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Polypeptide and phospholipid composition of the membrane of rat liver peroxisomes: comparison with endoplasmic reticulum and mitochondrial membranes

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Membranes were isolated from highly purified peroxisomes, mitochondria, and rough and smooth microsomes of rat liver by the one-step Na2CO3 procedure described in the accompanying paper (1982, J. Cell Biol. 93:97-102). The polypeptide compositions of these membranes were determined by SDS PAGE and found to be greatly dissimilar. The peroxisomal membrane contains 12% of the peroxisomal protein and consists of three major polypeptides (21,700, 67,700 and 69,700 daltons) as well as some minor polypeptides. The major peroxisomal membrane proteins as well as most of the minor ones are absent from the endoplasmic reticulum (ER). Conversely, most ER proteins are absent from peroxisomes. By electron microscopy, purified peroxisomal membranes are approximately 6.8 nm thick and have a typical trilaminar appearance. The phospholipid/protein ratio of peroxisomal membranes is approximately 200 nmol/mg; the principal phospholipids are phosphatidyl choline and phosphatidyl ethanolamine as in ER and mitochondrial membranes. In contrast to the mitochondria, peroxisomal membranes contain no cardiolipin. All the membranes investigated contain a polypeptide band with a molecular mass of approximately 15,000 daltons. Whether this represents an exceptional common membrane protein or a coincidence is unknown. The implications of these results for the biogenesis of peroxisomes are discussed.
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Polypeptide
and
Phospholipid
Composition
of
the
Membrane
of
Rat
Liver
Peroxisomes
:
Comparison
with
Endoplasmic
Reticulum
and
Mitochondrial
Membranes
YUKIO
FUJIKI,
STANLEYFOWLER,
HELEN
SHIO,
ANN
L
.
HUBBARD,
and
PAUL
B
.
LAZAROW
The
Rockefeller
University,
New
York
10021
;
and
Department
of
Cell
Biology
and
Anatomy,
The
Johns
Hopkins
University
School
of
Medicine,
Baltimore,
Maryland
21205
ABSTRACT
Membranes
were
isolated
from
highly
purified
peroxisomes,
mitochondria,
and
rough
and
smooth
microsomes
of
rat
liver
by the
one-step
Na
2
CO3
procedure
described
in
the
accompanying
paper
(1982,
J
.
Cell
Biol
.
93
:97-102)
.
The
polypeptide
compositions
of
these
membranes
were
determined
by
SDS
PAGE
and
found
to
be
greatly dissimilar
.
The
peroxisomal
membrane
contains
12%
of
the
peroxisomal
protein
and
consists
of
three
major
polypeptides
(21,700,
67,700
and
69,700
daltons)
as
well
as
some
minor
polypeptides
.
The
major
peroxisomal
membrane
proteins
as
well
as
most
of
the
minor
ones
are
absent
from
the
endoplasmic
reticulum
(ER)
.
Conversely,
most
ER
proteins
are
absent
from
peroxisomes
.
By
electron
microscopy,
purified
peroxisomal
membranes
are
-6
.8
nm
thick
and
have
a
typical
trilaminar
appearance
.
The
phospholipid/protein
ratio
of
peroxisomal
membranes
is
-200
nmol/mg
;
the
principal
phospholipids
are
phosphatidyl choline
and
phosphatidyl
etha-
nolamine,
as
in
ER
and
mitochondrial
membranes
.
In
contrast
to
the
mitochondria,
peroxisomal
membranes
contain
no
cardiolipin
.
All
the
membranes
investigated
contain
a
polypeptide
band
with
a
molecular
mass
of
15,000
daltons
.
Whether
this
represents
an
exceptional
common
membrane
protein
or a
coincidence
is
unknown
.
The
implications
of
these
results
for
the
biogenesis
of
peroxisomes
are
discussed
.
Knowledge
of
the
peroxisomal
membrane's
properties
is
essen-
tial
to
an
understanding
both
of
the
organelle's
functions
and
of
its
biogenesis
.
The
membrane
separates
the
peroxisomal
contents
from
the
cytosol
and
defines
the
peroxisomal
interior
as
a
distinct
intracellular
space
.
The
permeability
properties
of
the
membrane
determine
to
what
extent
the
peroxisome
func-
tions
as
a
separate
biochemical
compartment
.
Knowledge
of
how
the
membrane
is
formed
is
essential
to
an
understanding
of
the
biogenesis
of
the
organelle
as
a
whole
.
If
the
membrane
is
derived
from
some
other
intracellular
membrane,
for
exam-
ple the
endoplasmic
reticulum
(ER)
(as
is
widely
assumed),
then
one might
expect
to
see
some
similarity
in
composition
between
them
.
If,
on
the
other
hand,
the
peroxisomes
exist as
a
separate
intracellular
compartment,
as
has
recently
been
suggested
(1),
then
the
peroxisomal
membrane
needs
to
have
no
structural
similarity
to
the
ER
.
THE
JOURNAL
OF
CELL
BIOLOGY
"
VOLUME
93
APRIL
1982
103-110
©The
Rockefeller
University
Press
-
0021-9525/82/04/0103/08
$1
.00
We
have
applied
the
newly-developed
sodium
carbonate
procedure
described
in
the
accompanying
paper
(2)
to isolate
peroxisomal,
mitochondrial,
and
ER
membranes
.'
We
have
partially
characterized
these three
membranes,
and
found
that
their
polypeptide
compositions
are
almost
entirely
different,
but
their
phospholipid compositions are
similar
.
Some
of
these
results
have appeared
in
abstract
form
(3,
4)
.
'
Rat
liver
microsomes
were
subfractionated by isopycnic
centrifuga-
tion
in
linear
sucrose gradients
according
to
Beaufay
et al
.
(7)
.
The
fractions selected
as
the
"rough
microsomal
fraction"
have been
shown
by
these
authors
to
consist
almost
exclusively
of
vesicles
derived
from
the
rough
endoplasmic
reticulum
.
This
justifies
the use
of
the
term
"endoplasmic
reticulum
membranes"
rather
than
the
operational
expression
"microsomal
membranes
."
103
MATERIALS
AND
METHODS
Preparation
of
Membranes
by
Means
of
Sodium
Carbonate
Treatment
Peroxisomes
or
other
organelles
were
diluted
100-fold
with
ice-cold
100
mM
Na,M,
pH
11
.5,
kept
on
ice for
30 min,
and
centrifuged
for
I
h
at
50,000
rpm
in
a
Beckman
50
Ti
rotor
(Beckman
Instruments,
Inc
.,
Spinco
Div
.,
Palo
Alto,
CA)
as
described
previously
(2)
.
Isolation
of
Organelles
Peroxisomes
were
purified
from
rat
liver
by
sequential
differential
and
equi-
librium density
centrifugation
exactly
as
described
by
Leighton
et al
.
(5)
.
The
purity
of
the
peroxisomes
was
determined
by
measurement
of
specific
marker
enzymes
for the
various
organelles
.
We
selected
the
three
purest
peroxisome
preparations of 14
that
were
prepared,
and
used
fractions
on
the
dense
side
of
the
peroxisome
peak
to
further
minimize
contamination
by
other,
less
dense
organ-
elles
.
As
shown
in
Table
I,
the
relative specific activity
of
catalase
in
the
purified
peroxisomes
was
35,
which
in
comparison
to
the
results
of
Leighton
et at
.
(5)
implies
-93%
purity
.
The
ratio
of
catalase/cytochrome
oxidase
(a
mitochondrial
marker)
is
2,500,
and
the
ratio
of
catalase/esterase
(a
microsomal
marker)
is
65
.
From
the
relative
specific
activities
in
Table
I
and
the
fact that
mitochondria
and
ER
each
constitutes
-20%
of
total liver
protein
(5-7),
we
calculate
that
the
contamination
of
peroxisomes
by
mitochondria
is
0
.040
x
20
=0
.8%,
and
the
contamination
by microsomes
is
0
.175
x
20
=
3.5%
.
Free
peroxisome
cores,
which
are
known
to
be
concentrated
on
the
dense
side
of
the
peroxisome
peak
(5),
probably
contribute
the
bulk
of
the
remaining
protein
.
It
should
be
empha-
sized
that
microsomes
are
a
common
contaminant
of
peroxisomes
(because
rough
microsomes have a
density
similar
to
that
of
peroxisomes),
and
without
the
precautions
taken
here,
they
may
constitute
a
considerable
portion
of
the
protein
present
.
Mitochondria
were
also
purified
by
sequential
differential
and
isopycnic
centrifugation
(5)
and
were
-86%
to
93%
pure
.
The
preparation
of
rough
and
smooth
microsomal
subfractions
by
centrifugation
in
linear
sucrose gradients
and
the
removal
of
ribosomes
by
means
of
pyrophosphate
treatment
have been
described
elsewhere
(2,
7,
8)
.
Analytical
Methods
SDS
PAGE,
electron
microscopy
of
membranes,
enzyme
assays,
and
protein
determinations
were
carried
out
as
described
(2,
5)
.
Freshly
isolated
peroxisomes
were
prepared
for
electron
microscopy
according
to
Baudhuin
et al
.
(9)
but
with
modifications
(5)
to
prevent
osmotic
lysis
during
fixation
.
The
fixed
peroxisomes
were
collected
by
filtration
on
VC
Millipore
filters
(0
.1-Am
pore
size
;
Millipore
Corp
.,
Bedford,
MA)
.
Lipids
were
extracted
into
chloroform-methanol
by
the
procedure
of
Bligh
and
Dyer
(l0)
.
For
quantification, aliquots
of
the
chloroform phase
were
dried
down
and
assayed
for
organic
phosphate
after
Mg(N0
3
)
2
ashing
(11)
.
The
results
are
expressed
in
nanomoles
of
organic
phosphate
.
Phospholipid compositions
were
assessed
by
chromatography
on
250
gin
thick
Silica
Gel
G
plates
using
chloroform/methanol/water
(65
:25
:4,
vol/vol/vol) as
solvent
(l2)
.
After
chro-
TABLE
I
Properties of
Purified
Peroxisomes
obtained
by
Leighton
et
al
.
(5)
All
values
given
as
mean
and
standard
deviation
for
three
preparations
.
Peroxisomes
taken
from the dense
side
of
the peroxisome peak
in
the
sucrose
gradients
by
which
they
were
purified
.
Mean
density
=
1
.265
J-
0
.005
.
$
SA,
specific
activity
in
units/milligram
protein
;
units
defined
as in
reference
5
.
§ Glucose-6-phosphatase
as
microsomal marker
.
104
THE
JOURNAL
OF CELL
BIOLOGY
"
VOLUME
93,
1982
matography
the plates
were
sprayed
with
55%
H
2
SO
4
(wt/vol)
containing
0.6%
K
2
CrO
4
(wt/vol)
and
charred
at
180
°
C
for
15
min
in
order
to
detect
the
lipids
.
Materials
Thin
layer
chromatography
plates (Prekotes,
Silica
Gel
G)
were
purchased
from Applied
Science
Div
.,
Milton
Roy
Co
.,
Laboratories
Group
(State
College,
PA)
.
Phospholipid
standards
were from
Supelco,
Inc
.
(Bellefonte,
PA)
.
Other
materials
were
obtained
as
before
(2)
.
RESULTS
Characterization
of
Peroxisomal
Membranes
Peroxisomes
(Fig
.
1
a)
were
treated
with 100
mM
sodium
carbonate
and
centrifuged
.
Electron
microscope
examination
of
the
pellet
revealed
membranes,
mostly
in
the
form
of
un-
sealed
fragments
with
sizes
of
~0
.1
to
0
.5
pm
(Fig
.
1
b)
.
The
membranes
retained
a
trilaminar
appearance
(inset
to
Fig
.
1
b)
.
The
thickness
of
the
peroxisomal
membrane
was
7
.0
±
0
.8
nm
before,
and
6
.8
±
0
.8
nm
after
carbonate
treatment
.
The membrane
pellet
and
the
solubilized
proteins
were
compared
to
total
peroxisomal
proteins
by
SDS
PAGE
(Fig
.
2)
.
Nearly
all
of
the
proteins
were
found
in
soluble
form
(Fig
.
2,
lane
Q,
and
were
undetectable
in
the
pellet
(Fig
.
2,
lane B)
.
These
included
the
major
peroxisomal
protein
catalase,
located
in
the
organelle matrix,
and
urate
oxidase, located
in
the core
.
The
Na
2
CO
3
dissolved
the
cores
(13,
14),
and
quantitatively
released
the
matrix
proteins
.
One
polypeptide
band,
visible
among
the
total
peroxisomal
proteins
(indicated
with
an
arrow
in
Fig
.
2),
was
present
only
in
the
membranes
and
not
among
the
soluble proteins
.
Two
other
membrane
polypeptides
(ar-
rowheads)
could
not be discerned
among
the
total
proteins
due
to
the
large
amount
of
soluble
proteins
in
this
region
of
the
gel
.
The sum
of the
membrane
and
soluble
components
(Fig
.
2,
lanes
B
and
C)
was
similar
to
the
starting
material
(Fig
.
2,
lane
A),
indicating
apparently
quantitative
recovery
.
The
smaller
membrane
polypeptide
consistently
appeared
as
a sharp
band
in
SDS
PAGE
and
has
been
observed
in
all
of
our
membrane
preparations
.
The
two
larger
membrane
poly-
peptides
varied
in
their
appearance
:
occasionally
they
were
somewhat
fuzzy or not
resolved
from
one
another
in
SDS
PAGE
(Fig
.
6,
lanes
2
and
6)
.
The
same
pattern
of
membrane
proteins
as
that
shown
in
Fig
.
2
was
observed
when
peroxisomal
membranes
were
isolated in
the
presence
of
a
mixture
of
protease
inhibitors
(not
illustrated)
.
The
pattern
was
unaffected
by
a
second
extraction
with
carbonate
.
The
molecular
masses
of
the
peroxisomal
membrane
poly-
peptides
were
estimated
by
comparison
with
known
standards
in
SDS
PAGE
.
The
three
prominent bands
have
mean
masses
of
69,700
±
1,300,
67,700
±
1,000,
and
21,700
t
300
daltons
(means
and
standard
deviations
of
12
determinations)
.
Peroxisomal
membranes
contained
-12%
of the
total
per-
oxisomal
proteins
(Table
II)
.
The
membranes
were
highly
enriched
in
phospholipids
in
comparison
to
the
released
ma-
terial
.
The
phospholipid/protein
ratio
was
204
nmol
phos-
phate/mg
membrane
protein
(Table
II)
.
This
is
^-60%
of
the
phospholipid/protein
ratio
of
340
±
40
nmol/mg
that
we
found
for
two
preparations of
microsomal
membranes
(not
shown)
.
Characterization
of
Mitochondrial
Membranes
Application
of
the
carbonate
procedure
to
purified
mito-
chondria
yielded
membranes
(Fig
.
3)
which
contained
-21%
of
total
mitochondrial
proteins
(Table
III)
.
SDS
PAGE
analysis
revealed
about
a
dozen major
membrane
polypeptides,
as
well
Catalase
Cytochrome
oxidase
Esterase
Peroxisome
SA$
9
.46
±
1
.59
0
.0038
t
0
.0018
0
.146
t
0
.052
HomogenateSA
0
.273
±
0
.022
0
.105
t
0
.025
0
.847
±
0
.332
Relative
sp act 34
.7
t
4
.6
0
.040
±
0
.026
0
.175
±
0
.026
(Peroxisome
SA/homoge-
nate
SA)
Relative
SA
36
.3
±
6
.4
0
.11
t
0
.1
0
.09
±
0
.08§
FIGURE
1
Electron
micrographs
of
purified
peroxisomes
(a)
and
peroxisomal
membranes
prepared
by
sodium
carbonate
treatment
(b)
.
Arrow
indicates
a
free
peroxisomal core
.
Bar,
0
.5ILm
.
(a)
x
38,000
.
(b)
x
50,000
.
Inset
:
bar,
50
nm
.
x
234,000
.
as
many
minor
ones
(Fig
.
4)
.
These
are
presumably a
mixture
of
inner
and
outer
membrane
proteins
.
Comparison
of
the
Polypeptide
Composition
of
Peroxisomal
and
Mitochondrial
Membranes
The
final step in
the
purification
of
peroxisomes
and
mito-
chondria
is
centrifugation
in
a
sucrose
gradient
.
When
mem-
branes
were
prepared
from
each
fraction
of
the
gradient
(by
exposure
to
Na
2
CO
3
),
the
three
major
peroxisomal
membrane
polypeptides
were
clearly
visible,
peaking
in
the
peroxisomal
region
of
the
sucrose
gradient (Fig
.
5,
lanes
3
and
4,
arrows)
.
These
bands
decreased
in
intensity
as
the
density
decreased
into
the
mitochondrial
region
of
the
gradient
.
This
demon-
strates
that
these three
polypeptides
are
true
peroxisomal
pro-
teins
and
do
not belong
to
contaminating mitochondria
.
The
mitochondrial
membrane
proteins
were
observed
at
maximum
concentration
in
fraction
8,
but
could
still
be detected
in
small
amounts
in
the
peak
peroxisomal
fraction
(fraction
4)
.
This
illustrates
the
cross-contamination
that occurs,
and demon-
strates
the
need
to select
fractions
on
the
dense
side
of
the
peroxisomal
peak
to
obtain
sufficiently
pure
membranes
.
Comparison
of
the
Polypeptide
Composition
of
Peroxisomal,
Mitochondrial,
and
ER
Membranes
Equal
amounts
of
the
membranes
of
the
various
highly
purified
organelles
were
analyzed
by
SDS
PAGE
(Fig
.
6)
.
The
patterns
of
membrane
polypeptides
differed
strikingly
among
peroxisomes,
mitochondria,
and
microsomes,
whereas
the
two
subclasses
of
microsomal
membranes
were
similar
to
each
other
.
Cytochrome
P-450
and
other
majormicrosomal
proteins
were
absent
from
the
peroxisomal
membranes
.
Conversely,
the
three
main
peroxisomal
membrane
proteins
were
absent
from
the
microsomal
membranes
.
The
mitochondrial
pattern
was
also
unique
.
The
amount
of
peroxisomal
membrane
protein
analyzed
was
considerably
larger
in
this
experiment,
and
some
minor bands
were
visible
(Fig
.
6,
lanes
2
and
6)
.
A
few
of
these
may
be
present
owing
to
residual
contamination
by
mitochondria
and
ER,'
others
may
represent
traces
of
soluble
peroxisomal
pro-
teins,
and
still
others
may
be
genuine
constituents
of
the
peroxisomal
membrane
.
Although
the
polypeptide
compositions
of
the
three
organ-
elles'
membranes
were
distinctly different,
a few
polypeptides
with
the
same
apparent
size
were
present
in
the
three
mem-
branes
in
amounts
too
large
to
be
attributed
to
cross-contami-
nation
.
The
most
prominent
of
these
(labeled
with
an
asterisk)
had
a
molecular
mass
of
-
15,000
daltons,
and
a
similar
inten-
sity
in
all
samples
.
Whether
this
represents
a
common
mem-
brane
protein
in
all
three
organelles
remains
to
be
determined
by
methods
other
than one-dimensional
SDS
PAGE
.
In
addi-
tion,
both
mitochondrial
and
peroxisomal
membranes
con-
tained
small
amounts
of a
polypeptide
that
comigrated
with
microsomal cytochrome
b
5
(Fig
.
6)
.
This
is
consistent
with
the
presence of
this
cytochrome
in
outer
(but
not
inner)
mitochon-
dria)
membranes
(15-17)
and
its
reported
presence
in
small
amounts
in
the
peroxisomal
membrane
(18)
.
'The
traces
of
cytochrome
P-450
visible
in these
large
samples
of
peroxisomal
membranes
can
be
entirely
accounted
for
by
the
residual
3
.5%
contamination
of
the
purified
peroxisomes
by
endoplasmic
retic-
ulum
(see
Materials
and
Methods)
.
This
illustrates
the
limits
of
the
methodologies
and
the
need
for
quantitative
evaluation
of
membrane
purity
.
FU
J
iiu
ET
At
.
Peroxisomal
Membrane
105
FIGURE
2
Preparation
of
peroxisomal
membranes
by
sodium
car-
bonate
treatment
.
Peroxisomes
were
treated
with
Na
2
CO
3
and
cen-
trifuged
;
total
membranes
and
soluble
proteins
were
compared
with
the
starting
material
by
SDS
PAGE
.
(A)
Total
peroxisomal
protein-
100
Ftg
.
Membrane
(B)
and
soluble
proteins (C)
derived
from
100
wg
of
peroxisomal
protein
.
Molecular
mass
standards
:
bovine
serum
albumin
(BSA,68,000),oval
bumin
(OVAL,
45,000),trypsinogen
(TRY-
GEN,
24,000),
soybean
trypsin
inhibitor
(SBTI,
21,500),
,ß-lactoglob-
ulin
(ß-LG,
18,400),
lysozyme (LYSO,
14,300),
and
bovine
lung
tryp-
sin
inhibitor
(aprotinin)
(BTI,
6,500)
.
Arrows
and
arrowheads
point
to
the
membrane
proteins
.
Cat
.,
catalase
.
U
.Ox
.,
urate
oxidase
.
TABLE
II
Preparation
of
Peroxisome
Membranes
Protein
Phospholipid*
*
Expressed
in
nanomoles
of
organic
phosphate
.
$
In
two
other
experiments,
the
percentages
of
starting
material
were
12,
81,
and
93,
and
10,
104,
and
114
for
membranes,
soluble
proteins,
and
recovery,
respectively
.
Comparison
of
the
Lipid
Compositions
of
the
Membranes
All
membrane
preparations
investigated
contained
phospha-
tidyl
choline
and
phosphatidyl
ethanolamine
as
their
major
10
6
THE
JOURNAL
OF CELL
BIOLOGY
"
VOLUME
93,
1982
FIGURE
3 Electron
micrograph
of
mitochondrial
membranes
.
Bar,
0
.5
g,m
.
x
50,000
.
TABLE
III
Application
of
Carbonate Procedure
to
Various
Organelles
Protein
in
Phospholipid
in
membranes
membranes
Range
or
stand-
ard de-
n
Mean
viation*
n
Mean
Range
3
7
*
Range
where
n
=
2,
standard
deviation
where
n =
3
.
phospholipid
constituents (Fig
.
7)
.
Cardiolipin
was
found
only
in
the
mitochondrial
membranes
.
Some
sphingomyelin
may
be
present
in
microsomal
fractions,
possibly
owing
to
the
presence
of
plasma
membranes
in
these
preparations
(19)
.
Lyso
deriva-
tives
of
the
phospholipids
were
evident
in
various
amounts
in
different
membrane
preparations
and
were probably
the
result
of
endogenous
phospholipases
acting
on
the
preparations dur-
ing
organelle
isolation
and
storage
.
All
of
the
membrane
fractions
contained
some
cholesterol
and
cholesteryl
ester
.
These
appeared
as
rapidly
migrating
bands
near
the
solvent
front
in
the
thin
layer
chromatograms
shown
in
Fig
.
7,
and
were
identified
in
two
other
solvent
systems
(hexane
:diethyl
ether
:acetic
acid
(80
:20
:1,
vol/vol/vol)
and
isopropyl ether
:glacial
acetic
acid
(96
:4,
vol/vol),
not
illus-
trated)
.
The
amounts
of
cholesterol
found
were
much
smaller
%
of
membrane
and
soluble
%
of
membrane
and
soluble
Total
microsomes
2 53
±5
Stripped
rough
3
81
±15
2
94
microsomes
Mitochondria
3 21
±1
1
87
Peroxisomes
3 12
t3
2
83
Wg
starting
material$
nmol
starting
material
Phospholipid/
protein
nmol/mg
Peroxisomes 367
13
.3
36
Membranes
50 14 10
.2
77
204
Soluble
319
87
3
.2
24
10
Recovery 369
101 13
.4
101
FIGURE
4
Preparation
of
mitochondrial
membranes
by
sodium
car-
bonate
procedure
;
analysis
by
SIDS
PAGE
.
(A)
Total
mitochondrial
protein-100
Wg
.
Membrane
(8)
and
soluble
proteins
(C)
derived
from
100/g
of
mitochondrial
protein
.
Total (D),
membrane
(E),
and
soluble
proteins (F)
from
200
8g
of
mitochondrial
protein
.
STD,
molecular
weight
standards
.
than
what
was
present in
plasma
membranes
(analyzed
for
comparison
in
Fig
.
7,
lane
8)
.
It
has
been
shown
that
most of
the
cholesterol
in
microsomal
fractions
is
in
contaminating
plasma
membranes
(7,
8)
.
It
is
apparent
from
Fig
.
7
that
the
relative
abundances
of
the
various
lipids
is
not
identical
in
all
the
membranes
.
Thus
far,
insufficient
material
has
been
obtained
for
chemical
quantifi-
cation
.
Procedure
In
several control
experiments
we
found
that
the
most
im-
portant
parameter
for
the
successful
isolation
of
peroxisomal
and
mitochondrial
membranes
was
the
pH,
as
it
was
for
microsomal
membranes
(2)
.
Adjusting
the
pH
to
11
with
1
mM
K2B407
caused
a
release
of
proteins
similar
to that
produced
by
100
mM
Na
2
CO
3
(as
judged by
SDS
PAGE,
not
illustrated)
.
Lower
pHs
were
less
effective
or
ineffective
.
250
mM
NaHC03
or
NaCl
or
KCl
would
not
substitute
for
the
carbonate
.
A
second
application
of
Na
2
CO
3
to
isolated
peroxisomal
mem-
branes
did
not
change
the
polypeptide
composition
.
DISCUSSION
Comparison
of
Membranes
In this
investigation,
we
have
isolated
three
intracellular
membrane
systems
.
We
can
calculate
that
they vary
greatly
in
their
abundance,
ranging
from
0
.8
mg
of
peroxisomal
mem-
brane
protein/g
liver
to
28
mg
of
ER
membrane
protein/g
liver
(Table IV)
.
This
disparity,
and
the
similar
densities
of
rough
ER
and
peroxisomes,
are the
causes of the
difficulty
in
FIGURE
5
SIDS
PAGE
analysis
of
membranes
prepared
from each
fraction
of
a
sucrose
gradient
used
to
separate
peroxisomes
and
mitochondria
.
Membranes
were
prepared
from
6-AI
aliquots of
the
fractions
(5-570
wg
protein)
.
Numbers
at
the
top
indicate fraction
numbers,
starting
at
the
bottom
of
the
gradient
.
Peroxisomes
were
mainly
in
fractions
3
and
4
.
Mitochondria were
most abundant
in
fraction
8,
but
extended
down
as
far
as
fraction
4
.
The
three
main
peroxisomal
membrane
proteins
are
indicated
with
arrows
.
Two
mitochondrial
membrane
proteins
visible
in
lane
4
are
indicated
with dots
.
Standard
proteins
(STD)
as in
Fig
.
2
.
FIGURE
6
Polypeptide
composition
of
organelle
membranes
.
Equal
amounts
of
each type
of
membrane
(50ILg
protein)
were
analyzed
by
SDS
PAGE
.
Lanes
1
and
4,
mitochondria
;
2
and
6,
peroxisomes
;
3
and
7,
rough
microsomes
stripped
of
ribosomes
with
pyrophos-
phate
;
5
and
8,
smooth
microsomes
.
STD,
molecular
weight
stand-
ards
.
Identification
of
cytochromes
b
s
and
P-450
as
in
reference
2
.
Asterisk indicates a
stained
band
visible
in
all
the
membranes
.
FujIKI
ET
AL
.
Pemxisomal
Membrane
10
7
FIGURE
7
Thin
layer
chromatography
of
membrane
phospholipids
.
Membranes
were
prepared
by
the
Na2CO3
procedure
and
extracted
with
chloroform/methanol
(see
Materials
and
Methods)
.
Numbers
in
parentheses
indicate
the
milligrams
of
membrane
protein
from
which
the
analyzed
phospholipids
were
extracted
.
Lanes
:
3,
mito-
chondria
(0
.4)
;
4,
peroxisomes
(0
.7)
;
5,
smooth
microsomes
(0
.5)
;
6,
rough
microsomes
(1
.2)
;
7,
smooth
microsomes
(1
.0)
;
8,
plasma
membranes
(0
.6)
.
Standard
phospholipids
(0
.2
mg
except
where
noted)
:
Lane
1,
cholesterol
(CHOL),
phosphatidyl
ethanolamine
(PE),
phosphatidyl
serine
(PS,
0
.6
mg),
and
lysophosphatidyl
serine
(LPS,0
.4
mg)
.
Lane
2,cardiolipin
(CARD),
phosphatidyl choline
(PC),
sphingomyelin
(SPM),
and
lysophosphatidyl
choline
(LPC,
1
.6
mg)
.
Lane
9,
CHOL
and
PE
.
Lane
10,
PC
and
SPM
.
Open
arrowheads
point
to
standards
in
lanes
1
and
9,
closed
arrowheads
to
those
in
lanes
2 and
10
.
The
plasma
membranes
were
purified
30-fold
from
livers of
normal
rats
according
to
A
.
L
.
Hubbard
and
A
.
Ma
(manu-
script
in
preparation)
.
TABLE
IV
Amounts
of
Organelle
Membranes
in
Rat
Liver
Fraction
of
*
Assuming
260
mg
protein/g
fasted
liver,
of
which
ER,
mitochondria,
and
peroxisomes
contribute
20%,
20%,
and
2
.5%,
respectively
(5-7)
.
$
From
Table
111
.
obtaining
pure
peroxisomal
membranes
.
SDS
PAGE
analysis
(Fig
.
6)
revealed
very
different
patterns
for
the
three
membrane
types,
indicating
that
(a)
the
three
organelles
contain
mostly
different
proteins
in
their
membranes
and
(b)
the
three types
of
membrane
are
each
quite
pure
.
A
few
similarities
were
noted,
including
the
presence
of
a
15,000
dalton
polypeptide
in
about
the
same
abundance
in
all
three
membranes,
and
some
cytochrome
bs
in
each
.
It is
not
known
whether
the
15,000 dalton
band
is
the
same
polypeptide
in
the
three
membrane
types
.
These
results
do
not
exclude
the
pres-
ence
of
other
components
or
enzymes
in
trace
amounts
in
all
three
membranes
.
In
contrast
to
the
very
different
protein
phospholipid compositions
of
the
various
10
8
THE
JOURNAL
OF
CELL
BIOLOGY
"
VOLUME
93,
1982
compositions,
the
membranes
were
qualitatively
similar,
consisting
largely
of
phosphatidyl
choline
and
phosphatidyl
ethanolamine
.
Cardiolipin
was
found
only
in
mitochondria
.
Some
cholesterol
was
observed
in
the
membrane
prepara-
tions,
especially
in
the
microsomal
fractions
.
The
presence
of
cholesterol
in
microsomal
fractions
was
described
by
Dallner
and
Ernster
(20),
but
later
Beaufay
et
al
.
(7)
and
Amar-Costesec
et
al
.
(8)
reported
that
the
bulk
of
this
cholesterol
was
actually
in
fragments
of
the
plasma
membrane,
which
constitute
-7-
8%
of
the
protein
of
the
microsomal
fraction
(7),
and
which
are
known
to
be
very
rich
in
cholesterol (21)
.
Our
own
results
are
generally
compatible
with
this
view,
except
that
qualitatively
it
appears
that
we
have
more
cholesterol
in
the
rough
micro-
somal
fraction
than
would
be
expected
from
the
quantitative
results
of Beaufay
et al
.
(7)
.
The
explanation
may
be
that, to
purify
the
peroxisomes,
our
rats
were
pre-treated
with Triton
WR-1339,
which
is
known
to
produce
a
hypercholesterolemic
serum
(22, 23), as
well as
an
accumulation
of
cholesterol
in
hepatic
lysosomes
(24)
.
Since
the
livers
were
not
perfused,
cholesterol
might have
adsorbed
onto
the
membranes
during
homogenization
.
Cholesterol
was
also
observed
in
the
peroxisomal
and
mito-
chondrial
membrane
preparations
(Fig
.
7)
.
Considering
the
various
amounts
of
membrane
protein
analyzed
(see
legend
to
Fig
.
7),
we
estimate
that
the cholesterol/protein
ratio
is
of
the
order
of 10
to
20
times lower
in
these
preparations
than
in
the
purified
plasma
membranes
.
Whether
this
cholesterol
is
a
true
constituent
of
peroxisomal
and
mitochondrial
membranes
can-
not
be
decided
with
certainty
at
present
.
It
could
be
accounted
for
by a
5-10%
contamination
by
plasma
membranes,
or
could
originate
from
the
hypercholesterolemic
serum
.
Methodology
The
carbonate
procedure
(2)
has proved
successful
for
the
isolation
of
three types
of
endomembrane
in
this
investigation
.
It
may
be emphasized
that
the
method
is
nondestructive
(sol-
uble
+
membrane
proteins
=
starting
proteins)
and
efficient
(polypeptide
bands
are
generally
found
to be
either
soluble
or
in
the
membranes,
but not
both)
.
As
discussed
by
Fujiki
et
al
.
(2),
the carbonate
procedure
appears
to effectively
release
peripheral
membrane
proteins,
and
this
conclusion
is
further
corroborated
by
our
results
.
We
have
combined
the use
of
the
sodium
carbonate
proce-
dure with
isopycnic
centrifugation
and
SDS
PAGE
to
analyze
membrane
proteins
as
a
function of
their
size
and
of
the
density
of
their
host
organelle
(Fig
.
5)
.
This
procedure
may
prove
useful
in
other
studies
of
cell
architecture
.
The
Peroxisomal
Membrane
The
peroxisomal
membrane
contains
12%
of
the
total
per-
oxisomal
protein,
three
major
polypeptides
(21,700,
67,700,
and
69,700
daltons)
and
some
minor
polypeptides
.
None
of
its
major
proteins
are
present
in
the
ER
;
conversely,
the
peroxi-
some
lacks
most
ER
proteins
.
The
phospholipid/protein
ratio
of
the
peroxisomal
membrane
is
-200
nmol/mg
;
the
major
lipids
are
phosphatidyl
choline,
phosphatidyl
ethanolamine,
:'
3
Hajra
et al
.
(35)
have
demonstrated
that
the
first
enzyme
in
ether
glycerolipid biosynthesis,
acyl-CoA
:dihydroxyacetone
phosphate
acyl-
transferase,
is
located
in
peroxisomes
in
rat
liver
.
In
the
absence
of
any
information
on
the
comparative
mobilities
of
ether
glycerolipids
and
the
usual
phospholipids
in
the
solvent
system
used,
it
is
conceivable
that
some
of
the
peroxisomal
lipids
are
actually
ether
glycerolipids
.
Organelle
protein*
mg/g
liver
organelle
protein
in
membrane(s)$
Organelle
membrane
protein
mglg
liver
Endoplasmic
52
0
.53
28
reticulum
Mitochondria
52
0
.21
11
Peroxisomes
6
.5
0
.12
0
.8
and
perhaps
cholesterol
.
Thus
the
phospholipid composition
of
the
peroxisomal
membrane
is
qualitatively similar
to
that
of
the
ER
but
the
phospholipid/protein
ratio
appears
to
be
lower
.
Our
conclusions
differ
from
those of
Donaldson
et al
.
(25),
who
emphasized
the
similarity
of
peroxisomal
and
ER
mem-
branes
in
rat
liver
.
It
would
appear
that
this
is
due
to
contam-
ination
of
their
peroxisomal
membranes
with
ER
.
Those
au-
thors report
that
the
specific activity
of
glucose-6-phosphatase
in
their
purified
peroxisomes
was
35%
of
the
specific activity in
purified
microsomes
(their
Table
VIII)
;
this
means
that
35%
of
the
protein
in
their
purified
peroxisomes
was
actually
ER,
because
peroxisomes
lack
glucose-6-phosphatase
altogether
(5)
.
Since
the
membrane
represents
a
larger
percentage
of
the
total
organelle
protein
in
microsomes
than
in
peroxisomes
(53%
vs
.
12%),
we
calculate
that
-70%
of
the
membrane
protein
in
the
purified
peroxisomes
of
Donaldson
et al
.
(25)
was
derived
from
the
ER
.
Under
these
conditions,
it
is
not
surprising
that
their
"peroxisomal
membranes"
appeared
similar
to
ER
.
Peroxisome
Biogenesis
Peroxisomes
have
long
been
thought
to
formby
budding
from
the
ER
.
This theory
is
based
in
large
part
on
published
morphological
observations
showing
proximity
as
well
as
pos-
sible
connections
between
peroxisomes
and
ER
.
Some
investi-
gators,
especially
Novikoff
et
al
.
(26),
argue
that
connections
between
these
two
organelles
are
common,
whereas
other
scientists
report
not
finding
any
connections
after
careful
search
(27,
28),
including
serial
sections
(28)
.
Recent
experiments
of
Shio
and
Lazarow
(29)
found
no
diffusion
of
cytochemical
reaction
products
between
peroxisomes
and ER,
consistent
with
there
being
no
connections
.
Novikoff
et al
.
(26)
have
even
suggested
that
small
anucleoid
peroxisomes
(referred
to
by
them
as
"microperoxisomes")
are
"specialized
regions
of
smooth
ER,"
an
idea
disproven
by
our
results
(since
we
find
that
peroxisomes
are
not
bounded
by
ER
membranes)
.
Were
the
polypeptide
composition
of
the
peroxisomal
mem-
brane
to
resemble
that
of
the
ER,
this
would
strongly
support
the
budding
hypothesis
.
However,
such
is
not
the
case,
and
thus
our
results
support
and
extend
other
biochemical
investi-
gations
of the
past
15
years
that
have
similarly
found
no
evidence
for
a
role
of
the
ER
in
peroxisome
biogenesis
(re-
viewed
by
Lazarow
et al
.
[1])
.
For
example,
catalase,
the
principal
matrix
protein
of
the
peroxisome,
does
not
pass
through
the
ER
on
its
way
to
the
peroxisomes
(30-33)
.
If
there
are
any
connections
between
peroxisomes
andER,
they
mustbe
such
as
to
prevent
catalase
and
other
peroxisomal
matrix
proteins
from
diffusing
into
the
ER,
they
must
prevent
serum
albumin
and
other
secretory
proteins
from
entering
peroxisomes, they
do
not allow
peroxisomal
membrane
pro-
teins
to
diffuse
within
the
plane
of
the
membrane
into
the
ER
membrane,and
they
do
not allow
ER
membrane
proteins
to
enter
the
peroxisomal
membrane
.
In
addition,
any
such
con-
nections
play
no
role in
the
biogenesis
of
catalase
.
It is
possible
that
the
peroxisomal
membrane
proteins
are
synthesized
on
bound
polysomes
and
that
the
peroxisomal
membrane
could
formfrom
the
ER
by
"capping"
of
peroxi-
somal
membrane
proteins
within the plane of
the
ER
mem-
brane,
followed
by
outpouching
and
pinching
off
.
Such
a
process
occurs
in
the
formation
of
the
envelope
of
VSV
virus
fromplasma
membrane
(34)
.
However,
the
viral
core
provides
a
matrix
to
which
the
viral
envelope
proteins
may
bind
specif-
ically
as
the
virus
buds
out
through
the
plasma
membrane,
and
no
such
nucleation
mechanism
is
available
for the
budding
of
the
peroxisomal
membrane
(the
peroxisomal
core
protein,
urate
oxidase,
is
synthesized
on
free
polysomes
[33])
.
Also,
the
re-
modeling
of
the
peroxisomal
membrane
according
to
this
hypothesis
would
have
to
be
virtually
total,
since
we
detect
practically
no
overlap
between
the
polypeptide
compositions
of
the
ER
and
peroxisomal
membranes
.
Lazarow
et
al
.
(1)
have
recently
pointed
out
that
almost
all
of the
biochemical
data
in
the
literature,
and
most of
the
morphological
observations,
fit
a
model
in
which
peroxisomes
exist
in
the
cell
without
connections
to
the
ER
but with
transient
interconnections
among
themselves
.
It
was
suggested
that
newly
synthesized
peroxisomal
constituents
(including
soluble, core,
and
membrane
components)
could
be
added
to
pre-existing
peroxisomes,
and
perhaps
then
be
further
distrib-
uted
by
fission
and
fusion events
.
Our
results
on
the
unique
polypeptide
composition
of the
peroxisomal
membrane
are
compatible
with
this
hypothesis
.
The
authors
wish
to
thank
Dr
.
Christian
de
Duve
for
stimulating
discussions
and
criticism
of
this
manuscript
.
We
thank
Peter
Eisemann
for his
expert
and
dedicated
technical
assistance
throughout
the
course
of
these
experiments
.
This
work
was
supported
by
National
Institutes
of
Health
grants
AM
19394
and
GM
29185,
and by
National
Science
Foundation
grant
PCM
80-08713
.
Dr
.
Fowler
conducted
his
part
of
these
studies
during
the
tenure of
an
Otto
G
.
Storm
Established
Investigatorship
from
the
American
Heart
Association
.
Dr
.
Lazarow
was
supported
by an
Estab-
lished
Fellowship
of
the
New
York
Heart
Association
during the
latter
part
of
this
investigation
.
Received
for
publication
12
May
1981,
and
in
revised
form
2
October
1981
.
REFERENCES
1 .
Lazarow,
P
.
B
.,
H
.
Shin,
and
M
.
Robbi
.
1980
.
Biogenesis
of
peroxisomes
and
the
peroxisome
reticulum
hypothesis
.
In
31st
Mosbach
Colloquium
.
Biological
Chemistry
of
Organelle
Formation
.
H
.
Weiss,
T
.
Bucher,
and
W
.
Sebald,
editors
.
Springer-Verlag,
New
York
.
187-206
.
2
.
Fujiki
.
Y
.,
A
.
L
.
Hubbard,
S
.
Fowler,
and
P
.
B
.
Lazarow
.
1982
.
Isolatio
n
of
intracellular
membranes
by
means
of sodium
carbonate
treatment
:
application
to
endoplasmic
reticu-
lum
.
J
.
Cell Biol
.
93
:97-102
.
3
.
Lazarow,
P
.
B
.,
S
.
Fowler,
and
A
.
L
.
Hubbard
.
1979
.
Differen
t
composition
of
the
peroxisomal
and
endoplasmic
reticulum
membranes
of
rat
liver
.
J
Cell Biol
.
83(2,
Pt
.
2)
:263a(Abstr
.)
.
4
.
Fujiki,
Y
.,
S
.
Fowler,
A
.
L
.
Hubbard, and P
.
B
.
Lazarow
.
1981
.
Comparison of
rat
liver
peroxisome
membranes
with
endoplasmic
reticulum
and
mitochondrial
membranes
.
Fed
.
Proc
.
40
:1616(Abstr
.)
.
5
.
Leighton,
F
.,
B
.
Poole,
H
.
Beaufay,
P
.
Baudhuin,
J
.
W
.
Coffey,
S
.
Fowler,
and C
.
de
Dove
.
1968
.
The
large-scale
separation
of
peroxisomes,
mitochondria,
and
lysosomes
from
the
livers
of
rats
injected
with
Triton
WR-1339
.
J
.
Cell Biol
.
37
:482-512
.
6
.
Amar-Costesec
.
A
.,
H
.
Beaufay,
M
.
Wibo,
D
.
Thines-Sempoux,
E
.
Feytmans,
M
.
Robbi,
and
J
.
Berthet
.
1974
.
Analytical study
ofmicrosomes and
isolated
subcellular
membranes
from
rat
liver
.
II
.
Preparation
and
composition
of
the
microsomal
fraction
.
J
.
Cell Biol
.
61
:201-212
.
7
.
Beaufay,
H
.,
A
.
Amar-Costesec,
D
.
Thines-Sempoux,
M
.
W
ibo,
M
.
Robbi,
and
J
.
Berthet
.
1974
.
Analytical
study
ofmicrosomes and
isolated
subcellular
membranes
from
rat
liver
.
III
.
Subfractionation
of
the
microsomal
fraction
by
isopycnic
and
differential
centrifuga-
tion
in
density gradients
.
J
.
Cell
Biol
.
61
:213-231
.
8
.
Amar-Costesec,
A
.,
M
.
Wibo,
D
.
Thines-Sempoux,
H
.
Beaufay,
and
J
.
Berthet
.
1974
.
Analytical
study
of
microsomes and
isolated
subcellular
membranes
from
rat
liver
.
IV
.
Biochemical,
physical,
and
morphological
modifications
of
microsomal
components
in-
duced by
digitonin,
EDTA,
and
pyrophosphate
.
J
.
Cell Biol
.
62
:717-745
.
9
.
Baudhuin, P
.,
P
.
Evrard,
and J
.
Berthet
.
1967
.
Electron
microscopic
examination
of
subcelular
fractions
.
I
.
The
preparation
of
representative
samples
from
suspensions
of
particles
.
J
.
Cell Biol
.
32
:181-191
.
10
.
Bligh,
E
.
G
.,
and
W
.
J
.
Dyer
.
1959
.
A
rapid
method
of
total lipid
extraction
and
purification
.
Can
.
J
.
Biochem
.
Physiol
.
37
:911-917
.
I1
.
Ames, B
.
N
.,
and
D
.
T
.
Dubin
.
1960
.
Th
e
role
of
polyamines
in
the
neutralization of
bacteriophage deoxyribonucleic
acid
.
J
.
Biol
.
Chem
.
235
:769-775
.
12
.
Mangold,
H
.
K
.
1961
.
Thin-layer
chromatography of
lipids
.
J
.
Am
.
Oil
Chemists'
Soc
.
38
:708-727
.
13
.
Mahler,
H
.
R
.,
G
.
Hubscher,
and
H
.
Baum
.
1955
.
Studies
on
uricase
.
1 .
Preparation,
purification,
and
properties of
a
cuproprotein
.
J
.
Biol
.
Chem
.
216
:625-641
.
14
.
Leighton,
F
.,
B
.
Poole,
P
.
B
.
Lazarow,
and
C
.
de
Dove
.
1969
.
The
synthesis
and
turnover
of
rat
liver
peroxisomes
.
I
.
Fractionation
of
peroxisome
proteins
.
J
.
Cell Biol
.
41
:521-535
.
15
.
Sottocasa,
G
.
L
.,
B
.
Kuylenstierna,
L
.
Ernster,
and
A
.
Bergstrand
.
1967
.
An
electron-
transport
system
associated with
the
outer
membrane
of
liver
mitochondria
.
A
biochemical
and
morphological
study
.
J
.
Cell
Biol
.
32
:415-438
.
16
.
Parsons,
D
.
F
.,
G
.
R
.
Williams,
W
.
Thompson,
D
.
Wilson,
and B
.
Chance
.
1968
.
Improvements
in
the
procedure
for
purification
of
mitochondria!
outer
and
inner
mem-
FUJIKI
ET At
.
Peroxisomal
Membrane
10
9
brane
.
Comparison of
the
outer
membrane
with
smooth
endoplasmic
reticulum
.
In
Mitochondrial
Structure
and
Compartmentation
.
E
.
Quagiliariello,
S
.
Papa,
E
.
C
.
Slater,
and
J
.
M
.
Tager,
editors
.
Adriatica
Editrice,
Bari
.
29-70
.
17
.
Fukushima,
K
.,
A
.
Ito,
T
.
Omura,
and
R
.
Sato
.
1972
.
Occurrence
of
different
types
of
cytochrome
b
"
;
like
hemoprotein
in
liver
mitochondria
and
their
intramitochondrial
local-
ization
.
J
.
Biochem
.
(Tokyo)
.
71
:447-461
.
18
.
Fowler,
S
.,
J
.
Remacle,
A
.
Trouet,
H
.
Beaufay,
1
.
Berthet,
M
.
Wibo, and P
.
Hauser
.
1976
.
Analytical study
of
microsomes
and
isolated
subcellular
membranes
from
rat
liver
.
V
.
Immunological
localization
ofcytochrome
bs
by
electron
microscopy
:
methodology and
application
to
various
subcellular
fractions
.
J
.
Cell
Biol
.
71
:535-550
.
19
.
Thines-Sempoux,
D
.
1973
.
A
comparison
between
the
lysosomal
and
the
plasma
mem-
brane
.
In
Lysosomes
in
Biology
and
Pathology
.
J
.
T
.
Dingle,
editor
.
North-Holland,
Amsterdam
.
278-299
.
20
.
Dallner,
G
.,
and
L
.
Emster
.
1968
.
Subfractionation
and
composition
of
microsomal
membranes
:
a
review
.
J
.
Hisrochem
.
Cytochem
.
16
:611-632
.
21
.
DePierre,
J
.
W
.,
and
M
.
L
.
Kamovsky
.
1973
.
Plasma
membranes
of
mammalian
cells
.
A
review
of
methods
for
their
characterization
and
isolation
.
J
.
Cell Biol
.
56
:275-303
.
22
.
Friedman,
M
.,
and S
.
O
.
Byers
.
1953
.
Th
e
mechanism
responsible
for
the
hypercholester-
emia
induced
by
Triton
WR-1339
.
J
.
Exp
.
Med
.
97
:117-129
.
23
.
Frantz,
1 .
D
.,
Jr
.,
and B
.
T
.
Hinkelman,
1955
.
Acceleration
of
hepatic
cholesterol
synthesis
by
Triton
WR-1339
.
J
.
Exp
.
Med
.
101
:225-232
.
24
.
Hayashi,
H
.,
S
.
Niinobe,
Y
.
Matsumoto, and
T
.
Suga
.
1981
.
Effect
of
Triton
WR-1339
on
lipoprotein
lipolytic
activity
and
lipid
content
of
rat
liver
lysosomes
.
J
.
Biochem
.
(Tokyo)
.
89
:573-579
.
25
.
Donaldson,
R
.
P
.,
N
.
E
.
Tolbert,
and
C
.
Schnarrenberger
.
1972
.
A
comparison
of
microbody
membranes
with
microsomes and
mitochondria
from
plant
and
animal
tissue
.
11
0
THE
JOURNAL
Of
CELL
BIOLOGY
-
VOLUME
93,
1982
Arch
.
Biochem
.
Biophys
.
152
:199-215
.
26
.
Novikoff,
P
.
M
.,
A
.
B
.
Novikoff,
N
.
Quintana,
and
C
.
Davis
.
1973
.
Studies
on
micro-
peroxisomes
.
III
.
Observations
on
human
and
rat
hepatocytes
.
J
.
Hisrochem
.
Cytochem
.
21
:540-558
.
27
.
Legg,
P
.
G
.,
and
R
.
L
.
Wood
.
1970
.
New
observations
on
microbodies
:
a
cytochemical
study
on
CPIB-treated
rat
liver
.
J
.
Cell
Biol,
45
:118-129
.
28
.
Fahimi,
H
.
D
.,
B
.
A
.
Gray,
and V
.
K
.
Herzog
.
1976
.
Cytochemica
l
localization
of
catalase
and
peroxidase
in sinusoidal
cells
of
rat
liver
.
Lab
.
Invest
.
34
:192-201
.
29
.
Shin,
H
.,
and P
.
B
.
Lazarow
.
1981
.
Relationshi
p between
peroxisomes
and
endoplasmic
reticulum
investigated
by
combined
catalase
and
glucose-6-phosphatase
cytochemistry
.
J
.
Hisrochem
.
Cytochem
.
29
:1263-1272
.
30
.
Lazarow,
P
.
B
.,
and C
.
de
Duve
.
1973
.
The
synthesis
and
turnover
of
rat
liver
peroxisomes
.
V
.
Intracellular
pathway
of
catalane
synthesis
.
J
.
Cell Biol
.
59
:507-524
.
31
.
Redman,
C
.
M
.,
D
.
J
.
Grab,
and
R
.
Irukulla
.
1972
.
The
intracellular
pathway
of
newly
formed
rat
liver
catalase
.
Arch
.
Biochem
.
Biophys
.
152
:496-501
.
32
.
Robbi,
M
.,
and P
.B
.
Lazarow
.
1978
.
Synthesi
s
of catalase
in
two
cell-free
protein-synthe-
sizing
systems
and
in rat
liver
.
Proc
.
Nail
.
A
cad
.
Sci
.
U
.
S
.
A
.
75
:4344-4348
.
33
.
Goldman,
B
.
M
.,
and
G
.
Blobel
.
1978
.
Biogenesis
of
peroxisomes
:
intracellular
site
of
synthesis
of
catalase
and
uricase
.
Proc
.
Nail
.
Acad
.
Sci
.
U
.
S
.
A
.
75
:5066-5070
.
34
.
Lodish,
H
.
F
.,
W
.
A
.
Braell,
A
.
L
.
Schwartz,
G
.
J
.
A
.
M
.
Strous,
and
A
.
Zilberstein
.
1981
.
Synthesis
and
assembly
of
membrane
and
organelle
proteins
.
Int
.
Rev
.
Cylol
.
Suppl
.
12
:247-307
.
35
.
Hajra,
A
.
K
.,
C
.
L
.
Burke,
and
C
.
L
.
Jones
.
1979
.
Subcellula
r
localization
of
acyl
coenzyme
A
:dihydroxyacetone
phosphate
acyltransferase
in
rat
liver
peroxisomes
(microbodies)
.
J
.
Biol
.
Chem
.
254
:10896-10900
.
... To facilitate surfaceome analysis of colorectal cancer (CRC) cell-derived exosomes, we have developed, and report here, a procedure for the large-scale generation of mg quantities of highly purified exosomes secreted from the CRC cell line SW480. [16] Using sodium carbonate extraction [17,18] combined with Triton X-114 phase separation [19] and label-free mass spectrometry protein identification, we report a comprehensive profiling of 208 integral membrane proteins (IMPs) and 124 peripherally associated membrane proteins (PMPs). We next studied the SW480derived exosome surfaceome using gentle proteolytic digestion (proteinase K), which cleaved surface-exposed proteins while keeping the vesicle morphologically intact. ...
... SW480-GFP-Exos pellets suspended in ice-cold 100 mm Na 2 CO 3 , (pH, 11.5) containing protease inhibitor (Roche cOmplete) for 60 min, [17] and centrifuged at 100 000 × g for 1 h at 4°C to separate the pelleted exosomal IMP fraction from the peripheral membrane protein fraction (PMP) (supernatant). The exosomal PMP was precipitated using cold acetone; Triton X-114 (TX-114) phase partitioning was performed using multiple washing methods reported by Brusca et al. [24] Briefly, the carbonate-washed exosomal IMP was treated with 2% w/v TX-114, containing 10 nm Tris-HCl 7.5, and 150 mm NaCl. ...
... Membrane proteins (MPs) can be categorized either integral (IMPs), which span the lipid bilayer with up to 16 α-helical transmembrane domains (TMDs), or peripherally associated proteins (PMPs) which bind to IMPs or lipid head groups noncovalently through electrostatic or hydrophobic interactions. [55] To examine the SW80-GFP-Exos surfaceome, we first applied sodium carbonate extraction [17] -a canonical method to separate IMP from PMP [18] -IMP followed by Triton X-114 phase partitioning, which generates an IF, a DP, and an AP [19] (Figure 2A). It has been previously reported that both carbonate extraction and Triton X-114 phase partitioning increased the isolation of transmembrane and hydrophobic proteins, primarily through the depletion of soluble proteins and from membrane organelles. ...
Article
In localized tumours, basement membrane (BM) prevents invasive outgrowth of tumour cells into surrounding tissues. When carcinomas become invasive, cancer cells either degrade the BM or reprogram the stromal fibroblasts to breach the BM barrier and lead invasion of cancer cells into surrounding tissues in a process called fibroblast‐led invasion. However, tumour‐derived factors orchestrating fibroblast‐led invasion remains poorly understood. Here we show that although early‐stage primary colorectal adenocarcinoma (SW480) cells are themselves unable to invade MatrigelTM matrix, they secrete exosomes that reprogram normal fibroblasts to acquire de novo capacity to invade matrix and lead invasion of SW480 cells. Strikingly, cancer cells follow the leading fibroblasts as collective epithelial‐clusters, thereby circumventing the need for epithelial to mesenchymal transition, a key event associated with invasion. Moreover, acquisition of pro‐invasive phenotype by fibroblasts treated with SW480‐derived exosomes relied on exosome‐mediated MAPK pathway activation. Mass spectrometry‐based protein profiling revealed that cancer exosomes upregulated CRL1541 proteins implicated in focal adhesion (ITGA2/A6/AV, ITGB1/B4/B5, EGFR, CRK), regulators of actin cytoskeleton (RAC1,ARF1, ARPC3, CYFIP1,NCKAP1, ICAM1, ERM complex) and signalling pathways (MAPK, Rap1, RAC1, Ras) important in pro‐invasive remodelling of the extracellular matrix. Blocking tumour exosome‐mediated signalling to stromal fibroblasts could therefore represent an attractive therapeutic strategy in restraining tumours by perturbing stroma‐driven invasive outgrowth. This article is protected by copyright. All rights reserved
... Next, we isolated a mixture of mitochondrial inner and outer membranes from mitochondrial fractions of Huh7 cells employing sodium carbonate treatment that can isolate intracellular membranes of subcellular organelles (Fujiki et al, 1982). Following DFP treatment, a FTMT precursor form (~30-kDa) was detected in the isolated mitochondrial membrane along with voltage-dependent anion channel (VADC), an outer membrane protein. ...
... The mitochondrial membrane was isolated by means of sodium carbonate treatment, as described previously (Fujiki et al, 1982). In brief, the crude mitochondrial fractions were diluted 50-to 1,000fold with 100 mM sodium carbonate, pH 11.5, to bring the protein concentration to 0.02-1 mg/ml, and incubated for 30 min at 0°C. ...
Article
Mitochondrial quality is controlled by the selective removal of damaged mitochondria through mitophagy. Mitophagy impairment is associated with aging and many pathological conditions. An iron loss induced by iron chelator triggers mitophagy by a yet unknown mechanism. This type of mitophagy may have therapeutic potential, since iron chelators are clinically used. Here, we aimed to clarify the mechanisms by which iron loss induces mitophagy. Deferiprone, an iron chelator, treatment resulted in the increased expression of mitochondrial ferritin (FTMT) and the localization of FTMT precursor on the mitochondrial outer membrane. Specific protein 1 and its regulator hypoxia-inducible factor 1α were necessary for deferiprone-induced increase in FTMT. FTMT specifically interacted with nuclear receptor coactivator 4, an autophagic cargo receptor. Deferiprone-induced mitophagy occurred selectively for depolarized mitochondria. Additionally, deferiprone suppressed the development of hepatocellular carcinoma (HCC) in mice by inducing mitophagy. Silencing FTMT abrogated deferiprone-induced mitophagy and suppression of HCC. These results demonstrate the mechanisms by which iron loss induces mitophagy and provide a rationale for targeting mitophagic activation as a therapeutic strategy.
... Since c17orf80 was predicted to contain C-terminal TM helices, we sought to determine whether the protein is membrane-associated. For this, we subjected intact mitochondria isolated from HEK293 cells to sodium carbonate alkaline extraction (Fujiki et al., 1982a;Fujiki et al., 1982b). This method allows the separation of integral membrane proteins from peripheral and soluble ones. ...
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Molecular functions of many human proteins remain unstudied, despite the demonstrated association with diseases or pivotal molecular structures, such as mitochondrial DNA (mtDNA). This small genome is crucial for proper functioning of mitochondria, the energy-converting organelles. In mammals, mtDNA is arranged into macromolecular complexes called nucleoids that serve as functional stations for its maintenance and expression. Here, we aimed to explore an uncharacterized protein c17orf80, which was previously detected close to the nucleoid components by proximity-labelling mass spectrometry. To investigate the subcellular localization and function of c17orf80, we took an advantage of immunofluorescence microscopy, interaction proteomics and several biochemical assays. We demonstrate that c17orf80 is a mitochondrial membrane-associated protein that interacts with nucleoids even when mtDNA replication is inhibited. In addition, we show that c17orf80 is not essential for mtDNA maintenance and mitochondrial gene expression in cultured human cells. These results provide a basis for uncovering the molecular function of c17orf80 and the nature of its association with nucleoids, possibly leading to new insights about mtDNA and its expression.
... Un bilan de toutes les découvertes dans le domaine des membranes, commençant par les études de la perméabilité membranaire et aboutissant actuellement sur la compréhension du fonctionnement des domaines membranaires, a été réalisé par Eididin [52]. Ainsi, on sait que la membrane plasmique est hautement enrichie en cholestérol et glycosphingolipides, alors que ces composants sont peu présents dans la membrane du réticulum endoplasmique [53]. Malgré toutes ces différences de composition, il est possible de mettre en avant une structure générale à toutes les membranes. ...
Thesis
L’objectif de mon travail de thèse a été de caractériser l’adhésion de vésicules géantes lipidiques et de cellules vivantes. Dans le but d’obtenir des informations quantitatives sur l’adhésion, j’ai développé deux techniques de nanoscopie de fluorescence basées sur la microscopie TIRF (Total Internal Reflection Fluorescence). Cette technique repose sur création d’une onde évanescente à proximité d’une interface. J’ai développé pour cela un montage optique, qui permet de contrôler finement les caractéristiques de l’onde évanescente (longueur d’atténuation, état de polarisation, etc.). L’adhésion des vésicules a été étudiée par nTIRF (TIRF normalisé) : les images TIRF sont normalisées par des images en épi-fluorescence. J’ai pu ainsi caractériser l’adhésion non spécifique (interaction électrostatique) et spécifique (interaction biotine-streptavidine) de vésicules sur différentes surfaces fonctionnalisées. Pour quantifier l’adhésion des cellules, j’ai utilisé l’approche VA-TIRF (TIRF à angle variable). Cette dernière consiste à enregistrer une série d’images en régime évanescent à différents angles d’incidence. Ceci nous a permis d’établir une cartographie des distances entre la membrane ventrale d’une cellule et la surface pour différents comportements d’adhésion initiés sur divers substrats : chimiques ou protéiques. Ces deux techniques permettent de mesurer la distance membrane-surface avec une précision nanométrique, ≈20nm, ce qui est particulièrement adapté à l’étude du processus d’adhésion
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Mitochondria are complex organelles containing 13 proteins encoded by mitochondrial DNA and over 1,000 proteins encoded on nuclear DNA. Many mitochondrial proteins are associated with the inner or outer mitochondrial membranes, either peripherally or as integral membrane proteins, while others reside in either of the two soluble mitochondrial compartments, the mitochondrial matrix and the intermembrane space. The biogenesis of the five complexes of the oxidative phosphorylation system are exemplars of this complexity. These large multi-subunit complexes are comprised of more than 80 proteins with both membrane integral and peripheral associations and require soluble, membrane integral and peripherally associated assembly factor proteins for their biogenesis. Mutations causing human mitochondrial disease can lead to defective complex assembly due to the loss or altered function of the affected protein and subsequent destabilization of its interactors. Here we couple sodium carbonate extraction with quantitative mass spectrometry (SCE-MS) to track changes in the membrane association of the mitochondrial proteome across multiple human knockout cell lines. In addition to identifying the membrane association status of over 840 human mitochondrial proteins, we show how SCE-MS can be used to understand the impacts of defective complex assembly on protein solubility, giving insights into how specific subunits and sub-complexes become destabilized.
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The isolation and subsequent molecular analysis of extracellular vesicles (EVs) derived from patient samples is a widely used strategy to understand vesicle biology and to facilitate biomarker discovery. Expressed prostatic secretions in urine are a tumor proximal fluid that has received significant attention as a source of potential prostate cancer (PCa) biomarkers for use in liquid biopsy protocols. Standard EV isolation methods like differential ultracentrifugation (dUC) co‐isolate protein contaminants that mask lower‐abundance proteins in typical mass spectrometry (MS) protocols. Further complicating the analysis of expressed prostatic secretions, uromodulin, also known as Tamm‐Horsfall protein (THP), is present at high concentrations in urine. THP can form polymers that entrap EVs during purification, reducing yield. Disruption of THP polymer networks with dithiothreitol (DTT) can release trapped EVs, but smaller THP fibres co‐isolate with EVs during subsequent ultracentrifugation. To resolve these challenges, we describe here a dUC method that incorporates THP polymer reduction and alkaline washing to improve EV isolation and deplete both THP and other common protein contaminants. When applied to human expressed prostatic secretions in urine, we achieved relative enrichment of known prostate and prostate cancer‐associated EV‐resident proteins. Our approach provides a promising strategy for global proteomic analyses of urinary EVs.
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Organelles within the cell are highly dynamic entities, requiring dramatic morphological changes to support their function and maintenance. As a result, organelle membranes are also highly dynamic, adapting to a range of topologies as the organelle changes shape. In particular, peroxisomes—small, ubiquitous organelles involved in lipid metabolism and reactive oxygen species homeostasis—display a striking plasticity, for example, during the growth and division process by which they proliferate. During this process, the membrane of an existing peroxisome elongates to form a tubule, which then constricts and ultimately undergoes scission to generate new peroxisomes. Dysfunction of this plasticity leads to diseases with developmental and neurological phenotypes, highlighting the importance of peroxisome dynamics for healthy cell function. What controls the dynamics of peroxisomal membranes, and how this influences the dynamics of the peroxisomes themselves, is just beginning to be understood. In this review, we consider how the composition, biophysical properties, and protein-lipid interactions of peroxisomal membranes impacts on their dynamics, and in turn on the biogenesis and function of peroxisomes. In particular, we focus on the effect of the peroxin PEX11 on the peroxisome membrane, and its function as a major regulator of growth and division. Understanding the roles and regulation of peroxisomal membrane dynamics necessitates a multidisciplinary approach, encompassing knowledge across a range of model species and a number of fields including lipid biochemistry, biophysics and computational biology. Here, we present an integrated overview of our current understanding of the determinants of peroxisome membrane dynamics, and reflect on the outstanding questions still remaining to be solved.
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Late‐stage colorectal cancer (CRC) is still a clinically challenging problem. The activity of the tumor suppressor p53 is regulated via posttranslational modifications (PTMs). While the relevance of p53 C‐terminal acetylation for transcriptional regulation is well‐defined, it is unknown whether this PTM controls mitochondrially mediated apoptosis directly. We used wild‐type p53 or p53‐negative human CRC cells, cells with acetylation‐defective p53, transformation assays, CRC organoids, and xenograft mouse models to assess how p53 acetylation determines cellular stress responses. The topoisomerase‐1 inhibitor irinotecan induces acetylation of several lysine residues within p53. Inhibition of histone deacetylaces (HDACs) with the class I HDAC inhibitor entinostat synergistically triggers mitochondrial damage and apoptosis in irinotecan‐treated p53‐positive CRC cells. This specifically relies on the C‐terminal acetylation of p53 by CREB binding protein (CBP)/p300 and the presence of C‐terminally acetylated p53 in complex with the pro‐apoptotic BCL2 antagonist/killer (BAK) protein. This control of C‐terminal acetylation by HDACs can mechanistically explain why combinations of irinotecan and entinostat represent clinically tractable agents for the therapy of p53‐proficient CRC.
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A rapid and simple method for the isolation of membranes from subcellular organelles is described. The procedure consists of diluting the organelles in ice-cold 100 mM Na2CO3 followed by centrifugation to pellet the membranes. Closed vesicles are converted to open membrane sheets, and content proteins and peripheral membrane proteins are released in soluble form. Here we document the method by applying it to various subfractions of a rat liver microsomal fraction, prepared by continuous density gradient centrifugation according to Beaufay et al. (1974, J. Cell Biol. 61:213-231). The results confirm and extend those of previous investigators on the distribution of enzymes and proteins among the membranes of the smooth and rough endoplasmic reticulum. In the accompanying paper (1982, J. Cell Biol. 93:103-110) the procedure is applied to peroxisomes and mitochondria.
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Liver homogenates have been submitted to quantitative fractionation by differential centrifugation. Three particulate fractions: N (nuclear), ML (large granules), and P (microsomes), and a final supernate (S) have been obtained. The biochemical composition of the microsomal fraction has been established from the assay and distribution pattern of 25 enzymatic and chemical constituents. These included marker enzymes for mitochondria (cytochrome oxidase), lysosomes (acid phosphatase and N-acetyl-beta-glucosaminidase), and peroxisomes (catalase). The microsomal preparations were characterized by a moderate contamination with large cytoplasmic granules (only 6.2% of microsomal protein) and by a high yield in microsomal components. Enzymes such as glucose 6-phosphatase, nucleoside diphosphatase, esterase, glucuronyltransferase, NADPH cytochrome c reductase, aminopyrine demethylase, and galactosyltransferase were recovered in the microsomes to the extent of 70% or more. Another typical behavior was shown by 5'-nucleotidase, alkaline phosphatase, alkaline phosphodiesterase I, and cholesterol, which exhibited a "nucleomicrosomal" distribution. Other complex distributions were obtained for several constituents recovered in significant amount in the microsomes and in the ML or in the S fraction.
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