ltoh
of
ribosomes
the
the
correspond
the
the
show
close
activity more
distinguishable
than from
or
identical
indicated the
other
L44/45
in
that acidic its
to
proteins
it
is
proteins.
antigenic
125 i-labeled Figure 5. Radioimmunoassay. fraction Ax and anti-SPo 4, to Sepharose 4B, were allowed to react in the presence of increasing concentrations of unlabeled fraction A (0) or fraction Ax (B). Results obtained with fraction A were corrected for the 21% contamination of
289
bound
Ax.
Vol.
87,
No.
determinants, with
BIOCHEMICAL
1, 1979
and
characteristics
proteins
and phase.
proteins
The indicate
results
L44/45
L12
that
ti
L41 and
in
in
in
It
present
in
having
different
the
to
namely
A.
sal
ina
to
be
related
L45
and
Ax
results
from
ccl
with
Is
the
a protein to
the
change
indicate
in
in
that
stationary
changing
species having
proteins
L7/12 report).
one
or
Ax
growth ratio
“top
All
of
several
or
whether
polypeptide-two
and
band” them
these
P2
of
in are
A.
ways
system
(8),
(9),
and
ina
to
to
nine
in
proteins of
found
rat
are
ribosomes ccl
detail
1 iver
susceptible
eukaryotic
proteins
rat
sal
up
types
some
immunigenic
in
modified
several some
in or
derivatives, all
In
studied chemical
Pl
and
whether
proteins.
one
iminary
proteins,
known
L44,
eukaryotic
acidic
ribosomes
prel
agreement
four
(4),
yet
COMMUNICATIONS
(26).
cell,
acidic
duplicated
i
(this
not
In
suggested
proteins
ribosomes in
col
cells
been
three
in be
the
yeast
is
all
exist
1s
seem
to
in
bacterial
Is. Nothing
acidic on
L45
of
has
Our
originating
(5).
have
the
from
HeLa
Ax
of
E.
types
phosphorylation 1 iver
in
available
exist
and
Ax
RESEARCH
(9).
would
data
differences
protein.
L44/45
over
L7 and
be a different
preparations.
predominate These
BIOPHYSICAL
to
amounts
ribosomal
L44
L40
to
relative
different
to
similar
equivalent The
ccl
appears
AND
is
known
proteins. these
this
is
In
acidic so,
function
that
protein
species
about our
proteins
the
experience than
proteins
might
is
compatible
with
a more
of
eukaryotic bacterial
these
in
significance
sophisticated
have the
such
a diversity
ribosomes ribosomes
for
a modulator existance cell
are
less
functioning
effect of
of
a larger
on
ribosomal
dependent (15).
the
ribosome
number
of
If
acidic
machinery.
AC KNOWLEDG EHENTS This Biologia (Instituto Laboratories
work was supported by an institutional Molecular from ComisiBn Administradora National de PrevisiBn) and persanal and Lilly Indiana of Spain.
grant to the Centro de de1 Descuento Complementario grants from Essex
Vol. 87, No. 1, 1979
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
REFERENCES 1. 2.
M?iller, 25,5-12. Stgffler,
71,
W.,
Groene, G.,
Wool,
A.,
Terhorst,
I.G.,
Lin,
C. A.
and
and Rak,
Amons, K.H.
R.
(1372)
(1974)
Eur.J.Biochem. Proc.Nat.Acad.Sci.
4723-4726.
3. Mb'ller, 4.
5. 6. 7. 8. 3. 10. 11.
W., Slobin, L.I., Amons, R. and Richter, D. (1375) Proc.Nat.Acad. Sci. 72, 4744-4748. Horak, I. and Schiffmann, D. (1377) Eur.J.Biochem. 79, 375-380. Reyes, R., V6zquez, D. and Ballesta, J.P.G. (1377) Eur.J.Biochem. 73, 25-31. Leader, D.P., Coia, A.A. and Falmy, L.H. (1978) Biochem.Biophys.Res. Commun. 83, 50-58. Terhorst, C., Mgller, W., Laursen, R. and Wittmann-Liebold, B. (1973) Eur.J.Biochem. 34, 138-152. Tsurugi, K., Collatz, E., Todokoro, K., Ulbrich, N., Lightfoot, H.N. and Wool, 1-G. (1378) J.Biol.Chem. 253, 946-955. Van Agthoven, A., Maassen, J.A. and Mb'ller, W. (1977) Biochem.Biophys.Res. Cotrunun. 77, 989-998. Amons, R., van Agthoven, A., Pluijms, W., Mb'ller, W., Higo, K., Itoh, T. and Osawa, S. (1977) FEBS Letters 81, 308-310. Amons, R., van Agthoven, A., Pluijms, W. and Mb'ller, W. (1378)FEBS Letters
86, 282-284. 12. 13. 14. 15. 16. 17.
18. 19. 20. 21. 22. 23. 24. 25.
Mb'ller, W. (1374) In "Ribosomes" (M.Nomura, A.TissiEres and P.Lengyel, eds.), pp. 711-731. Cold Spring Harbor Laboratory, New York. Glick, B.R. (1377) FEBS Letters 73, l-5. Koteliansky, V.E., Domogatsky, S.P., Gudkov, A.T. and Spirin, A.S. (1377) FEBS Letters 73, 6-11. Ssnchez-Madrid, F., Reyes, R., Conde, P., VLzquez, D. and Ballesta, J.P.G. (1973) Eur.J.Biochem. (submitted). Rubin, G.M. (1873) J.Biol.Chem. 248, 3860-3875. Hamel, E., Koka, M. and Nakamoto, T. (1372) J.Biol.Chem. 247, 805-814. Greenwood, F.C., Hunter, W.M. and Glover, J.S. (1363) Bi0chem.J. 83, 114-123. Kaltschmidt, E. and Wittmann, H.G. (1370) Anal.Biochem. 36, 401-412. Howard, F.A. and Traut, R.R. (1374) Methods Enzymol. 30, 526-533. Martin, O.H. and Gould, H.J. (1571) J.Mol.Biol. 62, 403-405. Hardy,S.J.S. (1375) Mol.Gen.Genetics 140, 253-274. Kruiswijk, T. and Planta, R.J. (1375) FEBS Letters 58, 102-105. Zinker , S. and Warner, J.R. (1376) J.Biol.Chem. 251, 1739-1807. Kruiswijk, T., Planta, R.J. and Mager, W.H. (1378) Eur.J.Biochem. 83, 245-
252. 26.
Ramagopal,
S.
and
Subramanian,
A.R.
(1374)
291
Proc.Nat.Acad.Sci.
71,
2136-2140.