Initiation and processing in vitro of the primary translation products of guinea-pig caseins

Abstract

1. Guinea-pig caseins synthesized in a mRNA-directed wheat-germ cell-free protein-synthesizing system represent the primary translation products, even though they appear to be of lower molecular weight when analysed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis in parallel with caseins isolated from guinea-pig milk. 2. Identification of the N-terminal dipeptide of the primary translational product of caseins A, B and C and alpha-lactalbumin showed that all shared a common sequence, which was identified as either Met-Arg or Met-Lys. 3. Procedures utilizing methionyl-tRNAfMet or methionyl-tRNAMet in the presence or absence of microsomal membranes during translation provide a rapid method of distinguishing between N-terminal processing of peptides synthesized in vitro and other post-translational modifications (glycosylation, phosphorylation), which also result in a change in mobility of peptides when analysed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. 4. The results demonstrate that guinea-pig caseins, in common with most other secretory proteins, are synthesized with transient N-terminal 'signal'-peptide extensions, which are cleaved during synthesis in the presence of microsomal membranes.

Full text

Biochem.
J.
(1979)
184,
261-267
261
Printed
in
Great
Britain
Initiation
and
Processing
in
vitro
of
the
Primary
Translation
Products
of
Guinea-Pig
Caseins
By
Roger
K.
CRAIG,*t
Ponnamperuma
A.
J.
PERERA,*$
Andrew
M-ELLOR§
and
Alan
E.
SMITH§
*Courtauld
Institute
of
Biochemistry,
Middlesex
Hospital
Medical
School,
Londoni
WIP
7PN,
and
§Translation
Laboratory,
Imperial
Cancer
Research
Fund,
London
WC2A
3PX,
U.K.
(Received
10
April
1979)
1.
Guinea-pig
caseins
synthesized
in
a
mRNA-directed
wheat-germ
cell-free
protein-
synthesizing
system
represent
the
primary
translation
products,
even
though
they
appear
to
be of
lower
molecular
weight
when
analysed
by
sodium
dodecyl
sulphate/polyacryl-
amide-gel
electrophoresis
in
parallel
with
caseins
isolated
from
guinea-pig
milk.
2.
Iden-
tification
of
the
N-terminal
dipeptide
of
the
primary
translational
product
of
caseins
A,
B
and
C
and
a-lactalbumin
showed
that
all
shared
a
common
sequence,
which
was
iden-
tified
as
either
Met-Arg
or
Met-Lys.
3.
Procedures
utilizing
methionyl-tRNA'Met
or
methionyl-tRNAM"t
in
the
presence
or
absence
of
microsomal
membranes
during
trans-
lation
provide
a
rapid
method
of
distinguishing
between
N-terminal
processing
of
pep-
tides
synthesized
in
vitro
and
other
post-translational
modifications
(glycosylation,
phosphorylation),
which
also
result
in
a
change
in
mobility
of
peptides
when
analysed
by
sodium
dodecyl
sulphate/polyacrylamide-gel
electrophoresis.
4.
The
results
demonstrate
that
guinea-pig
caseins,
in
common
with
most
other
secretory
proteins,
are
synthesized
with
transient
N-terminal
'signal'-peptide
extensions,
which
are
cleaved
during
synthesis
in
the
presence
of
microsomal
membranies.
Most,
though
not
all,
secretory
proteins
(see
Palmiter
et
al.,
1978a)
are
synthesized
with
transient
N-terminal
signal
sequences.
The
signal
sequences
are
generally
removed
during
protein
synthesis,
a
membrane-requiring
event
(Blobel
&
Dobberstein,
1975a,b;
Palmiter
et
al.,
1978b;
Craig
et
al.,
1979).
In
addition
to
processing,
cell-free
synthesis
in
the
presence
of
microsomal
membranes
is
also
coupled
to
core
glycosylation
(Rothman
&
Lodish,
1977;
Lingappa
et
al.,
1978a,b).
Both
proteolytic
cleavage
and
glycosylation
of
peptides
may
give
rise
to
a
change
in
mobility
of
peptides,
when
analysed
by
SDS/polyacrylamide-gel
electrophoresis
(see
Craig
et
al.,
1976).
Although
the
glycosylated
peptides
can
be
identified
by
using
lectin
columns
(Lingappa
et
al.,
1978a,b),
the
identification
of
transient
N-terminal
signal
sequences
is
dependent
on
definitive
identification
of
the
primary
translation
products
by
using
sophisticated
micro-sequencing
techniques
(see
Kemper
et
al.,
1976).
We
have
previously
demonstrated
(Craig
et
al.,
1976)
that
the
guinea-pig
milk
protein
a-lactalbumin
is
synthesized
as
a
high-molecular-weight
precursor
polypeptide
modified
at
the
N-terminus
in
the
wheat-germ
cell-free
system,
and
that
this
may
be
Abbreviation
used:
SDS,
sodium
dodecyl
sulphate.
t
To
whom
reprint
requests
should
be
addressed.
t
Present
address:
Department
of
Biochemistry,
Uni-
versity
of
Sri
Lanka,
Peradeniya,
Sri
Lanka.
Vol.
184
processed
to
a-lactalbumin
by
the
Krebs-II
ascites-
cell-free
system,
a
membrane-requiring
event.
How-
ever,
a
similar
analysis
of
the
translation
products
of
guinea-pig
caseins
synthesized
in
the
wheat-germ
cell-free
protein-synthesizing
system
revealed
peptide
products
of
apparently
lower
molecular
weight
than
those
isolated
from
guinea-pig
milk
(Craig
et
al.,
1976).
Guinea-pig
caseins
are
heavily
phosphory-
lated,
and
to
a
lesser
extent
glycosylated
(Craig
et
al.,
1978).
It
seems
likely
that
the
presence
or
other-
wise
of
these
groups
may
well
contribute
to
the
anomalous
mobility
observed
on
polyacrylamide
gels
of
the
polypeptides
synthesized
in
vitro.
In
the
present
paper
we
describe
rapid
procedures
utilizing
formylated
and
unformylated
[35S]methi-
onine
donated
by
initiator
tRNA
which
demonstrate
that
guinea-pig
milk
proteins,
in
spite
of
this
anom-
alous
mobility,
are
synthesized
as
the
primary
translational
products
in
the
wheat-germ
cell-free
system.
Moreover,
the
caseins,
like
a-lactalbumin,
are
synthesized
with
transient
N-terminal
peptide
extensions,
which
are
processed
in
the
presence
of
added
membrane
fractions.
The
incorporation
of
formylmethionine
from
initiator
tRNA
has
been
used
to
study
the
initiation
of
protein
synthesis
in
several
eukaryotic
systems
(Housman
et
al.,
1970;
Smith
&
Marcker,
1970;
Smith,
1973).
The
procedure
has
proved
particularly
useful
in
experiments
designed
to
determine
whether

R.
K.
CRAIG,
P.
A.
J.
PERERA,
A.
MELLOR
AND
A.
E.
SMITH
certain
viral
proteins
result
from
initiation
of
protein
synthesis
from
single
sites
on
individual
mRNA
species,
or
are
derived
from
post-translational
cleavage
of
larger
polypeptides
(Clegg
&
Kennedy,
1975;
Glanville
et
al.,
1976;
Siddell
&
Smith,
1978).
Experimental
Preparation
of
solutions
and
chemicals
All
solutions
were
prepared
with
double-distilled
water.
Acrylamide
and
NN'-methylenebisacrylamide
were
recrystallized
in
chloroform
and
acetone
re-
spectively,
as
described
by
Loening
(1967).
NNN'N'-
Tetramethylenediamine
was
redistilled
in
vaci
o
immediately
before
use.
Materials
Unless
specifically
stated
otherwise,
all
chemicals
and
solvents
were
obtained
from
sources
previously
described
(Craig
et
al.,
1976,
1978,
1979;
Glanville
et
al.,
1976).
L-
[35S]Methionine
(700-1350
Ci/mmol)
was
obtained
from
The
Radiochemical
Centre,
Amersham,
Bucks.,
U.K.
mRNA
isolation
and
cell-free
protein
synthesis
The
isolation
of
poly(A)-containing
RNA
species
from
the
lactating
guinea-pig
mammary
gland
has
been
described
in
detail
elsewhere
(Craig
et
al.,
1976,
1979).
mRNA-directed
protein
synthesis
in
the
wheat-germ
cell-free
system
was
carried
out
in
assay
volumes
of
50,ul-1.Oml
for
75min
at
21°C
essentially
as
described
previously
(Craig
et
al.,
1976),
except
that
either
L-
[35S]methionine
(8OpCi/ml,
800-1350
Ci/mmol)
or
N-formyl[3SS]methionyl-tRNA'Met
(5
x
107
c.p.m./ml)
or
[35S]methionyl-tRNAMle
was
used
as
the
radiolabelled
precursor.
In
the
presence
of
initiator
tRNA,
unlabelled
methionine
(4pM)
was
also
included
in
the
assay
as
a
source
of
internal
methionine
residues.
Dog
pancreas
microsomal
membranes
were
prepared
as
described
by
Blobel
&
Dobberstein
(1975a).
Preparation
of
N-formyl[35S]methionyl-tRNAfMet,
[35S]methionyl-
tRNAfMet
and
[35S]methionyl-
tRNA
Met
tRNAfM't
and
tRNAM't
were
prepared
from
total
wheat-germ
tRNA
by
chromatography
on
BD-
cellulose
(benzoylated
DEAE-cellulose)
as
described
by
Smith
&
Marcker
(1970).
tRNAfM"t
was
charged
with
[35S]methionine
by
using
highly
purified
Escherichia
coli
methionyl-tRNA
synthetase
(Smith,
1973;
Mellor
&
Smith,
1978),
whereas
tRNAMCt
was
charged
by
using
crude
wheat-germ
synthetase
(Siddell
&
Smith,
1978).
Methionyl-tRNArM"e
was
charged
and
chemically
formylated
as
described
by
Glanville
et
al.
(1976).
Immunoprecipitation
and
polyacrylamide-gel
electro-
phoresis
Immunoprecipitation
was
carried
out
as
described
by
Craig
et
al.
(1976)
by
using
a
mixture
of
antisera
raised
in
rabbits
against
caseins
and
a-lactalbumin
(104lu/ml
of
initial
assay)
and
the
antibody-antigen
complex
recovered
after
precipitation
with
goat
anti-
(rabbit
immunoglobulin
G)
antiserum
(200#1/ml
of
initial
assay).
For
qualitative
analysis
these
were
dissolved
in
sample
buffer
and
used
directly
for
polyacrylamide-
slab-gel
electrophoresis
as
described
elsewhere
(Craig
et
al.,
1979)
by
the
procedure
of
either
Weber
et
al.
(1972)
or
Laemmli
(1970)
and
then
fluoro-
graphed
(Bonner
&
Laskey,
1974).
When
labelled
proteins
were
to
be
eluted
from
polyacrylamide
gels
for
subsequent
peptide
analysis,
the
total
antibody
precipitate
was
reduced,
and
then
carboxamido-
methylated
in
the
presence
of
urea
as
described
by
Crestfield
et
al.
(1963),
except
that
iodoacetamide
was
used
as
the
alkylating
agent.
The
position
of
the
peptides
was
determined
by
radioautography
of
the
wet
gel
at
4°C.
'Fingerprinting'
of
labelledpeptides
Proteins
labelled
with
[35S]methionine
were
eluted
from
polyacrylamide-gel
slices
by
vigorous
shaking
at
37°C
overnight
in
50mM-NH4HCO3
containing
0.1
%
(w/v)
SDS
and
0.1
%
(v/v)
2-mercaptoethanol,
and
the
eluted
protein
was
recovered
and
oxidized
with
performic
acid
in
the
presence
of
lOO,ug
of
the
appropriate
carboxamidomethylated
milk
protein
as
carrier
(see
Smith
et
al.,
1978).
The
recovered
protein
was
then
digested
overnight
with
trypsin
and
a
two-dimensional
analysis
of
the
peptide
products
performed
on
20cm
x
10cm
cellulose-coated
flexible
poly(ethylene
terephthalate)
supports
as
described
by
Craig
et
al.
(1978).
[35S]Methionine-containing
pep-
tides
were
detected
after
radioautography
at
room
temperature
for
1-6
days
by
using
Fuji
Rx
X-ray
film.
For
further
analysis
of
radiolabelled
peptides,
the
plates
were
incubated
for
2h
in
an
atmosphere
of
1202
and
performic
acid
(1:
20,
v/v),
the
areas
of
cellulose
corresponding
to
the
radiolabelled
peptides
removed,
and
the
peptides
extracted
with
l.Oml
of
0.1M-HCI
for
10min.
Cellulose
particles
were
re-
moved
by
centrifugation
at
lOOOgav
for
5
min
at
20°C,
and
the
supernatants
freeze-dried.
The
extracted
peptides
were
then
either
redissolved
in
20-40pl
of
water
and
re-analysed
directly,
or
subjected
to
Pronase
digestion
(140,ug
of
Pronase/mI,
for
2h
at
37°C)
before
further
two-dimensional
analysis.
The
dipeptide
fMet-['4C]Arg
was
prepared
as
described
by
Smith
(1973).
Results
Poly(A)-containing
RNA
isolated
from
the
post-
nuclear
supernatant
of
the
lactating
guinea-pig
1979
262

N-TERMINAL
PROCESSING
OF
CASEINS
mammary
gland
directs
the
almost
exclusive
syn-
thesis
in
the
wheat-germ
cell-free
system
of
caseins
A,
B
and
C
and
a-lactalbumin
polypeptides,
as
determined
by
antibody
precipitation
of
[35S]meth-
ionine-containing
peptides
synthesized
in
vitro
(Fig.
1).
Moreover,
if
N-formyl[35S]methionyl-
tRNAfMCt
replaces
[35S]methionine
as
the
radio-
labelled
precursor,
in
spite
of
a
decreased
incorpor-
ation
of
the
label
(2-3
%,
compared
with
35-40
%
of
the
free
amino
acid),
analysis
of
the
products
synthe-
sized
in
vitro,
by
immunoprecipitation
and
gel
electro-
phoresis,
reveals
a
pattern
identical
with
that
ob-
served
with
the
free
amino
acid
alone
(Fig.
l).
These
observations
suggest
that
the
labelled
polypeptides
represent
the
primary
translation
products
of
caseins
A,
B
and
C
and
a-lactalbumin,
in
spite
of
the
anomalous
mobility
of
the
caseins
when
compared
with
the
secreted
proteins
isolated
from
guinea-pig
milk.
In
order
to
justify
this
conclusion,
we
have
charac-
terized
the
N-terminal
peptides
of
the
primary
translation
products
of
[35S]formylmethionine-
N-
Formylq35S|
methionyl-tR
NA
II
-RNA
+RNA
+
-
+
[35S1NMethionine
Casein
A
-Casein
B
-Casein
C
:~~~~~
-ri-Lactalbumin
-RNA
*RNA
Antibody
precipitation
Fig.
1.
Initiation
of
mRNA-directed
milk-protein
synthesis
in
vitro
in
a
wheat-germ
cell-free
protein-sythesizing
system
Cell-free
protein
synthesis
with
either
[33S]methionine
or
N-formyl[35S]methionyl-tRNAfMe
as
the
radio-
labelled
precursor,
antibody
precipitation,
SDS/gel
electrophoresis
in
10
%
polyacrylamide
gels
(Weber
et
al.,
1972)
and
fluorography
were
carried
out
as
described
in
the
Experimental
section.
The
relative
electrophoretic
mobilities
of
the
authentic
caseins
and
a-lactalbumin
isolated
from
guinea-pig
milk
are
indicated.
Vol.
184
labelled
caseins
A,
B
and
C
and
a-lactalbumin,
syn-
thesized
on
a
large
scale
in
the
wheat-germ
cell-free
system.
The
radiolabelled
milk
proteins
were
re-
covered
from
the
cell-free
system
by
using
antibody
precipitation
and
the
polypeptides
then
separated
on
preparative
SDS/polyacrylamide
slab
gels
essen-
tially
as
described
in
the
legend
to
Fig.
I
(results
not
shown).
The
individual
peptides
were
then
extracted
from
the
gel
and
digested
with
trypsin
(see
the
Experimental
section).
Analysis
of
the
[S3]formylmethionine-containing
peptide
'fingerprints'
obtained
from
each
eluted
milk
protein
(Figs.
2a, 2b,
2c
and
2d),
revealed
that
each
contained
the
same
two
predominant
labelled
peptides,
a
result
confirmed
by
mixing
all
four
digests
before
fractionation
(Fig.
2f).
In
addition
to
these
two
peptides,
a
third
peptide
was
also
apparent
in
the
casein-B
preparation
(Fig.
2b).
After
further
treatment
with
performic
acid
to
ensure
that
all
peptides
were
oxidized
to
the
sulphone
derivative
(Clegg
&
Kennedy,
1975),
peptides
1,
2
and
3
were
extracted,
and
either
(i)
re-analysed
directly
or
(ii)
digested
with
Pronase
and
then
re-analysed.
Perfor-
mic
acid
treatment
demonstrated
that
spots
1
and
2
represent
different
oxidation
states
of
the
same
peptide,
peptide
I
now
co-migrating
with
peptide
2
(Figs.
3a
and
3c).
Pronase
digestion
had
no
effect
on
the
mobility
of
this
peptide,
whereas
performic
acid
treatment
did
not
affect
the
mobility
of
peptide
3
(Fig.
3d).
Peptide
3
was
not
further
characterized.
As
the
predominant
formylmethionine-containing
peptide
was
positively
charged
at
pH
3.5
(see
Figs.
2
and
3),
it
seemed
reasonable
to
predict
that
it
might
contain
either
lysine
or
arginine.
This
appeared
to
be
justified
(Fig.
3b)
as
the
mobility
after
two-dimen-
sional
analysis
was
similar
to
that
of
performic
acid-
treated
dipeptide
fMet-[14C]Arg.
As
no
fMet-Lys
standard
was
available,
we
were
unable
in
this
experi-
ment
to
identify
definitively
the
common
formyl-
methionine-containing
peptide
of
guinea-pig
caseins
A,
B
and
C
and
a-lactalbumin
as
either
fMet-Lys
or
fMet-Arg.
However,
since
these
experiments
were
performed,
we
have
found,
using
the
wheat-germ
cell-free
system,
that
if
[35S]methionyl-tRNAIMet
(unformylated)
is
added
as
the
radiolabelled
precursor,
the
N-terminal
initiating
methionine
residue
of
the
milk-protein-
mRNA
directed
peptides
is
not
removed.
Isolation
and
tryptic
digestion
of
these
peptides
followed
by
analysis
in
parallel
with
methionyl-lysine
o6tained
from
[Met,
Lys]bradykinin
showed
the
mobility
of
all
the
peptides
to
be
identical
after
two-dimensional
analysis
(A.
Mellor
&
A.
E.
Smith,
unpublished
work).
To
determine
whether
or not
the
primary
trans-
lational
products
of
the
caseins
were
synthesized
with
transient
N-terminal
peptide
sequences,
in
an
analogous
manner
to
pre-a-lactalbumin
(Craiget
al.,
-ve
263

R.
K.
CRAIG,
P.
A.
J.
PERERA,
A.
MELLOR
AND
A.
E.
SMITH
(a)
tb)
I
I
'a
a,
A
~~~~~~~~~~~~~~~~~~..
)
I'ii)
?*
I
*
-ve
2:*
Li
+ve:
(d)
(c)
-ve
.
LI
0
0
+ve
*
El
Ascending
chromatography
Fig.
3.
Identification
of
the
predominant
formylmethionine-
containing
peptides
Peptides
1,
2
and
3
were
extracted
from
the
cellulose
after
performic
acid
treatment
and
re-analysed
in
parallel
with
an
f
Met-['4C]Arg
marker
peptide.
(a)
Peptide
1,
(b)
f
Met-['4C]Arg,
(c)
peptide
2,
(d)
peptide
3.
In
practice
(a)
and
(b)
were
spotted
on
the
same
cellulose
thin-layer
plate
(see
legend
to
Fig.
2),
as
were
(c)
and
(d).
The
symbol
x
marks
the
point
of
application
of
each
sample.
Ascending
chromatography
Fig.
2.
Two-dimensional
analysis
of
peptides
labelled
with
fornmyl['5S],niethiontyl-tRNAfMet
after
niRNA-directed
eell-
free
protein
synthesis
in
the
wheat-germl
systent
Radiolabelled
milk
proteins
synthesized
in
the
wheat-germ
cell-free
system
(2
ml
assay
volume)
in
the
presence
of
N-formyl[35S]methionyl-tRNAIMeI
were
recovered
by
antibody
precipitation,
the
antibody-
antigen
complex
was
reduced
and
carboxamido-
methylated,
and
the
peptides
were
separated
on
10
%
SDS/polyacrylamide
gels
as
described
in
the
Experi-
mental
section.
The
radiolabelled
peptides
corre-
sponding
to
the
primary
translation
products
of
caseins
A,
B
and
C
and
a-lactalbumin
were
eluted,
oxidized
with
performic
acid,
digested
with
trypsin
(see
the
Experimental
section),
and
the
peptides
separated
by
electrophoresis
in
the
first
dimension
and
ascending
chromatography
in
the
second.
(a)
Casein
A,
(b)
casein
B,
(c)
casein
C,
(d)
a-lactal-
bumin,
(e)
:1:1
:1
mixture
of
all
four
peptide
digests.
Arrows
mark
the
positions
of
the
major
radiolabelled
peptides.
In
practice,
caseins
A
and
B
(a
and
b)
were
spotted
in
parallel,
one
on
the
edge,
and
the
other
in
the
centre
of
a
20cm
x
20cm
cellulose
thin-layer
sheet.
Electrophoresis
was
then
performed
simultaneously
on
both
samples.
The
sheet
was
then
divided
into
two,
dried
and
separated
in
the
second
dimension
by
ascending
chromatography,
performed
simultaneously
in
the
same
tank.
The
two
strips
were
then
re-aligned
and
radioautographed.
This
procedure
was
also
followed
for
casein
C
and
a-
lactalbumin
(c
and
d).
Where
a
mixture
of
digests
was
analysed
(e)
to
ensure
maximum
separation
of
the
peptides
in
the
second
dimension,
a
single
sample
only
was
spotted
on
the
edge
of
the
cellulose
sheet.
The
symbol
x
marks
the
point
of
application
of
each
sample.
1979
-ve
i
+ve
-ve
a)
p
0
0
Qy
t-ve
-ve
1
2-
264
.2
aU
c
-C
r
c
t
(L
LL

N-TERMINAL
PROCESSING
OF
CASEINS
-ve
-Caseins
A
and
B
w
w
_
~~~~Casein
C
-(X-Lactalbumin
+ve
)
(2)(3)
(4)
(5)
(6)
(7)
(8)
(9)
(10)'(1
1
Fig.
4.
N-Terminal
cleavage
of
the
primary
translation
products
of
guinea-pig
caseins
by
a
dog
microsomal
nzem-
brane
fraction
during
mRNA-directed
protein
synthesis
in
the
wheat-germ
cell-free
system
Guinea-pig
milk
proteins
were
synthesized
in
a
mRNA-directed
wheat-germ
cell-free
protein-syn-
thesizing
system,
by
using
[35Slmethionine,
[35S]-
methionyl-tRNAfM'e
or
[3aS]methionyl-tRNAMeI
as
the
radiolabelled
precursors.
Synthesis
was
in
the
presence
or
absence of
a
dog
microsomal
membrane
fraction.
The
[35S]methionine-containing
peptides
were
then
separated
on
a
SDS/polyacrylamide
(15
%)
gel
(Laemmli,
1970).
The
relative
electrophoretic
mobilities
of
the
authentic
caseins
and
a-lactalbu-
min
isolated
from
guinea-pig
milk
are
indicated.
Gels:
(1)
-mRNA,
+
[3
SSmethionyl-tRNAfMet;
(2)
-mRNA,
+[3sS]methionyl-tRNAMet;
(3)
-mRNA,
+
[35S]methionine;
(4)
+mRNA,
+[35S]methionyl-
tRNAfMCt;
(5),
as
(4),
+
membranes;
(6)
and
(7),
as
(4)
and
(5),
but
twice
the
radioactivity
loaded
on
the
gel;
(8)
+mRNA+[3SS]methionyl-tRNAMeI;
(9),
as
(8),
+
membranes;
(10)
and
(11),
as
(8)
and
(9),
but
with
[35S]methionine
as
the
radiolabelled
precursor.
1976),
we
have
synthesized
milk
proteins
in
the
wheat-germ
cell-free
system
by
using
[35S]meth-
ionine,
[35S]methionyl-tRNAfMet
or
[35S]methionyl-
tRNAMCt
as
the
radiolabelled
precursors,
each
in
the
presence
and
absence
of
a
dog
pancreas
stripped-
microsomal-membrane
preparation.
The
results
(Fig.
4)
demonstrate
firstly
a
very
marked
change
in
distribution
of
those
peptides
with
methionine
residues
donated
by
the
free
amino
acid
or
methionyl-
tRNAM"C
as
a
result
of
added
membranes
(tracks
9
and
11).
Processing
of
the
primary
translation
product
of
casein
C
to
a
smaller
peptide
was
parti-
cularly
marked.
However,
the
primary
translation
Vol.
184
products
of
caseins
A
and
B,
although
not
well
separated
by
the
Laemmli
(1970)
polyacrylamide-gel
system,
clearly
underwent
processing
in
the
presence
of
microsomal
membranes,
as
judged
by
the
appear-
ance
of
two
additional
peptides,
one
of
slightly
increased
and
the
other
of
slightly
decreased
electro-
phoretic
mobility.
Examination
of
the
fate
of
peptides
labelled
only
at
the
N-terminal
methionine
by
using
[35S]methionyl-tRNAfTM'
but
synthesized
in
the
presence
of
microsomal
membranes
(tracks
5
and
7)
demonstrated
that
the
effect
of
membranes
was
to
eliminate
[35S]methionine-labelled
peptides
corre-
sponding
to
the
processed
casein
polypeptides,
results
consistent
with
an
N-terminal
cleavage
event
as
a
result
of
adding
microsomal
membranes.
Throughout
these
experiments
evidence
for
the
processing
of
pre-a-lactalbumin
was
less
evident,
as,
although
these
particular
separation
conditions
(15
%
polyacrylamide) revealed
processing
of
the
caseins,
a-lactalbumin
and
pre-a-lactalbumin
were
not
resolved.
Moreover,
the
disparate
mobility
of
the
caseins
isolated
from
guinea-pig
milk
when
compared
with
those
synthesized
in
vitro
under
different
electrophoretic
conditions
(see
Figs.
1
and
4),
emphasizes
the
inconsistency
that
can
result
from
molecular-weight
estimations
by
using
SDS/poly-
acrylamide-gel
electrophoresis
alone.
Discussion
Our
observations
using
N-formyl[35S]methionyl-
tRNAMC"
as
the
radiolabelled
precursor
for
cell-free
protein
synthesis,
followed
by
extensive
character-
ization
of
the
resulting
formylmethionine-containing
peptides,
demonstrate
that,
in
spite
of
their
anom-
alous
mobility
on
SDS/polyacrylamide
gels
when
compared
with
the
purified
milk
proteins,
each
represents
the
primary
translation
product
of
the
appropriate
mRNA
species.
Analysis
of
the
formylmethionine-containing
pep-
tides
identified
either
fMet-Arg
or
fMet-Lys
as
the
N-terminal
sequence
of
the
primary
translation
products
of
caseins
A,
B
and
C
and
a-lactalbumin.
Casein
B
exhibited
some
heterogeneity,
in
keeping
with
our
previous
observations
(Craig
et
al.,
1978).
Comparison
of
the
known
N-terminal
amino
acid
of
the
proteins
isolated
from
guinea-pig
milk
(Table
1)
with
those
of
the
primary
translation
products
demonstrates
that
the
primary
translation
product
of
casein
B,
in
addition
to
a-lactalbumin
(known),
is
also
synthesized
with
N-terminal
amino
acids
(f
Met-
Arg
or
fMet-Lys)
not
present
in
the
purified
secreted
protein.
Similar
comparison
of
the
remaining
pep-
tides
was
less
definitive,
as
all
possess
lysine
as
the
N-terminal
residue
in
the
secreted
form
(see
Brew,
1972;
Craig
et
al.,
1978).
Identification
of
the
formylmethionine-containing
peptides
is
essential
in
all
experiments
involving
265

R.
K.
CRAIG,
P.
A.
J.
PERERA,
A.
MELLOR
AND
A.
E.
SMITH
Table
1.
Comparison
of
the
amino
acids
found
at
the
N-terminus
of
the
primary
translation
products
and
the
authentic
guinea-
pig
milk
proteins
Casein
A*
Casein
B*
Casein
C*
a-Lactalbumint
N-Terminal
amino
acid
of
caseins
and
oc-lactal-
bumin
isolated
from
milk
N-Terminal
amino
acid
of
the
primary
trans-
lation
products
*
From
Craig
et
al.
(1978).
t
From
Brew
(1972).
Lys
Met
Lys
Lys
Lys/Arg
Lys/Arg
Lys/Arg
Lys/Arg
formylmethionyl-tRNAfM"e
in
eukaryote
systems,
as,
although
the
presence
of
the
formyl
group
prevents
the
removal
of
the
N-terminal
methionine
residue
after
initiation
of
peptide
synthesis
(Houseman
et
al.,
1970),
it
is
not
a
physiological
initiator
in
eukaryote
systems,
and
so
competes
poorly
with
endogenous
initiator
tRNA
(Smith,
1973).
Consequently,
as
we
used
wheat-germ
tRNA
as
the
starting
material,
it
was
important
to
ensure
that
incorporation
was
not
due
to
small
amounts
of
contaminating
methionyl-
tRNAMCt
in
the
formylmethionyl-tRNArMet
prep-
aration,
resulting in
the
incorporation
at
internal
methionine
residues.
An
assessment
of
methionyl-
tRNA'Met
preparations
showed
them
to
be
over
95
%
pure,
as
judged
by
analysis
of
the
primary
translation
product
of
the
SV40
capsid
protein
VP1,
which
has
an
N-terminal
sequence
of
Met-Lys-Met.
Peptide
analysis
of
protein
VP1
labelled
in
vitro
by
using
[35S]methionyl-tRNAfMet
demonstrated
that
over
95
%
of
the
incorporated
label
was
in
the
N-terminal
methionine
(A.
Mellor,
unpublished
work).
Our
observation
that
the
unformylated
initiating
methionine
was
not
removed
from
the
nascent
polypeptide
chain
was
not
unexpected,
as
sequencing
studies
of
many
different
proteins
have
shown
that
in
vitro
the
initiating
N-terminal
methionine
is
retained
(Burstein
et
al.,
1977;
Palmiter
et
al.,
1978b;
Mellor
&
Smith,
1978).
Our
experiments
with
a
membrane-supplemented
cell-free
system,
using
[31S]methionyl-tRNAM't
or
the
free
amino
acid,
demonstrate
that
the
addition
of
microsomal
membranes
results
in
the
processing
of
all
the
milk-protein
primary
translation
products.
In
these
experiments
we
cannot
exclude
changes
in
electrophoretic
mobility
of
the
caseins,
caused
in
part
by
other
post-translational
modifications,
a
supposition
supported
by
reports
of
coupled
cell-free
synthesis,
segregation
and
core
glycosylation
of
integral
membrane
(Rothman
&
Lodish,
1977)
and
secretory
proteins
(Lingappa
et
al.,
1978a,b).
How-
ever,
our
experiments,
using
methionyl-tRNAM"e
in
the
presence
and
absence
of
microsomal
mem-
branes,
suggest
that
the
addition
of
membranes
results
in
a
proteolytic
cleavage
at
the
N-terminus
of
all
the
caseins
synthesized
in
vitro.
Interpretation
of
this
result
depends
very
much
on
the
assumption
that
the
initiating
methionine,
whether
formylated
or
otherwise,
is
not
removed
by
eukaryote
aminopeptidases
(Housman
et
al.,
1970;
Mellor
&
Smith,
1978),
particularly
in
the
present
experiment
by
a
membrane-associated
enzyme.
Evidence
concerning
the
timing
of
this
event
is
limited,
but
is
consistent
with
a
rapid
removal
of
the
initiator
methionine.
Thus
it
is
removed
from
globin
when
the
polypeptide
is
15-20
residues
long
(Jackson
&
Hunter,
1970),
and
from
ovalbumin
when
the
nascent
chain
is
19-22
residues
long
(Palmiter
et
al.,
1978b).
Ovalbumin
is
a
secreted
protein,
which,
although
devoid
of
a
transient
N-terminal
peptide
(Palmiter
et
al.,
1978a),
is
sequestered
in
an
identical
manner
to
those
proteins
containing
such
a
sequence
(Lingappa
et
al.,
1978a).
Thus
it
seems
reasonable
to
speculate
that
N-terminal
specific
methionyl
aminopeptidases
capable
of
removing
the
initiating
methionine
are
not
present
in
the
membrane
preparation,
as
calculations
suggest
that
attachment
of
the
nascent
polypeptide
chain
of
secretory;
proteins
to
the
endoplasmic
reticulum
occurs
after
the
addition
of
at
least
40-50
residues
(Rothman
&
Lodish,
1977;
Palmiter
et
al.,
1978b;
Craig
et
al.,
1979).
These
observations
tend
to
pre-
clude
any
possibility
that
the
evidence
that
we
have
presented
represents
only
the
removal
of
the
initi-
ating
methionine
residue
from
guinea-pig
caseins
as
a
result
of
added
microsomal
membranes.
We
conclude
that,
in
spite
of
the
anomalous
electrophoretic
mobility
on
a
variety
of
SDS/poly-
acrylamide-gel
systems
of
the
caseins
synthesized
in
vitro
when
compared
with
their
counterparts
isolated
from
guinea-pig
milk
(see
Craig
et
al.,
1976,
1978,
1979;
Zehavi-Willner
&
Lane,
1977),
it
is
apparent
that
the
primary
translation
products
of
guinea-pig
caseins,
like
oc-lactalbumin,
are
synthesized
with
transient
N-terminal
peptide
extensions
of
un-
determined
length,
and
that
these
are
removed
when
1979
266

N-TERMINAL
PROCESSING
OF
CASEINS
267
synthesis
occurs
in
the
presence
of
added
microsomal
membranes,
an
observation
consistent
with
current
concepts
concerning
the
segregation
of
secretory
proteins
(Blobel
&
Dobberstein,
1975a,b).
The
rationale
behind
our
approach
has
been
substantiated
by
reports
from
several
laboratories
which
demon-
strate,
by
sequence
analysis
of
milk
proteins
synthe-
sized
in
vitro
in
the
absence
of
membrane
prepara-
tions,
that
rat
a-lactalbumin
(Lingappa
et
al.,
1978b),
sheep
a-lactalbumin
(Mercier
et
al.,
1978a),
fl-lacto-
globulin
(Mercier
et
al.,
1978b),
as,-,
aS2-,
0-
and
K-caseins
(Gaye
et
al.,
1977)
are
all
synthesized
with
N-terminal
peptide
extensions.
It
is
also
noteworthy
that
the
N-terminal
peptides
were
found
to
be
Met-
Lys
for
sheep
fl-lactoglobulin,
aSl-,
aS2-
and
,B-caseins,
Met-Arg
for
sheep
K-caseins,
and
Met-Met
for
sheep
a-lactalbumin
and
rat
a-lactalbumin.
Our
approach
therefore
provides
a
simple
rapid
method
that
may
beused
to
distinguishrapidly
between
proteolytic
cleavage
of
transient
N-terminal
'signal'
sequences,
and
co-translational
or
post-translational
modifications
of
secretory
proteins,
both
of
which
may
affect
the
mobility
of
the
resulting
peptides
when
analysed
by
SDS/polyacrylamide-gel
electro-
phoresis.
This
conclusion
is
supported
by
similar
experiments
which
have
recently
demonstrated
in
an
identical
manner
that
the
glycoprotein
(G)
of
ves-
icular
stomatitis
virus
is
synthesized
with
a
nascent
N-terminal
peptide
extension,
which
is
removed
during
cell-free
protein
synthesis
in
the
presence
of
membranes
(Irving
et
al.,
1979).
We
thank
Professor
P.
N.
Campbell
and
Dr.
A.
P.
Boulton
for
many
helpful
discussions,
and
Mr.
D.
Parker
for
valuable
technical
assistance.
P.
A.
J.
P.
was
supported
by
a
Commonwealth
Medical
Fellowship
from
the
Commonwealth
Scholarship
Commission.
References
Blobel,
G.
&
Dobberstein,
B.
(1975a)
J.
Cell
Biol.
67,
835-851
Blobel,
G.
&
Dobberstein,
B.
(1975b)
J.
Cell
Biol.
67,
852-862
Bonner,
W.
M.
&
Laskey,
R.
A.
(1974)
Eur.
J.
Bioclheim.
46,
83-88
Brew,
K.
(1972)
Eur.
J.
Biochemt.
27,
341-353
Burstein,
Y.,
Zemell,
R.,
Kantor,
F.
&
Schechter,
1.
(1977)
Proc.
Natl.
Acad.
Sci.
U.S.A.
74,
3157-3161
Clegg,
J.
C.
S.
&
Kennedy,
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T.
(1975)
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