ArticlePDF Available

Variant forms of mitochondrial translation products in yeast: evidence for location of determinants on mitochondrial DNA

Authors:
  • Texas Science Education Foundation

Abstract and Figures

Products of mitochondrial protein synthesis in yeast have been labeled in vivo with 35SO42-. More than 20 polypeptide species fulfilling the criteria of mitochondrial translation products have been detected by analysis on sodium dodecyl sulfate-exponential polyacrylamide slab gels. A comparison of mitochondrial translation products in two wild-type strains has revealed variant forms of some polypeptide species which show genetic behavior consistent with the location of their structural genes on mtDNA. Our results demonstrate the feasibility of performing genetic analysis on putative gene products of mtDNA in wild-type yeast by direct examination of the segregation and recombination behavior of specific polypeptide species.
Content may be subject to copyright.
Proc.
Nat.
Acad.
Sci.
USA
Vol.
73,
No.
4,
pp.
1083-1086,
April
1976
Biochemistry
Variant
forms
of
mitochondrial
translation
products
in
yeast:
Evidence
for
location
of
determinants
on
mitochondrial
DNA
(mitochondrial
protein
synthesis/cytoplasmic
inheritance/polyacrylamide
slab
gel
electrophoresis)
M.
G.
DOUGLAS*
AND
R.
A.
BUTOWt
Department
of
Biochemistry,
The
University
of
Texas
Health
Science
Center,
Dallas,
Texas 75235
Communicated
by
Lester
J.
Reed.
January
15,1976
ABSTRACT
Products
of
mitochondrial
protein
synthesis
in
yeast
have
been
labeled
in
vivo
with
35S042-.
More
than
20
polypeptide
species
fulfilling
the
criteria
of
mitochondrial
translation
products
have
been
detected
by
analysis
on
sodi-
um
dodecy
sulfate-exponential
polyacrylamide
slab
gels.
A
comparison
of
mitochondrial
translation
products
in
two
wild-type
strains
has
revealed
variant
forms
of
some
polypep-
tide
species
which
show
genetic
behavior
consistent
with
the
location
of
their
structural
genes
on
mtDNA.
Our
results
demonstrate
the
feasibility
of
performing
genetic
analysis
on
putative
gene
products
of
mtDNA
in
wild-type
yeast
by
di-
rect
examination
of
the
segregation
and
recombination
be-
havior
of
specific
polypeptide
species.
The
function
of
the
mitochondrial
genome
in
the
biogenesis
of
the
organelle
has
received
a
great
deal
of
attention
in
re-
cent
years.
In
yeast,
considerable
emphasis
has
been
given
to
the
problem
of
identification
and
localization
of
genes
on
mtDNA.
Essentially
three
approaches
have
been
taken:
(i)
molecular
hybridization
studies
together
with
physical
map-
ping
(1,
2),
(ii)
in
itro
transcription
and
translation
(3,
4),
and
(iii)
genetic
analysis
of
cytoplasmically
inherited
muta-
tions
(5).
The
latter
include
mutants
resistant
to
mitochon-
dria-specific
antibiotics
and
inhibitors
(6)
and,
more
recent-
ly,
cytoplasmic
inheritance
mutants
which
are
unable
to
grow
on
nonfermentable
carbon
sources
but
still
retain
the
capability
of
transcribing
and
translating
their
mtDNA
(7,
8).
An
alternative
approach
to
the
identification
and
localiza-
tion
of
mitochondrial
polypeptide
structural
genes
stems
from
consideration
of
data
suggesting
that
mtDNAs
from
different
grande
(wild-type)
yeast
strains
are
not
identical
(9,
10).
Such
considerations
thus
raise
the
possibility
that
wild-type
mitochondrial
genomic
variability
may
be
mani-
fested
as
variations
in
mitochondrial
translation
products
which
might,
in
principle,
be
detectable
by
the
appropriate
techniques.
This
report
describes
a
sensitive
procedure
for
the
labeling
and
analysis
of
products
of
mitochondrial
protein
synthesis
in
yeast.
This
methodology
has
enabled
us
to
visualize
at
least
21
polypeptide
species
fulfilling
the
criteria
of
mito-
chondrial
translation
products
and
also
to
detect
differences
in
several
of
these
polypeptides
among
various
grande
strains.
These
strain-dependent
differences
(polymor-
phisms?)
have
allowed
us
to
determine
that
some
mitochon-
drial
translation
products
behave
genetically
in
a
manner
consistent
with
the
location
of
their
structural
genes
on
mtDNA.
Abbreviations:
mtDNA,
mitochondrial
DNA;
CAP,
chlorampheni-
col;
CHX,
cycloheximide.
*
Present
address:
Abteilung
Biochemie,
Biozentrum,
CH-4056
Basel,
Switzerland.
I
To
whom
requests
for
reprints
should
be
sent.
1083
MATERIALS
AND
METHODS
Strains.
Haploid
grande
strains
of
Saccharomyces
cerev-
isiae
55R5-3C/221-2
a
ura
w-
Cs
ER
OS
ps,
41-6/19
a
ade
Iys
W-
CR
Es
oR
pR
and
4810
a/a
Iys-l/lys-l
w+
Cs
Es
os
pR
were
kindly
provided
by
Dr.
Philip
Perlman,
Ohio
State
University.
Strain
48-Si
is
a
spontaneous
cytoplasmic
petite
obtained
from
4810.
The
p°$
petite
strains
55E46
and
EK
41-8
used
in
this
study
were
generated
by
extensive
ethi-
dium
bromide
mutagenesis
of
55R5-3C
a
ura
w
Cs
Es
Os
Ps
and
41-6/19,
respectively,
under
conditions
we
have
de-
termined
sufficient
to
generate
petites
lacking
detectable
mtDNA
according
to
the
method
of
Grossman
et
al.
(11).
In
addition,
these
petite
strains
exhibited
no
suppressivity
of
p+
phenotype
in
crosses
with
grande
tester
strains.
Growth
and
Labeling
Conditions.
All
cultures
were
grown
at
300
in
liquid
YPgal
media
(5
ml),
which
contains
1%
Bacto
yeast
extract,
1%
BactoPeptone,
0.1%
KH2PO4,
0.12%
(NH4)20S4,
and
2%
galactose.
After
harvesting
in
late
logarithmic
phase,
the
cells
were
washed
twice
with
5
ml
of
cold
water
and
suspended
in
2
ml
of
a
modified
G
medium
(12)
containing
0.3%
glucose
with
all
SO42-
replaced
by
Cl-.
After
30
min
incubation
at
300,
the
cells
in
the
presence
or
absence
of
antibiotics
were
labeled
with
200
gCi/ml
H23'04
(New
England
Nuclear,
carrier-free).
After
1
hr
of
labeling
(incorporation
is
essentially
complete
after
20-30
min),
Na2SO4
and
casamino
acids
were
added
10
min
prior
to
harvesting
to
final
concentrations
of
1
mg/ml
and
1%,
re-
spectively.
The
cells
(0.1
ml
packed
volume)
were
washed
twice
with
Na2SO4-casamino
acids
as
above
and
suspended
in
0.4
ml
of
0.25
M
mannitol,
0.02
M
Tris-H2SO4,
0.001
M
EDTA,
pH
7.1.
Small
scale
multisample
mitochondrial
prep-
arations
were
performed
essentially
as
described
by
Needle-
man
and
Tzagoloff
(13).
Once-washed
mitochondrial
pellets
(80-150
,g
of
protein)
were
immediately
suspended
in
2%
sodium
dodecyl
sulfate,
0.002
M
EDTA,
0.150
M
2-mercap-
toethanol,
0.05
M
Tris-HCI
(pH
6.8),
and
10%
(vol/vol)
glyc-
erol,
then
heated
at
900
for
2
min.
35SO42-
incorporation
was
determined
as
hot
trichloroacetic
acid
precipitable
ra-
dioactivity.
Slab
Gel
Autoradiography.
Resolution
of
mitochondrial
protein
on
15
cm
gel
slabs
under
dissociating
conditions
was
performed
according
to
Studier
(14)
using
the
discontinuous
buffer
system
described
by
Laemmli
(15)
except
that
0.002
M
EDTA
was
included
in
the
stacking
and
resolving
gels.
The
stacking
gels
were
5%
acrylamide
and
the
resolving
gels
were 10-15%
exponential
gradients
of
acrylamide.
In
order
to
insure
even
drying
of
the
gels
for
autoradiography,
0.5%
t
p
is
a
cytoplasmic
factor
synonymous
with
mtDNA
whose
muta-
tion
generally
leads
to
a
pleiotropic
loss
of
mitochondrial
func-
tions.
1084
Biochemistry:
Douglas
and
Butow
Table
1.
Comparison
of
3
S042-
and
[1
4C
jleucine
incorporation
into
mitochondrial
protein
35
S04
2-
[14C]
Leucine,
Conditions
cpm/pg
cpm/,lg
No
antibiotics
78,620
726
+
CAP
72,940
505
+
CHX
6,390
77
+
CAP
+
CHX
880
7
Mitochondria
from
55R5-3C
were
labeled
and
prepared
exactly
as
described
in
Materials
and
Methods.
[14C]Leucine
and
35SO42-
were
2.0
1uCi/ml
and
100
MCi/ml,
respectively.
Where
in-
dicated
CAP
(3
mg/ml)
was
added
20
min
and
CHX
(0.6
mg/ml)
4
min
prior
to
the
addition
of
label.
Protein
concentrations
were
de-
termined
according
to
the
Lowry
et
al.
method
(16).
polyacrylamide
(BDH
laboratories)
was
included
in
the
re-
solving
gel.
After
overnight
aging
of
the
gel,
a
stacking
gel
was
poured
and
up
to
25
samples
of
mitochondrial
protein
(25,000-30,000
cpm
each)
were
run
in
the
cold
at
30
mA
constant
current
until
the
blue
tracking
dye
reached
the
bot-
tom
of
the
slab
(about
5'
hr).
Following
electrophoresis,
gels
were
fixed
for
30
min
in
50%
trichloroacetic
acid,
stained
for
20
min
in
40%
trichloroacetic
acid
plus
0.1%
Coomassie
brilliant
blue,
and
destained
overnight
in
7%
acetic
acid.
Gels
dried
to
Whatman
3
MM
paper
were
exposed
3-4
days
to
Kodak
RP.R2
x-ray
film
which
was
subsequently
devel-
oped
in
Kodak
liquid
x-ray
developer
for
5
min
at
700.
RESULTS
35so4a-
labeling
of
mitochondria
In
order
to
analyze
and
compare
products
of
mitochondrial
protein
synthesis
in
a
large
number
of
samples
at
high
level
of
resolution,
it
was
necessary
to
develop
procedures
to
label
protein
to
high
specific
activity
at
reasonable
cost.
Table
1
compares
the
in
vivo
incorporation
of
s15SO42-
and
['4C]leu-
cine
into
the
mitochondrial
fraction
from
a
wild-type
yeast
strain.
Clearly,
under
the
labeling
conditions
employed,
35so4a,
either
in
the
presence
or
absence
of
antibiotics,
can
be
incorporated
into
trichloroacetic
acid
precipitable
mate-
rial
to
high
specific
activity.
By
contrast
to
['4C]leucine,
35SO42-
incorporation
is
only
slightly
inhibited
by
chloram-
phenicol
(CAP)
alone,
a
mitochondria-specific
translation
inhibitor,
whereas
the
incorporation
of
both
labels
shows
about
the
same
degree
of
sensitivity
to
cycloheximide
(CHX),
a
specific
inhibitor
of
cytoplasmic
protein
synthesis,
and
the
combination
of
CHX
plus
CAP.
This
combination
of
inhibitors
serves
as
a
measure
of
protein
synthesis
directed
by
mitochondrial
ribosomes.
Control
experiments
(not
shown)
have
also
demonstrated
that,
in
the
presence
of
CHX,
cytoplasmic
petites
incapable
of
carrying
out
mito-
chondrial
protein
synthesis
do
not
incorporate
35SO42-
into
the
mitochondrial
fraction.
Thus,
by
these
criteria,
"5SO42-
can
be
utilized
to
specifically
label
mitochondrial
translation
products
in
vio
to
high
specific
activity.
The
high
extent
of
incorporation
of
`5SO42-
into
organelle
proteins
makes
possible
better
resolution
of
polypeptides
by
sodium
dodecyl
sulfate-polyacrylamide
slab
gel
electropho-
resis.
Autoradiography
of
a
dried
slab
gel
(Fig.
1)
shows
rep-
resentative
profiles
of
mitochondrial
polypeptides
from
a
grande
and
cytoplasmic
petite
labeled
in
the
presence
and
absence
of
antibiotics.
Of
the
many
polypeptides
resolved
in
the
absence
of
antibiotics,
only
a
relatively
small
number
are
decreased
or
eliminated
by
labeling
in
the
presence
of
CAP.
_
D
16.9
13.9
13.4
FIG.
1.
Influence
of
antibiotics
on
polyacrylamide
gel
profiles
of
mitochondrial
proteins.
Mitochondrial
proteins
from
p+
(a-c)
and
p-
(d-f)
strains
of
4810
were
labeled
with
ssSO42-
as
described
in
Table
1.
Fractionation
and
visualization
of
these
labeled
pep-
tides
by
sodium
dodecyl
sulfate-polyacrylamide
gel
electrophoresis
is
given
in
Materials
and
Methods.
(a)
4810
p+
without
antibiotics;
(b)
p+
plus
CAP;
(c)
p+
plus
CHX;
(d)
p-
without
antibiotics;
(e)
p-
plus
CAP;
(f)
p-
plus
CHX.
Comparison
of
the
gel
patterns
in
Fig.
1
illustrates
that
those
better
resolved
species
in
the
grande
strain
which
are
inhibited
by
CAP
(Fig.
lb)
or
absent
in
the
cytoplasmic
pe-
tite
(Fig.
ld),
appear
preferentially
labeled
in
the
presence
of
CHX
(Fig.
lc).
That
these
polypeptides
are
intrinsic
mito-
chondrial
translation
products
is
supported
further
by
our
observations
that
these
species
are
not
found
in
cytoplasmic
petites
labeled
in
the
presence
of
CHX
(Fig.
lf),
or
wild-
type
labeled
in
the
presence
of
CHX
together
with
CAP,
ethidium
bromide
or
acriflavine
(data
not
shown).
The
ap-
parent
molecular
weights
for
these
particular
CHX-insensi-
tive
polypeptides
(about
21)
range
from
about
9300
to
43,000.
Although
the
experimental
procedure
described
here
has
enabled
the
detection
of
more
discrete
polypeptide
species
fulfilling
the
criteria
of
mitochondrial
translation
products
than
has,
to
our
knowledge,
been
reported
thus
far,
it
would
be
premature
to
conclude
that
they
represent
unique
gene
products;
polypeptide
aggregates,
specific
and/
or
nonspecific
cleavage
products
cannot
be
ruled
out,
partic-
ularly
for
the
minor
components.
Variant
forms
of
mitochondrial
translation
products
Fig.
2a
and
b
shows
the
profile
of
mitochondrial
translation
products
in
two
wild-type
strains.
It
is
evident
that
a
number
of
differences
in
the
profiles
exist.
Particularly
obvious
is
the
difference
in
mobility
of
Band
A
migrating
in
a
molecular
weight
region
of
40,000-43,000.
This
mobility
difference,
corresponding
to
about
2500
daltons,
is
reproducibly
ob-
tained
for
these
two
strains
and
can
be
demonstrated
as
well
when
the
strains
are
labeled
separately
and
mixed
prior
to
the
isolation
of
mitochondria
(Fig.
2c).
We
shall
refer
to
these
polypeptide
species
as
variant
1
fast
(VAR1f)
for
strain
41-6/19
and
variant
1
slow
(VARls)
for
55R5-3C-221-2.
Other
variants,
although
less
obvious,
are
consistently
seen
a
bc
,or
__
U'
...w
-IV
d
e
f
iI
.......
..Veix
...
-130
1
92.5
4.
68
-;.57
K
53
_
i
CD
49
0
_41
:
2.
x
o
22.5-
Proc.
Nat.
Acad.
Sci.
USA
73
(1976)
Proc.
Nat.
Acad.
Sci.
USA
73
(1976)
1085
a
b
c
d
e
1
2
I'
o
bc
dabcd
'S
X~p
VAR3
immail-a.7
VAR,
.-VA
R,
-
-VAR
-VAR2
FIG.
2.
Polyacrylamide
gel
profiles
of
mitochondrial
transla-
tion
products
in
two
p+
strains.
Labeling
of
strains
with
35SO42-
in
the
presence
of
CHX
was
carried
out
as
described
in
Material;
and
Methods.
(a)
55R5-3C/221-2;
(b)
41-6/19;
(c)
equal
portions
of
55R5-3C/221-2
and
41-6/19
were
mixed
prior
to
breakage
and
iso-
lation
of
mitochondria;
(d)
total
diploid
progeny
from
the
cross
55E46
p0
X
41-6/19;
and
(e)
total
diploid
progeny
from
the
cross
EK41-8
p0
X
55R5-3C/221-2.
for
these
two
strains:
in
the
apparent
molecular
weight
re-
gion
around
24,000-25,000,
designated
as
VAR2,
and
the
presence
or
absence
of
a
band
at
46,000,
designated
as
VAR3.
Cytoplasmic
inheritance
of
the
variant
forms
Since
the
variant
forms
are
present
in
strains
of
opposite
mating
type,
their
transmission
in
crosses
as
well
as
mitotic
and
meiotic
segregation
patterns
can
be
readily
determined.
Fig.
2c
and
d
compares
the
profiles
of
mitochondrial
transla-
tion
products
in
two
crosses
between
41-6/19
and
55R5-3C-
221-2
where
one
haploid
parent
in
each
case
has
been
con-
verted
to
a
p0
petite.
These
data
show
that
the
variant
forms
observed
in
the
total
diploid
progeny
of
the
cross
are
those
exclusively
of
the
p+
partner,
data
consistent
with
the
loca-
a
b
c
d
e
f
g
h
i
j
k
I
m
n
o
p
q
r
FIG.
3.
Mitotic
segregation
of
variant
proteins.
Strains
55R5-
3C/221-2
and
41-6/19
were
mated
in
liquid
culture
(1%
yeast
ex-
tract,
1%
BactoPeptone,
2%
dextrose)
for
2
hr.
Aliquots
of the
mat-
ing
mixture
were
then
plated
on
minimal
2%
dextrose
medium.
In
order
to
obtain
pure
diploid
clones,
following
3
days
growth,
sterile
water
was
added
to
a
plate
containing
about
200
diploid
colonies,
respread,
then
diluted
and
replated
on
minimal
2%
dextrose
medi-
um.
After
three
days
growth,
diploid
colonies
were
randomly
se-
lected
and
labeled
with
3SO42-
in
the
presence
of
CHX,
mito-
chondria
were
isolated,
and
slab
gel
autoradiographic
analysis
was
performed
as
described
in
Materials
and
Methods.
A
representa-
tive
sample
of
the
diploids
is
shown
here.
Only
a
portion
of
the
gel
is
displayed.
FIG.
4.
Meiotic
segregation
of
variant
proteins.
Two
purified
diploid
clones
from
Fig.
3
which
showed
parental
phenotypes
for
VAR1f,
VAR1',
VAR2,
and
VAR3
were
made
to
sporulate
and
tet-
rads
were
dissected.
One
complete
tetrad
from
each
(indicated
in
the
figure
as
1,
abcd
and
2,
abcd)
showing
proper
segregation
of
all
nuclear
markers
was
processed
as
described
in
Fig.
3.
Only
a
por-
tion
of
the
gel
is
displayed.
tion
of
determinants
for
these
polypeptide
species
on
mtDNA.
Further
support
for
this
view
is
obtained
from
the
data
presented
in
Fig.
3.
The
mitochondrial
translation
products
shown
for
18
randomly
selected
diploids
(recloned
twice)
of
the
wild-type
by
wild-type
cross
illustrate
that
the
variant
forms
segregate
mitotically:
in
this
case
three
diploid
clones
show
VARis
(Fig
Sc,
f,
and
n)
and
the
remaining
15
show
VAR1f.
Moreover,
apparent
recombinants
exist
be-
tween
VARI
and
VAR3,
as
shown
in
Fig.
3d
and
1.
It
should
also
be
noted
that
of
the
total
number
of
diploids
we
have
analyzed
in
this
cross
(about
300)
we
have
not
observed
a
strain
carrying
both
VAR1
forms
or
one
completely
lacking
VAR1
polypeptide.
When
purified
diploid
clones
were
made
to
sporulate
and
the
mitochondrial
translation
products
from
a
complete
tetrad
were
analyzed
(Fig.
4)
all
of
the
variant
forms
for
that
particular
diploid
clone
showed
4:0
segrega-
tion,
while,
as
expected,
all
nuclear
markers
segregated
2:2.
Taken
together
these
data
are
consistent
with
the
location
of
determinants
for
the
VAR
polypeptides
on
mtDNA.
Results
of
more
complete
transmission
and
recombination
analysis
of
the
variant
forms
will
be
presented
in
a
separate
commu-
nication.
DISCUSSION
The
sensitivity
with
which
mitochondrial
translation
prod-
ucts
can
be
displayed
by
the
procedures
described
here
has
permitted
us
to
detect
more
than
20
polypeptide
species
ful-
filling
the
criteria
of
mitochondrial
translation
products.
While
we
cannot
be
certain
that
all
of
these
reflect
unique
structural
gene
products,
an
important
observation
has
emerged:
namely,
variant
forms
of
some
of
these
polypep-
tides
exist
among
populations
of
wild-type
cells.
Considering
the
limited
number
of
strains
we
have
analyzed
and
the
number
of
variant
species
we
have
been
able
to
detect,
we
would
not
be
surprised
if
a
systematic
analysis
of
other
grande
strains
would
reveal
many
more
differences
in
the
pattern
of
mitochondrial
translation
products.
Presently,
we
can
only
speculate
on
the
molecular
basis
for
the
variant
forms:
additions
or
deletions at
the
structural
gene
level;
secondary
chemical
modifications
which
give
rise
to
altered
mobility;
specific
variations
in
processing
ac-
tivities
at
the
transcriptional
or
translational
level;
strain
spe-
cific
polypeptide
aggregation
or
cleavages
resulting
in
the
appearance
or
disappearance
of
bands,
etc.
Notwithstand-
ing,
whatever
explanations
may
eventually
obtain,
it is
ap-
parent
from
our
data
that
the
variant
forms
behave
geneti-
Biochemistry:
Douglas
and
Butow
wvv
C22
I
VA
Rn
-
VAR2
1086
Biochemistry:
Douglas
and
Butow
cally
in
a
manner
consistent
with
the
location
of
determi-
nants
on
mtDNA
(5):
(1)
they
segregate
mitotically
in
dip-
loids,
(ii)
they
show
4:0
segregation
of
the
meiotic
products
obtained
from
stable
diploid
clones
displaying
a
particular
variant
form,
and
(iii)
transmission
of
a
variant
-form
in
crosses
is
eliminated
by
prior
conversion
of
the
respective
haploid
parent
to
a
p0
petite.
Moreover,
it
is
also
apparent
that
the
variant
forms
recombine,
suggesting
that
separate
alleles
are
involved.
Although
it
is
not
possible
at
this
time
to
state
that
the
segregation
behavior
for
a
particular
polypep-
tide
species
reflects
the
segregation
of
its
structural
gene,
the
methodology
and
phenomena
we
have
described
offer
a
new
approach
to
the
study
of
genes
on
mtDNA.
In
particu-
lar,
analyses
can
be
carried
out
on
cells
that
are
wild-type
and
the
segregation
and
recombination
behavior
of
specific
polypeptide
species
is
determined
directly.
Ultimately,
it
will
be
important
to
relate
such
species
to
components
of
the
inner
mitochondrial
membrane
which
are
known
to
contain
subunits
synthesized
on
mitchondrial
ribosomes
(17,
18).
It
is
clear,
however,
that
the
variant
forms
of
mitochondrial
translation
products,
irrespective
of
the
molecular
basis
for
the
variations,
can
be
exploited
to
determine
linkage
rela-
tionships
between
the
forms
themselves
as
well
as
to
known
mitochondrial
markers.
We
are
grateful
to
Ms.
Elaine
Kendrick
for
expert
technical
assis-
tance,
to
Dr.
David
Finkelstein
for
help
in
developing
the
labeling
and
gel
procedures,
and
to
Dr.
Philip
Perlman
for
helpful
discus-
sions.
This
investigation
was
supported
by
Grant
GM
19090
from
the
U.S.
Public
Health
Service
and
Grant
NP-128E
from
the
Amer-
ican
Cancer
Society.
M.G.D.
is
the
recipient
of
USPHS
National
Re-
search
Service
Award
GM
01673.
1.
Casey,
J.
W.,
Huey-Juang,
H.,
Rabinowitz,
M.,
Getz,
G.
&
Fu-
kuhara,
H.
(1974)
J.
Mol.
Biol.
88,717-733.
2.
Faye,
G.,
Kujawa,
C.
&
Fukuhara,
H.
(1974)
J.
Mol.
Blol.
88,
185-203.
3.
Scragg,
A.
H.
&
Thomas,
D.
(1975)
Eur.
J.
Biochem.
56,
183-192.
4.
Halbreich,
A.,
Franco,
A.
D.,
Groudinsky,
O.,
Cosson,
J.
&
Slo-
nimski,
P.
P.
(1975)
Biochem.
Biophys.
Res.
Commun.
64,
1286-1292.
5.
Coen,
D.,
Deutsch,
J.,
Netter,
P.,
Petrochilo,
E.
&
Slonimski,
P. P.
(1970)
in
Control
of
Organelle
Development,
ed.
Miller,
P.
L.
(Symp.
Soc.
Exp.
Biol.,
Cambridge
University
Press,
Cambridge);
pp.
449-496.
6.
Linnane,
A.
W.,
Saunders,
G.
W.,
Gingold,
E.
B.
&
Lukins,
H.
B.
(1968)
Proc.
Nat.
Acad.
Scd.
USA
59,903-910.
7.
Flury,
U.,
Mahler,
H.
R.
&
Feldman,
F.
(1974)
J.
Biol.
Chem.
249,6130-6137.
8.
Tzagoloff,
A.,
Akai,
A.,
Needleman,
R.
B.
&
Zulch,
G.
(1975)
J.
Biol.
Chem.
250,8236-8242.
9.
Michaelis,
G.,
Douglas,
S.,
Tsai,
M.,
Burchiel,
K.
&
Criddle,
R.
S.
(1972)
Biochemistry
11,
2026-2036.
10.
Bernardi,
G.,
Prunell,
A.
&
Kopecka,
H.
(1975)
in
Molecular
Biology
of
Nucle'coytoplasmic
Relationships
(Elsevier,
Am-
sterdam),
pp. 85-90.
11.
Grossman,
L.
I.,
Goldring,
E.
S.
&
Marmur,
J.
(1969)
J.
Mol.
Biol.
46,367-376.
12.
Galzy,
P.
&
Slonimski,
P. P.
(1957)
Compt.
Rend.
245,
2423-
2426.
13.
Needleman,
R.
B.
&
Tzagoloff,
A.
(1975)
Anal.
Biochem.
64,
545-549.
14.
Studier,
F.
W.
(1973)
J.
Mol.
Biol.
79,237-248.
15.
Laemmli,
U.
K.
(1970)
Nature
227,680-685.
16.
Lowry,
0.
H.,
Rosebrough,
N.
J.,
Farr,
A.
L.
&
Randall,
R.
J.
(1951)
J.
Biol.
Chem.
193,
265-275.
17.
Mason,
T.
L.
&
Schatz,
G.
(1973)
J.
Biol.
Chem.
248,
1355-
1360.
18.
Tzagoloff,
A.
&
Meagher,
P.
(1972)
J.
Biol.
Chem.
247,
594-
603.
Proc.
Nat.
Acad.
Sci.
USA
73
(1976)
... The high hydrophobicity of the translated membrane proteins together with the difficulty to purify translationally active mitoribosomes has prevented the design of functional in vitro assays [2]. Radioactive labeling of mitochondrial translation products has been the classical approach to study mitochondrial translation [3,4]. Depending on the research question asked, either whole cells (in vivo) or isolated mitochondria (in-organello) are incubated with radioactive [ 35 S]-methionine which is then incorporated into newly synthesized polypeptide chains. ...
Chapter
The mitochondrial genome encodes only a handful of proteins, but methods to track their synthesis are highly limited. Saccharomyces cerevisiae is a model organism that offers possibilities to expand the classical systems to analyze mitochondrial translation. In this chapter, we present two approaches of monitoring mitochondrial protein synthesis. Labeling of mitochondrially translated products with radioactive amino acids can be performed either in intact cells or in isolated mitochondria. However, these classical methods have disadvantages that can affect cell physiology and hence are not suitable for all types of research questions. Some of these limitations can be overcome by the use of reporter genes that are inserted into yeast genetic screens mitochondrial DNA via biolistic transformation. These reporter genes can be used for yeast genetic screen and to monitor regulation and efficiency of mitochondrial translation with a variety of methods.
Article
A family of mitochondrial RNAs hybridizes specifically to the var1 region on Saccharomyces cerevisiae mitochondrial DNA (Farrelly et al., J. Biol. Chem. 257:6581-6587, 1982). We constructed a fine-structure transcription map of this region by hybridizing DNA probes containing different portions of the var1 region and some flanking sequences to mitochondrial RNAs isolated from var1-containing petites. We also report the nucleotide sequence of more than 1.2 kilobases of DNA flanking the var1 gene. Our primary findings are: (i) The family of RNAs we detect with homology to var1 DNA is colinear with the var1 gene. Their direction of transcription is olil to cap, as it is for most other mitochondrial genes. (ii) Extensive hybridization anomalies are present, most likely due to the high A-T (A-U) content of the hybridizing species and to the asymmetric distribution of their G-C residues. An important conclusion is that failure to detect transcripts from A-T-rich regions of the yeast mitochondrial genome by standard blot transfer hybridizations cannot be interpreted to mean that such sequences, which are commonly supposed to be spacer DNA, are noncoding or lack direct function in the expression of mitochondrial genes.
Article
Translation of the Saccharomyces cerevisiae mitochondrial COX3 mRNA, encoding subunit III of cytochrome c oxidase, specifically requires the action of the nuclear gene products PET54, PET122, and PET494 at a site encoded in the 612-base 5' untranslated leader. To identify more precisely the site of action of the translational activators, we constructed two large deletions of the COX3 mRNA 5' untranslated leader. Both deletions blocked translation without affecting mRNA stability. However, one of the large deletions was able to revert to partial function by a small secondary deletion within the remaining 5' leader sequences. Translation of the resulting mutant (cox3-15) mRNA was still dependent on the nuclear-encoded specific activators but was cold sensitive. We selected revertants of this mitochondrial mutant at low temperature to identify genes encoding proteins that might interact with the COX3 mRNA 5' leader. One such revertant carried a missense mutation in the PET122 gene that was a strong and dominant suppressor of the cold-sensitive defect in the mRNA, indicating that the PET122 protein interacts functionally (possibly directly) with the COX3 mRNA 5' leader. The cox3-15 mutation was not suppressed by overproduction of the wild-type PET122 protein but was very weakly suppressed by overproduction of PET494 and slightly better suppressed by co-overproduction of PET494 and PET122.
Article
We have examined the possible role of adenosine 3′,5′-phosphate (cAMP) in functions associated with the plasma membranes of Saccharomyces cerevisiae. Purified membranes from this source contained an adenylate cyclase which was insensitive to activation by fluoride or guanine nucleotides, only weakly responsive to changes of carbon source in the growth medium, and strongly stimulated by vanadate. They also contained at least two classes of receptor proteins for guanine nucleotides (as measured by binding of labeled 5′-guanylyl methylene diphosphate) with apparent dissociation constants equal to 1.0 × 10 ⁻⁷ and 3 × 10 ⁻⁶ M, a protein kinase capable of phosphorylating added histones, the activity of which was stimulated by cAMP, and cAMP receptors that may function as regulatory subunits for this kinase. Membrane proteins were also susceptible to phosphorylation by endogenous kinase(s), with polypeptides of apparent molecular weights equal to 160 × 10 ³ , 135 × 10 ³ , 114 × 10 ³ , and 58 × 10 ³ as the major targets. Of these, the 114,000-molecular-weight polypeptide was probably identical to the proton-translocating ATPase of the membranes. However, the cAMP-dependent protein kinase did not appear to be involved in these reactions. Intact ( rho ⁺ or rho ⁰ ) cells responded to dissipation of the proton electrochemical gradient across their plasma membranes by rapid and transient changes in their intracellular level of cAMP, as suggested earlier (J. M. Trevillyan and M. L. Pall, J. Bacteriol., 138 :397-403, 1979). Thus, although yeast plasma membranes contain all the essential components of a stimulus-responsive adenylate cyclase system, the precise nature of the coupling device and the targets involved remain to be established.
Article
We transformed Saccharomyces cerevisiae with a high-copy-number plasmid carrying either the wild-type gene coding for a repressible cell surface acid phosphatase or two modified genes whose products lack a 13- or 14-amino-acid segment spanning or immediately adjacent to the signal peptidase cleavage site. The wild-type gene product underwent proteolytic cleavage of the signal peptide, core glycosylation, and outer chain glycosylation. The deletion spanning the signal peptidase cleavage site led to an unprocessed protein. This modified protein exhibited core glycosylation, whereas its outer chain glycosylation was severely inhibited. Secretion of the deleted protein was impaired, and active enzyme accumulated within the cell. The deletion immediately adjacent to the signal peptidase cleavage site exhibited only a small decrease in the efficiency of processing and had no effect on the efficiency of secretion.
Article
Full-text available
Oxidative phosphorylation 1 (OP1), a nuclear gene of yeast Saccharomyces cerevisiae which is required for the expression of a functional mitochondria, has been isolated on a recombinant plasmid. The gene was selected from a recombinant plasmid pool which contained wild type yeast genomic DNA by transformation of the yeast nuclear mutant (op1) followed by a two-stage screening procedure. A recombinant plasmid containing a 2.6 kilobase Bam HI fragment of genomic yeast DNA inserted in either orientation into the single Bam HI site of yeast vector YEp 13 could complement the op1 mutation. Analysis of the gene product of this inserted DNA by three independent methods, 1) in vivo expression in Escherichia coli maxicells, 2) cell-free translation of plasmid selected RNA, and 3) expression analysis in yeast, revealed that its gene product is a protein of Mr = 30,000-32,000, which cross-reacts with specific anti-serum to the adenine nucleotide translocator of the mitochondrial inner membrane. The selection procedure is efficient and can be used for the isolation of any defined yeast nuclear gene which participates in mitochondrial development.
Article
Full-text available
Incubation of the reconstituted H⁺-ATPase from chromaffin granules on ice resulted in inactivation of the proton-pumping and ATPase activities of the enzyme. Inactivation was dependent on the presence of Mg²⁺, Cl⁻, and ATP during the incubation at low temperature. Approximately 1 mM ATP, 1 mM Mg²⁺, and 200 mM Cl⁻ were required for maximum inactivation. Incubation for about 10 min on ice was required to achieve 50% inactivation. A much smaller decline in activity was observed when the enzyme was incubated at room temperature with the same chemicals. Inactivation in the cold resulted in the release of five polypeptides from the membrane with apparent molecular masses of 72, 57, 41, 34, and 33 kDa on sodium dodecyl sulfate gels. Three of the polypeptides of 72, 57, and 34 kDa were identified as subunits of vacuolar H⁺-ATPases by antibody cross-reactivity. Similar results were obtained with several other vacuolar H+-ATPases including those from plant sources. It was concluded that the catalytic sector of the enzyme is released from the H⁺-ATPase complex by cold treatment, resulting in inactivation of the enzyme.
Article
Full-text available
The gamma-aminobutyric acid/benzodiazepine receptor from bovine cerebral cortex was solubilized with sodium deoxycholate and purified by affinity chromatography on benzodiazepine-agarose and ion exchange chromatography. The benzodiazepine binding protein was enriched 1800-fold. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and dithiothreitol showed the presence of two major bands of Mr = 57,000 and 53,000. [3H]Flunitrazepam, after UV irradiation, was incorporated irreversibly into both bands of the isolated protein. A high affinity binding site for gamma-aminobutyric acid was co-purified with the benzodiazepine binding site and the two sites were shown to reside on the same physical structure. The dissociation constants were 10 +/- 4 nM for [3H] flunitrazepam and 12 +/- 3 nM for the gamma-aminobutyric acid agonist [3H]muscimol. The maximum specific activity for [3H] muscimol binding was 4.3 nmol/mg of protein. The ratio of [3H]muscimol to [3H]flunitrazepam binding sites was between 3 and 4. Gel filtration and sucrose density gradient sedimentation studies gave a Stokes radius of 7.3 +/- 0.5 nm and a sedimentation coefficient of 11.1 +/- 0.3 S, respectively. The purified complex had a pharmacological profile that corresponds to the receptor specificity found in membranes and crude soluble extracts.
Article
Full-text available
Golgi apparatus was prepared from rat liver, and enzymatic properties and the subunit structure of the H⁺-ATPase were characterized. GTP (and also ITP) was found to drive H⁺-transport with about 20% of the initial velocity as that of ATP. Bafilomycin, a specific inhibitor for vacuolar H⁺-ATPase, inhibited the activity at 2.5 nM. The H⁺-ATPase was completely inhibited in the cold in the presence of MgATP (5 mM) and NaNO3 (0.1 M). The cold inactivation of the H⁺-ATPase resulted in release of a set of polypeptides from Golgi membrane, with molecular masses almost identical to that of the hydrophilic sector of chromaffin granule H⁺-ATPase (72, 57, 41, 34, and 33 kDa). Three of these polypeptides (72, 57, and 34 kDa), cross-reacted with antibodies against the corresponding subunits of the chromaffin granule H⁺-ATPase. A counterpart of the 39-kDa hydrophobic component of chromaffin granule H⁺-ATPase was identified in the membrane, but no 115-kDa component was found. Hence, the Golgi H⁺-ATPase shows typical features of vacuolar H⁺-ATPase, in relatively low substrate specificity, its response to inhibitors, inactivation by cold treatment in the presence of MgATP, and subunit composition judged by antibody cross-reactivity.
Article
Full-text available
Mitochondrial RNA from two cytoplasmic ϱ− mutants ofS. cerevisiae, which have kept the mitochondrial DNA segment including the ATPase-oligomycin resistance-conferring gene, stimulates protein synthesis in anE. coli cell-free system. SDS-acrylamide gel electrophoresis of the protein product revealed one major peak and two minor ones with apparent molecular weights of around 11,000, 13,500 and 17,000 respectively. The effect is specific since no stimulation is observed with RNA from a ϱ− mutant devoid of detectable mitochondrial DNA. These results are interpreted to mean that the mitochondrial DNA of these mutants codes for anin vitro translatable mRNA.
Article
Full-text available
Mutants of Saccharomyces cervisiae with defects in enzymes of the electron transfer chain and in the rutamycin-sensitive ATPase have been isolated. Some of the mutants are specifically affected in either cytochrome oxidase, coenzyme QH2-cytochrome c reductase or ATPase. Other strains are deficient in both cytochrome oxidase and coenzyme QH2-cytochrome c reductase but still have rutamycin-sensitive ATPase. All the mutants reported in this study fail to be complemented by a rho0 tester derived from a respiratory competent strain. The meiotic spore progeny obtained by mating the mutants to a respiratory competent haploid yeast, when scored for growth on glycerol, show a non-Mendelian segregation of the phenotype. These two genetic tests indicate the mutations to be cytoplasmically inherited.
Article
A procedure is described for the rapid preparation of mitochondria and the soluble cell fraction of yeast. The method makes use of an adaptor for the Braun homogenizer which allows 16 samples to be processed at once.
Article
The optimum conditions for transcription in vitro of yeast mtDNA into biologically relevant RNA by Escherichia coli RNA polymerase holoenzyme and yeast mitochondrial RNA polymerase was found to critically depend on salt concentration. RNA was transcribed (at 0.25 M KCl concentration) from high-molecular-weight mtDNA which was then translated in an E. coli (S-30) cell-free protein synthesising system. Efficient translation of mitochondrial RNA was achieved using conditions which had also been determined to be optimal in other systems. Identification of the polypeptides produced in the translation system was made using antiserum raised against mitochondrial membranes. Electrophoresis of the completely dissociated antigen-antibody complexes using dodecylsulphate-polyacrylamide gels revealed that the system in vitro produced polypeptides of similar molecular weight to those synthesised in vivo by cycloheximide-inhibited whole cells.
Article
A novel class of respiration-deficient (RD) yeast mutants has been isolated by a procedure designed to enrich for mitochondrial mutations. Four such independently isolated mutants exhibit a phenotype that differs from the classical petite mutant and does not show complementation with a DNA-less (p0) member of the latter class. One of these mutants, strain 73/1 was subjected to further analysis and found to exhibit mitochondrial inheritance on the basis of: (a) lack of this complementation; (b) non-Mendelian segregation of the RD trait after meiosis of diploids formed by mating with standard auxotrophic testers; (c) segregation on mitosis of these diploids, with the RD trait exhibiting vegetative dominance; (d) elimination of the trait and of c upon conversion to a p0 mutant by prolonged growth in the presence of ethidium bromide. Examination of the phenotype disclosed the absence in cells and mitochondria of cytochrome oxidase, cytochrome aa3, and of NADH- and succinate-cytochrome c reductases, and the presence of significant amounts of cytochromes b and c1. These two cytochromes appear reducible by succinate to a small and by NADH to a considerable extent. The mitochondrial ATPase in the mutant is sensitive to the specific inhibitor Dio-9 but resistant to oligomycin at concentrations that inhibit the wild type enzyme ≥50%. Unlike cytoplasmic petites, mitochondria of mutant 73/1 contain a functional protein-synthesizing system. Its cells appear capable not only of mitochondrial polypeptide chain initiation as measured by the formation of formylmethionyl puromycin, but also of incorporating labeled amino acids into mitochondrial proteins in the presence of cycloheximide, a process inhibited by chloramphenicol and acriflavin. The presence of a functional mitochondrial system of protein synthesis is confirmed by the elimination of the cytochrome b peak at 560 nm upon prolonged growth (six generations) in the presence of chloramphenicol. This conversion to a petite phenocopy is reversed completely upon removal of the inhibitor.
Article
Mitochondrial and nuclear DNAs from various wild-type and petite yeast strains have been highly purified by hydroxylapatite chromatography. Yeast mitochondrial, nuclear, and Escherichia coli DNA-dependent RNA polymerases were used to transcribe mitochondrial DNA of yeast into RNA. The RNA produced by these enzymes was used to study both the nature of the enzyme products and the properties of the template DNAs. The E. coli enzyme and mitochondrial enzyme transcribed the ribosomal genes of mitochondrial DNA as shown by competition with cold mitochondrial ribosomal RNA. The labeled in vitro synthesized RNAs were hybridized to various DNAs in an attempt to compare the extent of homology among these DNAs. Very little homology was indicated between nuclear and mitochondrial DNA of yeast. Total cell DNA from two petite mutants lacking the mitochondrial satellite band showed the same low degree of homology to mitochondrial DNA. Wild-type mitochondrial DNAs differed in their homologies to each other and appeared to contain different amounts of repetitive DNA. Mitochondrial DNAs from two petite mutants showed reduced homology to wild-type mitochondrial DNA. However, no additional sequences which differed from those of wild-type mitochondrial DNA could be detected in the mitochondrial DNA of one petite mutant studied, thus indicating a simple deletion mechanism for the origin of this strain. This petite mutant lacks 50-60% of the wild-type mitochondrial genome transcribed in vitro.
Article
Hybridization saturation analyses of mitochondrial DNA from 11 petite clones genetically characterized with respect to chloramphenicol and erythromycin resistance markers, have been carried out with 11 individual mitochrondrial transfer RNAs. Mitochondrial tRNA cistrons were lost, retained, or amplified in different petite strains. In some cases hybridization levels corrected for kinetic complexity of the mtDNA‡ were two- to threefold greater than that for grande mtDNA indicating selective amplification, or increased number of copies, of the segment of mtDNA containing that tRNA cistron. Hybridization levels corrected for reduced kinetic complexity of petite mtDNAs in many cases were only 1 to 10% of that for grande mtDNA suggesting a low level of intracellular molecular heterogeneity of mtDNA with respect to tRNA cistrons. Some petite clones that retained tRNA genes continued to transcribe mitochondrial tRNAs, since tRNA isolated from these strains could be aminoacylated with Escherichia, coli synthetases and hybridized with mtDNA. Hybridization data allow us to order several of the tRNA cistrons on the mitochondrial genome with respect to the chloramphenicol and erythromycin antibiotic resistance markers.