BIOCHEMICAL
Vol. 91, No. 2, 1979 November
AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 643-650
28, 1979
AN ACIDIC
PROTEIN
Saccharomyces
cerevisiae
F. lnstituto
de
August
and
de
TO RIBOSOMES
. CHANGES
Sanchez-Madrid
Biochimica C.S.I.C.
Received
ASSOCIATED
DURING
CELL
CYCLE
J.P.G.Ballesta
Macromoleculas,Centro
,Canto
OF
de
Blanco,Madr
id-34,Spa
Biologia
Molecular,
in
lo,1979
SUMMARY A large proportion of protein Ax, one of the three acidic proteins detected in S.cerevisiae is found in the cell cytoplasm. The fraction of Ax associated with the ribosomes is partially released by treatment of these particles in conditions that remove the bound inttiation factors but a residual amount remains tightly bound to the ribosomes. Protein Ax is associated with the large ribosomal subunit and the amount of protein in the particles duplicates as the celle enters the stationarv phase. The amino acid composition of protein Ax is similar to those of other acidic proteins from the ribosomes of yeast and other species being very rich in alanine (21 .3%) ,glycine (12.2%),aspartic acid (14.7%) and glumatic acid
(14.3%). INTRODUCTION We have visae
reported
of
bilities cells
phosphorylated
similar
to
been
ribosomes (5)
or at
ribosome
(11).
previous
factor
Al
and
inducing
part
The
from
It
the
to
in
typical
ribosomal
protein
some
two
this
conditions
in
the
yeast
ref.
10)
have
been
other
and
make
it
is
called
as
not
and
EF-2
Ax,
was
with not
wel I as related
with
L44/45
authors
named
eukaryotic
factors
factor
than
(1)
moand
elongation
of
phosphorylated
proteins
electrophoretic
the
,by
have
cere--
prokaryotic
interaction we
Saccharomyces
other of
from
which
more
to
they
(8), are
the
mentioned
immunogenical
ly
elongation
results) protein
further
presented
in
is
of
explore
in in
ished
results
although
proteins
ly,
of with
interaction
protein,
other
(unpubl
the
that
(mentioned
third
ribosomes proteins
proteins in
ial
characteristics us
of
these A2
least
activtty The
of
in acidic
implicated
reports.
different
existance
those
(2-7).Two
involved,
in
the
several
have
~35/36
(1)
Ax its
report but
relation seem
somewhat it
is
highly with
to
the
indicate
bound
ribosome.
that
resembles
firmly
interesting,
Ax
The is
a supernatant to
the
not
a factor
ribosome.
0006-291X/79/220643-08$01.00/0 643
Copyright All rights
@I 1979 by Academic Press, Inc. of reproduction in any form reserved.
Vol. 91, No. 2, 1979
BIOCHEMICAL
MATERIALS Preparation
of
AND BIOPHYSICAL
AND
RESEARCH COMMUNICATIONS
METHODS
r ibosomes.
Cells of Saccharomyces cerevisiae Y166 grown in YEPD medium (11) in a Lab-Line/S.M.S. Hi-density fermentor were broken by sea sand grinding in Buffer 1 (80 mM #Cl, 12.5 mM MgCl I mM DTT and 100 mM Tris-HCI, pH 7.4), the suspension was centrifiied at low speed (5.000 rpm for 20 min.) in order to remove sand and the larger cell debris and the supernatant was centrifuged again at 30.000 x g to obtain an S-30 fraction and a membrane-enriched pellet (P-30). The ribosomes were pelleted from the S-30 supernatant at 150.000 x g, resuspended in Buffer 2(500 mM HN Acetate, 100 mM MgCl 5 mM B-mercaptoethanol, 20 mtl Tris-HCl pH 7.41j and f’ rnally washed 8’ y centrifugation through two layers of 20% and 40% sucrose in buffer 2. The particles were kept at -80”~ in buffer 1, Ribosomes attached to membrane fragments were recovered by resuspending the P-30 fraction ?n buffer ‘1 containing 1% sodium deoxicholate (DOC). After 30 min. continuous shaking at O”C, the sample was centrifuged for 15 min. at 30.000 x g to remove the precipitated mater ial, which was discarded, the supernantant was centrtfuged at 150.000 x g and the pelleted particles were washed as described above and resuspended In buffer 2 and kept at -80”~. Rtbosomal subunits were prepared by dissociating the particles by dialysis against Buffer 3 (500 mM KCI, 5 mM MgCl 10 mM B-mercaptoethanol, 50 rnM Tris-HCl, pH 7.4) for 12 h.The par Q!rcles were separated by centrifugation through a 7-35% sucrose gradient in buffer 3 at 17°C in a zonal rotor. After 5 l/2 h. at 32.000 rpm. 12 ml fractions were collected over 1M MgC12 to give a final concentration of 50 mM and those containing the subunits were p&led and centrifuged at 150.000 x g. The particles were kept in buffer 1 at -80”~. The and thg 2.7~10 High
salt
A2 Onm of mo 0 ecular and 1.3x10 treatment
a solution IS’ei g Rt of respectively. of
containing 1 mg/ml is taken the 8Ds, 60s and 40s particles (12).
to
be 14.00 to be 4.0~10~.
ribosomes.
In order to release the acidic proteins the ribosomes were treated in several ways: a) Ammonium-ethanol washing. The particles were treated With 0.5 M NH,+Cl in the presence of 50% ethanol as previously reported (11) ’ b) Removal of i‘nitiation factors. A suspension of ribosomes in buffer 2 (20-30 mg/ml) was adjusted to 0.5 M KCl, held at O’C for 30 minutes and then centrifuged at 150.000 g for 3 hours. The supernatant contain Ing the crude initiation factor preparation was fractionated by ammonium sulphate precipitation. Material which precipitated between 30% and 70% ammonium sulphate saturation was used as the initiatlon factor preparatfon (13). Estimation of Ax. The amount of protein Ax present in the different samples tested was estimated by radial immunodiffusion (14). 0.3 ml of antiserum to Ax was mixed with 5 ml of 1% agar in 10 mM Tris-HCl pH 7.4 and 150 mH NaCl at 50°C and spread on glass plates and increasing concentration of Ax were placed In 5 mm diameter holes punched in the agar layer. After developing for 18h at room temperature the plates were photographed. the diameter of the preciptin Measurements were made on enlarged prints; ring is a linear function of the logarithm of the antigen concentration. Amino acid composition of protein’ Ax. 0.1 mf of oroteln Ax was hydrolyzed in 6N HCI for 24h at 1lO’C. The amino acid’composition was.determlned in a Durrum D-500 analyzer. The
644
BIOCHEMICAL
Vol. 91, No. 2, 1979
determinations ment, Universidad
were
performed Autonoma,Madrld.
by
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Dr.
M.Ugarte
at
the
Biochemistry
Depart-
RESULTS
tn. were
80s
dissociated
extracted 67% by
and
from
acetic
similar
acid
radial
separated amounts
and
the
of
the
40s
particle
is
almost
top
fraction
is
lower
during
amount
Presence bind
strongly
high
salt
removed
to
to
Ax
high
Protein prepare
with
the
in
the
washi’ng
initiation
TABLE
1.
used from
and
the
of
pg
80s
ribosome
amount
of
Ax
of
the
can
in associated
ribosome
mol
Ax/ml
40s
0.41
0.04
fraction
with
washing
preparations used
2).
particle
--
0.26’a)
top
by
dissociation.
0.47
the
factors
activity(Table
2.04
of
lost
only
60s
volumen
is
conditions
0.96
The
in
factor
3.14
top
present
proteins
80s
Gradient
the
initiation
fractionation
initiating
particles
about
while
protein
removed
ribosomes
after
retains
Ax
intitiation
is
estimated
protein
be
crude
were with
was
subunit
preparation.
having
Ax/mg
the
a “sticky”
moreover,
protein
samples
large
sulfate as
the
treatment
the
The
proteins,
by
in
part
they
fraction
Distribution
Fraction
(a)
are
released
sul’phate
is
ammonfum
factors
ammonium
it.
factors which
After
50%
Ax
initiatLon from
in
Probably
that
subunits
present that
of
expected.
rtbosomes
is
Ax
devoid
Total
and
present
totally than
salt
Ax
ribosomes
ori’ginally
concentrations. by
(13).
Ax
centrifugation.
1 shows
suggesting
of
of
Table
of
manipulation
zonal
amount’fprotein
immumodiffusion.
50%
by
ribosomes
used
particles.
645
corresponded
to
1 mg of
dissociated
80s
the
Vol.
91,
No.
2, 1979
TABLE
2.
BIOCHEMICAL
Presence
of
protein
AND
Ax
In the
Fraction
control KC1 washed
80s
Supernatant
(O-30%
Supernatant
(jO%-70%
Lines
4 correspond
of
3 and
ribosomes
of
indicated
the
late
exponential
detected of
the
amount enter
probability
of
in
dtscarded
30.000
TaGle
xg the 4,
protein
change
with
an
increase
in
above
0
0
0.75
0.23
wash
proteins
sulphate
The easy
to
from
the
free
protein
in
the
of
roughly
to
remove
some
cytoplasm
bound
lmg
data
at
in
the
previous
exchange
of
in
from
an
a pool the
derived
ribos.
preclpltation,
cytoplasm.
of
the
extracted
cells
twice
ribosomes
step cytoplasm.
collected
as
(7.2
Ax cell
mount
the
ribosomes
the
total
cell
the
much
and
at
was
3.0
pg
loss
cells.
Most
preparation
and
Fn this
cell
fraction.
lysate
was
extracted
ribosomes of
protein
were Ax
646
in
of
(Table of
indicate
doubles amount
cycle
synthesis
growtng
material the
the
results
r Fbosome
protein
while
0.66
although
not
the
from
Ax phase
exponenttally
of
Ax/mol
of
respectively),
durtng
presence
from
low,
as
mol
2.15
estimated,
supernatant
of
does
present
was
stationary
cytoplasm
is
Ax
preparation.
1.2
possibility
protein
ribosomes
The
cells
of
factor
ribos.
relatively
wtth
COMMUNlCATlONS
3.9
salt
the
is
the
phase
in
in
Ax
synthesis
amount
high
Ax
protein
protein
When
to
RESEARCH
inltiation
Ax/mg
by ammonium
suggesting
during
cells
NH4S04)
protein
that
ribosomes,
NH,,S041
fractionated
Presence
Ax/mg
crude
pg
80s 0.94
BIOPHYSICAL
of
part
of
with recovered, ri6osomes
in
the stationary
the
protein
obtained
is
for
the
pelleted
deoxycholate As
Ax
materlal
tested material
the
in
membrane
therefore The
Ax
the
Since
a protein
cell
we
3).
as
at (DOC)
indicated from
and in
either
Vol. 91, No. 2, 1979
TABLE
Optical
BIOCHEMICAL
3.
Distribution
of
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
protein
Ax throughout
Protein
Density
of
the
cell
Ax
cycle,
In:
the
culture
Ribosomes
(Asso) pg Ax/mg
r i bos.
mol
Supernatant Ax/mol
pg Ax/mg
ribos,
5
3.1
0.95
5.8
10
3.05
0.93
6.7
15
4.09
1.25
5.64
16
6.9
2.12
6.2
the
cytoplasm
fraction
or is
about
Chemical clearly
the 50%
similar
Al
and
of
Al
analysis
and
of
Ax
that
protein
Ax
Neverthless the
other
A2
in protein
to A2 of
similar,
of
protein.
is
t-i-cher
is
composition
indicate
ribosomal
membrane
has
4.
(see
reported Ax
the
standard
Ax.
The
results
reference
in
of
Table
protein
Sample
form
so
behave
far
like
a typical
L44/45
10).
The
chemical
(10)
and
the
5 indicates
Cytoplasmic
membrane
Membrane Cytoplasmic
7.3
ribosomes
395
ribosomes
3,l
647
composition
similarity
fractfons.
11,8
supernatant
(protelns
a close
Protein
supernatant
It
aminoacld
pg Ax/mg
Hembrane
presented
respects
proteins
Ax with
the
supernatant.
physFcochemical
acidic
prevfously
detailed
Association
not
some
ribosomal
authors been
protein
TABLE
typical
supernatant
than
does in
the
ribos.
Ax r i bosom.
membrane
Vol.
91,
No.
BIOCHEMICAL
2, 1979
Table
5.
Amino
acid
AND BIOPHYSICAL
composition
of
protein
Ax
between
the
acidic Al
aminoacids and
A2
in
percent)
5.45 14.3 0.15 2.1 12.2 21.3
7.2 2.6 7Y5 0.5 2.6 Undetected
5.3 Undetected
determinations and cysteine
three
(moles
14.7 3.2
Asp Thr Ser Glu Glr Pro GUY Ala Val Ile Leu Tv Phe His LYS Arg
Amino acid Tryptophan
RESEARCH COMMUNICATIONS
were carried out were not determined.
proteins,
Protein
Ax
has
and
content
of
bask
lower
agreement
with
its
higher
in
triplicate
samples.
a slightly
higher
amlnoacids
acFdic
pK
content
than
of
protefns
(I).
DISCUSSION Protein
Ax
associated
to
Saccharomyces
is
a highly
the
ribosomes
cerevi
phosphorylated as
s iae
ccl
chemical
compositfon
it
?s
ribosomal
proteins
equivalent
protein
well
I s.
as
In
its
similar
to
to
free
the
that
in
the
appears
cytoplasm
electrophoretic proteins
mobility L44/45,
bacterial
of
the
proteins
L7
and yeast and
L12
Ct,lO). In
other
respects
determinants types
of
LJ),
being
Ax
in it Fat ion released
Prel
imfnary
from
the
there
is
i.e. no
the
functional
role
slmilarrty
and
between
immunogenfc
these
two
proteins,
Protein the
however,
is
y released
factors
but
in
tions
results ribosomes
part1
cond? seem fs
from
50%
to
related
of
that
ft
totally
that its
648
degree
in bound
remains
also
fndicate to
rlbosomes
remove
the
easy of
association to
the
wlth 60s
proteins
removal
phosphorFlation.
subunits, L44/45
of
Ax
(11).
BIOCHEMICAL
Vol. 91, No. 2, 1979
A substantial fractions,
probably
growing
cells.
removed
not
synthesis
protein
ribosomes date
are
no
direct
has
so
effect far
been
Taken a typical
in
far
In any prepared
case,
synthesis clari’fy
The
as
of
Ax
more
the
Ax
detergent.
Although to
ribosomes involved
removable tfghtly
fraction to
phase.
protein
vitro”
in
bound
stationary of
as
protein
ribosomes
easily
“in
of
and/or
an
membrane
bound
amount
in
Ax.
These
However,
ribosomal
the
the
recent
funct,lons
antisera
functional
to
It
its
role of
to
Ax
provide
of
this
of
protein
these Ax
649
with
protein
“in
protelns
to
the
vitro” mRNA
wbth in
experiments the
is
molecule.
exogenous us
(15)
chemical
the
an
proteins
function.
not
of
naturai
of
those
ribosome.
tools the will
to Ax
acidic
acidic
related
translates
results
most
whether
describind
protein
rlbosome
not
bound
systems
in
stability
protein
the
is
a
tightly of
contractile
requirements
(16)
be
to
Ax
like
composition
in
of
protein
behaves can
similarity
sfgnificance The
that
resemblance
question
report
that
that
as
clue
physicochemical
suggest
chemical
well
the
relationship
of
conditions,
Its
structural
machinery. the
some
are
yeast
amount
that
rather
chemical
raises
of the
close
the
from
but
interesting.
be
to
exponentialy
membrane
results
in
and
but
in
with
role on
present
(10)
analyzed
to
with
(10,).
subunit.
the
availabtlity explore
protein
might
same
enter
this
which,
characteristics function
cells
a regulatory
ribosomes
Nevertheless,
amount
with
protein
more
associated
disruption
enriched
the
ribosomal
noteworthy
so
suggest
the
even
present
results
the
ribosomal
it
the
detected
factor
large
makes
Ax
be
RESEARCH COMMUNICATIONS
larger
protein
together,
supernatant the
of
the
of
as
compatible
have
a cons?derably
Moreover,
duplicates
Is
polysomes,
membrane
might
Ax.
Ax
by
binding
be excluded,
protein of
but
stmply
unspecific
protein
ribosomes
particles
easily
can
of
membrane-bound
These
cytoplasmFc
an
fraction
AND BIOPHYSICAL
system and
the to
protein probably
Vol. 91, No. 2, 1979
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
ACKNOWLEDMENTS This work was supported by an Institutional grant to the Centro de BiologTa Molecular from Comision Administradmora de1 Descuento Complementario (.lnstituto National de PrevistGn) and personal grants from Comision Asesora de Investigacien Cientifica y TGcnlca, We thank Dr. D, Ya’zquez for reading and commenting the manuscript and Dra, M. Ugarte for the analysis of amino aci’ds.
REFERENCES 1. 2.
3. 4. Z:
Sanchez-Madrid, F., Conde, P., Vdzquez, D. and Bal lesta, J .P.G. (1979) Blochem. Biophys. Res. Commun. 87, 281-291. Mbller, W., Groene, A., Terhorst, C. and Amons, R. (1972) Eur.-J. Biochem. 25, 5-12. St^dffler, G., Wool, I.G., Lin, A. and Rak. K.H. (1974) Proc. Nat. Acad.Sci. 71, 4723-4726. Mdller, W., Slobin, L.I., Amons, R. and Richter, D. (1975) Proc, Nat. Acad. Sci. 72, 4744-4748. Horak, I. and Schiffmann, D. (1977) Eur. J. Blochem. 79, 375-380. Reyes, R., Va’zquez, 0. and Ballesta, J.P.G. (1977) Eur. J. Biochem.
73, 7. 8. 9. 10. 11. 12. 13.
68, 14.
25-31.
Leader, D.P., Coiia, A.A. and Falmy, L.H. (1978) Biochem. Biophys. Res. Commun, 83, 50-58. Kruiswijk, I. and Planta, R.J. (1975) FEBS Lett. 58, 102-105. Zinker, S. and Warner, J.R. (1976) J. Biol. Chem. 251, 1799-1807. Tsurugi, K., Collatz, E.,Todokoro, K.,Ulbrich, N., Lightfoot, H!N. and Woll, I.G. (1978) J. Biol. Chem. 253, 946-955. Sanchez-Madrid, F., Conde, P. and Ballesta, J.P.G. (1979) Eur. J. . 98, 409-416 Biochem. Petterman, M.L. (1964). In the Physical and Chemical propetles of ribosomes, pp. 121-133. Elsevier, Amsterdam. Fresno, M. Carrasco, L. and VLzquez, D. (.I9761 Eur. J. Biochem.
355-364.
Mancini,
G.,
Carbonara,
A.O.
and
Heremans,
J.F.
(1965)
Immunochemistry
2, 235-254. 15, 16.
Amons,,R., Van Agthoven,A. FEBS Lett. 86, 282-284. Gaslor, E., Herrera, F., K. (1979) J. Biol. Chem.
PluiJms, Sadnik, I., 254, 3965-
650
W. and
Mijller,
MacLaughlin, 3976.
W. C.S.
(19781, and
Moldave,
Vol. 91, No. 2, 1979 November
AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 643-650
28, 1979
AN ACIDIC
PROTEIN
Saccharomyces
cerevisiae
F. lnstituto
de
August
and
de
TO RIBOSOMES
. CHANGES
Sanchez-Madrid
Biochimica C.S.I.C.
Received
ASSOCIATED
DURING
CELL
CYCLE
J.P.G.Ballesta
Macromoleculas,Centro
,Canto
OF
de
Blanco,Madr
id-34,Spa
Biologia
Molecular,
in
lo,1979
SUMMARY A large proportion of protein Ax, one of the three acidic proteins detected in S.cerevisiae is found in the cell cytoplasm. The fraction of Ax associated with the ribosomes is partially released by treatment of these particles in conditions that remove the bound inttiation factors but a residual amount remains tightly bound to the ribosomes. Protein Ax is associated with the large ribosomal subunit and the amount of protein in the particles duplicates as the celle enters the stationarv phase. The amino acid composition of protein Ax is similar to those of other acidic proteins from the ribosomes of yeast and other species being very rich in alanine (21 .3%) ,glycine (12.2%),aspartic acid (14.7%) and glumatic acid
(14.3%). INTRODUCTION We have visae
reported
of
bilities cells
phosphorylated
similar
to
been
ribosomes (5)
or at
ribosome
(11).
previous
factor
Al
and
inducing
part
The
from
It
the
to
in
typical
ribosomal
protein
some
two
this
conditions
in
the
yeast
ref.
10)
have
been
other
and
make
it
is
called
as
not
and
EF-2
Ax,
was
with not
wel I as related
with
L44/45
authors
named
eukaryotic
factors
factor
than
(1)
moand
elongation
of
phosphorylated
proteins
electrophoretic
the
,by
have
cere--
prokaryotic
interaction we
Saccharomyces
other of
from
which
more
to
they
(8), are
the
mentioned
immunogenical
ly
elongation
results) protein
further
presented
in
is
of
explore
in in
ished
results
although
proteins
ly,
of with
interaction
protein,
other
(unpubl
the
that
(mentioned
third
ribosomes proteins
proteins in
ial
characteristics us
of
these A2
least
activtty The
of
in acidic
implicated
reports.
different
existance
those
(2-7).Two
involved,
in
the
several
have
~35/36
(1)
Ax its
report but
relation seem
somewhat it
is
highly with
to
the
indicate
bound
ribosome.
that
resembles
firmly
interesting,
Ax
The is
a supernatant to
the
not
a factor
ribosome.
0006-291X/79/220643-08$01.00/0 643
Copyright All rights
@I 1979 by Academic Press, Inc. of reproduction in any form reserved.
Vol. 91, No. 2, 1979
BIOCHEMICAL
MATERIALS Preparation
of
AND BIOPHYSICAL
AND
RESEARCH COMMUNICATIONS
METHODS
r ibosomes.
Cells of Saccharomyces cerevisiae Y166 grown in YEPD medium (11) in a Lab-Line/S.M.S. Hi-density fermentor were broken by sea sand grinding in Buffer 1 (80 mM #Cl, 12.5 mM MgCl I mM DTT and 100 mM Tris-HCI, pH 7.4), the suspension was centrifiied at low speed (5.000 rpm for 20 min.) in order to remove sand and the larger cell debris and the supernatant was centrifuged again at 30.000 x g to obtain an S-30 fraction and a membrane-enriched pellet (P-30). The ribosomes were pelleted from the S-30 supernatant at 150.000 x g, resuspended in Buffer 2(500 mM HN Acetate, 100 mM MgCl 5 mM B-mercaptoethanol, 20 mtl Tris-HCl pH 7.41j and f’ rnally washed 8’ y centrifugation through two layers of 20% and 40% sucrose in buffer 2. The particles were kept at -80”~ in buffer 1, Ribosomes attached to membrane fragments were recovered by resuspending the P-30 fraction ?n buffer ‘1 containing 1% sodium deoxicholate (DOC). After 30 min. continuous shaking at O”C, the sample was centrifuged for 15 min. at 30.000 x g to remove the precipitated mater ial, which was discarded, the supernantant was centrtfuged at 150.000 x g and the pelleted particles were washed as described above and resuspended In buffer 2 and kept at -80”~. Rtbosomal subunits were prepared by dissociating the particles by dialysis against Buffer 3 (500 mM KCI, 5 mM MgCl 10 mM B-mercaptoethanol, 50 rnM Tris-HCl, pH 7.4) for 12 h.The par Q!rcles were separated by centrifugation through a 7-35% sucrose gradient in buffer 3 at 17°C in a zonal rotor. After 5 l/2 h. at 32.000 rpm. 12 ml fractions were collected over 1M MgC12 to give a final concentration of 50 mM and those containing the subunits were p&led and centrifuged at 150.000 x g. The particles were kept in buffer 1 at -80”~. The and thg 2.7~10 High
salt
A2 Onm of mo 0 ecular and 1.3x10 treatment
a solution IS’ei g Rt of respectively. of
containing 1 mg/ml is taken the 8Ds, 60s and 40s particles (12).
to
be 14.00 to be 4.0~10~.
ribosomes.
In order to release the acidic proteins the ribosomes were treated in several ways: a) Ammonium-ethanol washing. The particles were treated With 0.5 M NH,+Cl in the presence of 50% ethanol as previously reported (11) ’ b) Removal of i‘nitiation factors. A suspension of ribosomes in buffer 2 (20-30 mg/ml) was adjusted to 0.5 M KCl, held at O’C for 30 minutes and then centrifuged at 150.000 g for 3 hours. The supernatant contain Ing the crude initiation factor preparation was fractionated by ammonium sulphate precipitation. Material which precipitated between 30% and 70% ammonium sulphate saturation was used as the initiatlon factor preparatfon (13). Estimation of Ax. The amount of protein Ax present in the different samples tested was estimated by radial immunodiffusion (14). 0.3 ml of antiserum to Ax was mixed with 5 ml of 1% agar in 10 mM Tris-HCl pH 7.4 and 150 mH NaCl at 50°C and spread on glass plates and increasing concentration of Ax were placed In 5 mm diameter holes punched in the agar layer. After developing for 18h at room temperature the plates were photographed. the diameter of the preciptin Measurements were made on enlarged prints; ring is a linear function of the logarithm of the antigen concentration. Amino acid composition of protein’ Ax. 0.1 mf of oroteln Ax was hydrolyzed in 6N HCI for 24h at 1lO’C. The amino acid’composition was.determlned in a Durrum D-500 analyzer. The
644
BIOCHEMICAL
Vol. 91, No. 2, 1979
determinations ment, Universidad
were
performed Autonoma,Madrld.
by
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Dr.
M.Ugarte
at
the
Biochemistry
Depart-
RESULTS
tn. were
80s
dissociated
extracted 67% by
and
from
acetic
similar
acid
radial
separated amounts
and
the
of
the
40s
particle
is
almost
top
fraction
is
lower
during
amount
Presence bind
strongly
high
salt
removed
to
to
Ax
high
Protein prepare
with
the
in
the
washi’ng
initiation
TABLE
1.
used from
and
the
of
pg
80s
ribosome
amount
of
Ax
of
the
can
in associated
ribosome
mol
Ax/ml
40s
0.41
0.04
fraction
with
washing
preparations used
2).
particle
--
0.26’a)
top
by
dissociation.
0.47
the
factors
activity(Table
2.04
of
lost
only
60s
volumen
is
conditions
0.96
The
in
factor
3.14
top
present
proteins
80s
Gradient
the
initiation
fractionation
initiating
particles
about
while
protein
removed
ribosomes
after
retains
Ax
intitiation
is
estimated
protein
be
crude
were with
was
subunit
preparation.
having
Ax/mg
the
a “sticky”
moreover,
protein
samples
large
sulfate as
the
treatment
the
The
proteins,
by
in
part
they
fraction
Distribution
Fraction
(a)
are
released
sul’phate
is
ammonfum
factors
ammonium
it.
factors which
After
50%
Ax
initiatLon from
in
Probably
that
subunits
present that
of
expected.
rtbosomes
is
Ax
devoid
Total
and
present
totally than
salt
Ax
ribosomes
ori’ginally
concentrations. by
(13).
Ax
centrifugation.
1 shows
suggesting
of
of
Table
of
manipulation
zonal
amount’fprotein
immumodiffusion.
50%
by
ribosomes
used
particles.
645
corresponded
to
1 mg of
dissociated
80s
the
Vol.
91,
No.
2, 1979
TABLE
2.
BIOCHEMICAL
Presence
of
protein
AND
Ax
In the
Fraction
control KC1 washed
80s
Supernatant
(O-30%
Supernatant
(jO%-70%
Lines
4 correspond
of
3 and
ribosomes
of
indicated
the
late
exponential
detected of
the
amount enter
probability
of
in
dtscarded
30.000
TaGle
xg the 4,
protein
change
with
an
increase
in
above
0
0
0.75
0.23
wash
proteins
sulphate
The easy
to
from
the
free
protein
in
the
of
roughly
to
remove
some
cytoplasm
bound
lmg
data
at
in
the
previous
exchange
of
in
from
an
a pool the
derived
ribos.
preclpltation,
cytoplasm.
of
the
extracted
cells
twice
ribosomes
step cytoplasm.
collected
as
(7.2
Ax cell
mount
the
ribosomes
the
total
cell
the
much
and
at
was
3.0
pg
loss
cells.
Most
preparation
and
Fn this
cell
fraction.
lysate
was
extracted
ribosomes of
protein
were Ax
646
in
of
(Table of
indicate
doubles amount
cycle
synthesis
growtng
material the
the
results
r Fbosome
protein
while
0.66
although
not
the
from
Ax phase
exponenttally
of
Ax/mol
of
respectively),
durtng
presence
from
low,
as
mol
2.15
estimated,
supernatant
of
does
present
was
stationary
cytoplasm
is
Ax
preparation.
1.2
possibility
protein
ribosomes
The
cells
of
factor
ribos.
relatively
wtth
COMMUNlCATlONS
3.9
salt
the
is
the
phase
in
in
Ax
synthesis
amount
high
Ax
protein
protein
When
to
RESEARCH
inltiation
Ax/mg
by ammonium
suggesting
during
cells
NH4S04)
protein
that
ribosomes,
NH,,S041
fractionated
Presence
Ax/mg
crude
pg
80s 0.94
BIOPHYSICAL
of
part
of
with recovered, ri6osomes
in
the stationary
the
protein
obtained
is
for
the
pelleted
deoxycholate As
Ax
materlal
tested material
the
in
membrane
therefore The
Ax
the
Since
a protein
cell
we
3).
as
at (DOC)
indicated from
and in
either
Vol. 91, No. 2, 1979
TABLE
Optical
BIOCHEMICAL
3.
Distribution
of
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
protein
Ax throughout
Protein
Density
of
the
cell
Ax
cycle,
In:
the
culture
Ribosomes
(Asso) pg Ax/mg
r i bos.
mol
Supernatant Ax/mol
pg Ax/mg
ribos,
5
3.1
0.95
5.8
10
3.05
0.93
6.7
15
4.09
1.25
5.64
16
6.9
2.12
6.2
the
cytoplasm
fraction
or is
about
Chemical clearly
the 50%
similar
Al
and
of
Al
analysis
and
of
Ax
that
protein
Ax
Neverthless the
other
A2
in protein
to A2 of
similar,
of
protein.
is
t-i-cher
is
composition
indicate
ribosomal
membrane
has
4.
(see
reported Ax
the
standard
Ax.
The
results
reference
in
of
Table
protein
Sample
form
so
behave
far
like
a typical
L44/45
10).
The
chemical
(10)
and
the
5 indicates
Cytoplasmic
membrane
Membrane Cytoplasmic
7.3
ribosomes
395
ribosomes
3,l
647
composition
similarity
fractfons.
11,8
supernatant
(protelns
a close
Protein
supernatant
It
aminoacld
pg Ax/mg
Hembrane
presented
respects
proteins
Ax with
the
supernatant.
physFcochemical
acidic
prevfously
detailed
Association
not
some
ribosomal
authors been
protein
TABLE
typical
supernatant
than
does in
the
ribos.
Ax r i bosom.
membrane
Vol.
91,
No.
BIOCHEMICAL
2, 1979
Table
5.
Amino
acid
AND BIOPHYSICAL
composition
of
protein
Ax
between
the
acidic Al
aminoacids and
A2
in
percent)
5.45 14.3 0.15 2.1 12.2 21.3
7.2 2.6 7Y5 0.5 2.6 Undetected
5.3 Undetected
determinations and cysteine
three
(moles
14.7 3.2
Asp Thr Ser Glu Glr Pro GUY Ala Val Ile Leu Tv Phe His LYS Arg
Amino acid Tryptophan
RESEARCH COMMUNICATIONS
were carried out were not determined.
proteins,
Protein
Ax
has
and
content
of
bask
lower
agreement
with
its
higher
in
triplicate
samples.
a slightly
higher
amlnoacids
acFdic
pK
content
than
of
protefns
(I).
DISCUSSION Protein
Ax
associated
to
Saccharomyces
is
a highly
the
ribosomes
cerevi
phosphorylated as
s iae
ccl
chemical
compositfon
it
?s
ribosomal
proteins
equivalent
protein
well
I s.
as
In
its
similar
to
to
free
the
that
in
the
appears
cytoplasm
electrophoretic proteins
mobility L44/45,
bacterial
of
the
proteins
L7
and yeast and
L12
Ct,lO). In
other
respects
determinants types
of
LJ),
being
Ax
in it Fat ion released
Prel
imfnary
from
the
there
is
i.e. no
the
functional
role
slmilarrty
and
between
immunogenfc
these
two
proteins,
Protein the
however,
is
y released
factors
but
in
tions
results ribosomes
part1
cond? seem fs
from
50%
to
related
of
that
ft
totally
that its
648
degree
in bound
remains
also
fndicate to
rlbosomes
remove
the
easy of
association to
the
wlth 60s
proteins
removal
phosphorFlation.
subunits, L44/45
of
Ax
(11).
BIOCHEMICAL
Vol. 91, No. 2, 1979
A substantial fractions,
probably
growing
cells.
removed
not
synthesis
protein
ribosomes date
are
no
direct
has
so
effect far
been
Taken a typical
in
far
In any prepared
case,
synthesis clari’fy
The
as
of
Ax
more
the
Ax
detergent.
Although to
ribosomes involved
removable tfghtly
fraction to
phase.
protein
vitro”
in
bound
stationary of
as
protein
ribosomes
easily
“in
of
and/or
an
membrane
bound
amount
in
Ax.
These
However,
ribosomal
the
the
recent
funct,lons
antisera
functional
to
It
its
role of
to
Ax
provide
of
this
of
protein
these Ax
649
with
protein
“in
protelns
to
the
vitro” mRNA
wbth in
experiments the
is
molecule.
exogenous us
(15)
chemical
the
an
proteins
function.
not
of
naturai
of
those
ribosome.
tools the will
to Ax
acidic
acidic
related
translates
results
most
whether
describind
protein
rlbosome
not
bound
systems
in
stability
protein
the
is
a
tightly of
contractile
requirements
(16)
be
to
Ax
like
composition
in
of
protein
behaves can
similarity
sfgnificance The
that
resemblance
question
report
that
that
as
clue
physicochemical
suggest
chemical
well
the
relationship
of
conditions,
Its
structural
machinery. the
some
are
yeast
amount
that
rather
chemical
raises
of the
close
the
from
but
interesting.
be
to
exponentialy
membrane
results
in
and
but
in
with
role on
present
(10)
analyzed
to
with
(10,).
subunit.
the
availabtlity explore
protein
might
same
enter
this
which,
characteristics function
cells
a regulatory
ribosomes
Nevertheless,
amount
with
protein
more
associated
disruption
enriched
the
ribosomal
noteworthy
so
suggest
the
even
present
results
the
ribosomal
it
the
detected
factor
large
makes
Ax
be
RESEARCH COMMUNICATIONS
larger
protein
together,
supernatant the
of
the
of
as
compatible
have
a cons?derably
Moreover,
duplicates
Is
polysomes,
membrane
might
Ax.
Ax
by
binding
be excluded,
protein of
but
stmply
unspecific
protein
ribosomes
particles
easily
can
of
membrane-bound
These
cytoplasmFc
an
fraction
AND BIOPHYSICAL
system and
the to
protein probably
Vol. 91, No. 2, 1979
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
ACKNOWLEDMENTS This work was supported by an Institutional grant to the Centro de BiologTa Molecular from Comision Administradmora de1 Descuento Complementario (.lnstituto National de PrevistGn) and personal grants from Comision Asesora de Investigacien Cientifica y TGcnlca, We thank Dr. D, Ya’zquez for reading and commenting the manuscript and Dra, M. Ugarte for the analysis of amino aci’ds.
REFERENCES 1. 2.
3. 4. Z:
Sanchez-Madrid, F., Conde, P., Vdzquez, D. and Bal lesta, J .P.G. (1979) Blochem. Biophys. Res. Commun. 87, 281-291. Mbller, W., Groene, A., Terhorst, C. and Amons, R. (1972) Eur.-J. Biochem. 25, 5-12. St^dffler, G., Wool, I.G., Lin, A. and Rak. K.H. (1974) Proc. Nat. Acad.Sci. 71, 4723-4726. Mdller, W., Slobin, L.I., Amons, R. and Richter, D. (1975) Proc, Nat. Acad. Sci. 72, 4744-4748. Horak, I. and Schiffmann, D. (1977) Eur. J. Blochem. 79, 375-380. Reyes, R., Va’zquez, 0. and Ballesta, J.P.G. (1977) Eur. J. Biochem.
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Leader, D.P., Coiia, A.A. and Falmy, L.H. (1978) Biochem. Biophys. Res. Commun, 83, 50-58. Kruiswijk, I. and Planta, R.J. (1975) FEBS Lett. 58, 102-105. Zinker, S. and Warner, J.R. (1976) J. Biol. Chem. 251, 1799-1807. Tsurugi, K., Collatz, E.,Todokoro, K.,Ulbrich, N., Lightfoot, H!N. and Woll, I.G. (1978) J. Biol. Chem. 253, 946-955. Sanchez-Madrid, F., Conde, P. and Ballesta, J.P.G. (1979) Eur. J. . 98, 409-416 Biochem. Petterman, M.L. (1964). In the Physical and Chemical propetles of ribosomes, pp. 121-133. Elsevier, Amsterdam. Fresno, M. Carrasco, L. and VLzquez, D. (.I9761 Eur. J. Biochem.
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Mancini,
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