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The ribosomal subunits of hamster cell mitochondria
Vol. 41, No. 2, 1970
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
THE RIBOSOMALSUBUNITSOF HAMSTERCELLMITOCHONDRIA Bland S. Montenecourt and Donald T. Dubin Department of Microbiology,
Received
September
Rutgers Medical School, New Brunswick, N.J.
15, 1970
Summary. The "13s" and 1'17s" RNA's previously shown to be specifically associated with the mitochondrial fraction of cultured hamster cells have been shown to occur as components of submitochondrial particles, presumably mitochondrial ribosomal subunits, sedimenting at approximately 25s and 33s, respectively. Reconstruction experiments indicate that these particles are not artifacts of the isolation procedure. Wehave described two discrete and 17s, specifically hamster cells.
RNAspecies, sedimenting at about 13s
associated with the mitochondrial
These RNA's were present in approximately
amounts, and we proposed that they were the structural drion-specific
fraction
ribosomes.
of cultured
equimolar
RNA's of mitochon-
However, they appeared unusually small for ribo-
somal RNA's, and were unusually low in methylated nucleotides and in GC content (1,2,3).
Furthermore, similar
RNAspecies observed in HeLa cell
mitochondria did not appear to be present in equimolar amounts, and were thought not to be ribosomal (4), The aim of the present studies was to examine this question further; the results
indicate
that the 13s and 17s RNA's of hamster mitochondria are
indeed components of ribosomal subunits. METHODS Cultured hamster (BHK-21) cells were labeled with 14C-uridine for 18-22 hours, as previously routinely
described (2).
added to preferentially
Actinomycin D, 0.1 pg/ml, was
suppress incorporation
ribosomes (5) and unlabeled deoxycytidine prevent labeling of DNA.
458
into cytoplasmic
and thymidine were added to
Vol. 41, No. 2, 1970
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
To minimize mitochondria,
for
an abbreviated Cells
was used. 10,000
opportunity
were
xg pellet
version
harvested
a pellet,
when dissolved similar
purified
In addition,
vinyl cles figures
sulfate (cf. -
ref.
to all 6).
and in the
solutions Further
of an earlier
to those it
isolation
procedure
('C"
but
obtained was found
are described
of of ref.21
the crude
of submitochondrial
in 1% SDS, was found
used in processing details
during
as before,
as the source
13s and 17s RNA patterns mitochondria.)
action
and disrupted
was used directly
(Such
particles.
ribonuclease
using
to yield
more rigorously
necessary
to add pol-y-
submitochondrlal in the legends
partito the
text.
800 -
600 z. P 5, *400-
A
B
Fraction Number 14
with C-uridine (0.2 )rc/O.Ol umole Legend to Figure 1. Cells were labeled /ml, 18 hrs,) and a crude mitochondrial pellet was obtained, as described This pellet was treated at 5' with 2% Triton X-100 in buffer in Methods. containing 0.5 NaCl, 0.01 g Tris, pH 7.4, 0.0015 E MgC12 and 20 ug/ml polyvinylsulfate. The preparation was then layered onto a gradient of 5-20X sucrose in the same buffer, and centrifuged for 90 min. at 56,000 Samples were obtained and assayed rev /min. at 5O in a Spinco SW56 rotor. as previously described (2).
459
BIOCHEMICAL
Vol. 41, No. 2, 1970
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
RJlSULTSANDDISCUSSION Figure 1 shows the pattern chondrial pellet
obtained when a 14C-uridine-labeled
was disrupted with 2% Triton
a sucrose density gradient,
X-100, and sedimented through
both in the presence of 0.5N NaCl.
of the labeled RNAsedimented as two fairly
mito-
discrete
About 30%
peaks, whose estimated
sedimentation constants were 25s and 33s (range, 24-26s and 32-34s). markers, either
28s RNAin "acetate-NaCl"
buffer
(7) or 50s cytoplasmic
ribosomal subunits in 0.02 M EDTA (8), were run in parallel The yield of labeled
RNA
in these particles
13s and 17s RWAobtained when mitochondrial Similar particles
For
tubes.
approximated the yield of
pellets were lysed In 1% SDS.
were obtained when mitochondria were disrupted and
sedimented in buffer
containing
a low concentration
However, under these conditions the yield
(0.01 N) of NaCl.
of particles
was greatly
Et
18s 200 .E E Y) E "2 100 xxx k 2 I
‘0
20 , Fraction Number
lines of Figure 1 Legend to Figure 2. The fractions within the vertical The resulting were pooled, and precipitated wfth 2 volumes of ethanol. pellets were dissolved in buffer containing 0.5% SDS, 0.01 E NaCl, 0.01% Tris, pH 7.4 and centrifuged through a 5-20X sucrose gradient containing the same0.5% SDSbuffer at 27,000 rev/min. for 17 hours in an SW41rotor at 23'C. The position of the 18s RNAmarker is indicated by the arrow.
Abbreviation:
SDS, sodium dodecylsulfate 460
Vol. 41, No. 2, 1970
BIOCHEMICAL
diminished,with density
increased
gradient
density
particle
labeled
low
ment is
some
tein.
and the
just
or high
this
prior salt
shown in Fig.
these,
than
3.
of,the
were
poss$bUty,
to or just
after
free
amounts
addition
A representative
The added standard
(ref.
RNA (cf. -
ref.
RNA did 7)
RNA
and protein
mitochondrial of Triton-X, "high
conditions,
perhaps association
and using
salt"
somewhat
proportion
but
of 31+-uridine-
sediment
9) or limited
a significant
The 25s
structures,
to 14 C-uridine-labeled
added
in no case did
native
between trace
2.
17's, RNA.
not
association
conditions.
under
aggregation However,
after
25s and 33s peaks
shown in Fig.
33s particle
the particles
13s and 17s RNA were
either
under
13s,
that
To test
either
in the pellet
to the
such as that
from artifactual
8).
pellets,
corresponding
patterns
largely
resulted ref.
fractions
gradient
was possible
rather (cf. -
from
yielded It
of RNA appearing
centrifugation.
RNA extracted yielded
amounts
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
experi-
faster because with
of the added
of proRNA
500
f
400 0 E E
2 s s
300
200
100
Fraction Number
Legend to chondrial Immediately plus 13s fractionated 100 min.
Figure 3. Cells were labeled with 14C-uridine, and a crude mitopellet was obtained and treated with Triton-X, as for Figure 1. after addition of the Triton-X, trace amounts of purified 17s RNA, labeled with 3H-uridine, were added and the mixture was as described for Fig, 1, except that centrifugation was for 14c,
(x-x)
; 3H,
(-e-o-),
461
Vol. 41, No. 2, 1970
sediment
with
trates
BIOCHEMICAL
the 25s or 33s particles.
another
property
of our
25s and 33s particles Although
is not
are
system.
Namely,
somewhat
33s particle
situation
several
from
of Fig.
the relative
one experiment
The reason
3 also
illus-
amounts
of
to another.
was the more heavily
was reversed.
described
"pulsed"
in liver
radioactive
labeled
(Fig.
this
variability
for
subunits. Xenopis
Swanson
l),
results,
about
95s,
as well
as "45s"
and ours
In summary, are
mately
we have
intimately
Together
that,
the mitochondrial
compared
cell
with
mitochondria
to ribosomal
(14-19). 33s particles
that
is
at present
shown
other
particles
evidence
in
sediment
reasonable (13) that the bulk
agreement have
described
sedimented
at
of their
mito-
relationship
between
clear.
the 13s and 17s ENA's of hamster tith
that recent rfbosomal
have
A more detailed is
that
con-
presented
and subunits
contained
not
particle
of our putative
and Penman
RNA; the
were
low sedimentation
monomer
Perlman
at about
this
in HeLa mitochondria
assoctated
subunits,
animal
hand,
its
also
(11)
mitochondria
have recently
--values
particles
25s and 33s respectively,
like
(12)
high-molecular-weight
particles
chondrfa
respectively
sedimented
low values
ribosomal
structure"
chondrion-specific these
and Dawid
On the other
a "protein-synthesizing
monomer,
particles
and Work
as was suggested,
unusually
the mitochondrial
at 6Os, and 43s and 329, our
If,
the
that
when isolated
ribosomal with
ribosome-like
and Ashwell
mitochondria
leucine.
be in accord
(10)
labeled
the mitochondrial
ribosomal
of distinctive
and Kalf
was preferentially
would
in
reports
Obrien
a particle
with
represents stant
other
mitochondria.
55s and that
with
The pattern
known,
in animal
that
the
this
There
have
varied
generally
occasionally
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
partfcles, appear
sedfmentfng
to be analogous these
RNA (3),
the ri,bosomal
low
sedimentation
from mitochondria
physicochemical
of lower
characterization
in progress,
462
at approxito rrbosomal
observations,
unusually
mito-
findtngs
suggest
partLcles
values,
of
even
organisms of the
25s and
Vol. 4 1, No. 2, 1970
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
This work was supported by grant No. GM 14957 of the National Institute of General Medical Sciences, U.S. Public Health Service,
and a grant
from the Moy Mel1 Foundation.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19.
Dubin, D. T. and Brown, R.E., Biochem. Biophys. Acta 145:538 (1967). Dubin, D. T. and Montenecourt, B. S., J. Mol. Biol. 48:279 (1970). Montenecourt, B. S., Langsam, M., and Dubin, D. T., J. Cell Biol. 46:241 (1970). Vesco, C. and Penman, S., Proc. Natl. Acad. Sci., Wash. 62:218 (1969). Dubin, D. T., Biochem. Biophys. Res. Commun.29:655 (1967). Rifkin, M. R., Wood, D. D. and Luck, D. 3. L., Proc. Natl. Acad. Sci., Wash. 58:1025 (1967). Dubin, D. T., J. Mol. Biol. 8:749 (1964). Girard, M. and Baltimore, D., Proc. Natl. Acad. Sci., Wash. 56:999 (1966). Vaughan, M. H., Warner, J. R., and Darnell, J. E., J. Mol. Biol. 25:235 (1967). Obrien, T. W. and Kalf, G. F., J. Biol. Chem. 242:2180 (1970). Ashwell, M. A. and Work, T. S., Biochem. Biophys. Res. Commun.39:204 (1970). Swanson, R. F. and Dawid, I. B., Proc. Natl. Acad. Sci., Wash. 66:117 (1970). Perlman, S., and Penman, S., Nature 227:133 (1970). Kiintzel, H. and Nell, H., Nature 215:1340 (1967). Kiintzel, H., J. Mol. Biol. 40:315 (1967). Vignais, P. V., Huet, J., and Andre, J., FEBSLetters 3:177 (1969). Schmitt, H., FEBSLetters 4:234 (1969). Stegeman, W. J., Cooper, C. S. and Avers, C. J., Biochem. Biophys. Res. Commun.39:69 (1970). Edelman, M., Verma, I. M. and Littauer, U. Z., J. Mol. Biol, 49:67 (1970).
463
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
THE RIBOSOMALSUBUNITSOF HAMSTERCELLMITOCHONDRIA Bland S. Montenecourt and Donald T. Dubin Department of Microbiology,
Received
September
Rutgers Medical School, New Brunswick, N.J.
15, 1970
Summary. The "13s" and 1'17s" RNA's previously shown to be specifically associated with the mitochondrial fraction of cultured hamster cells have been shown to occur as components of submitochondrial particles, presumably mitochondrial ribosomal subunits, sedimenting at approximately 25s and 33s, respectively. Reconstruction experiments indicate that these particles are not artifacts of the isolation procedure. Wehave described two discrete and 17s, specifically hamster cells.
RNAspecies, sedimenting at about 13s
associated with the mitochondrial
These RNA's were present in approximately
amounts, and we proposed that they were the structural drion-specific
fraction
ribosomes.
of cultured
equimolar
RNA's of mitochon-
However, they appeared unusually small for ribo-
somal RNA's, and were unusually low in methylated nucleotides and in GC content (1,2,3).
Furthermore, similar
RNAspecies observed in HeLa cell
mitochondria did not appear to be present in equimolar amounts, and were thought not to be ribosomal (4), The aim of the present studies was to examine this question further; the results
indicate
that the 13s and 17s RNA's of hamster mitochondria are
indeed components of ribosomal subunits. METHODS Cultured hamster (BHK-21) cells were labeled with 14C-uridine for 18-22 hours, as previously routinely
described (2).
added to preferentially
Actinomycin D, 0.1 pg/ml, was
suppress incorporation
ribosomes (5) and unlabeled deoxycytidine prevent labeling of DNA.
458
into cytoplasmic
and thymidine were added to
Vol. 41, No. 2, 1970
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
To minimize mitochondria,
for
an abbreviated Cells
was used. 10,000
opportunity
were
xg pellet
version
harvested
a pellet,
when dissolved similar
purified
In addition,
vinyl cles figures
sulfate (cf. -
ref.
to all 6).
and in the
solutions Further
of an earlier
to those it
isolation
procedure
('C"
but
obtained was found
are described
of of ref.21
the crude
of submitochondrial
in 1% SDS, was found
used in processing details
during
as before,
as the source
13s and 17s RNA patterns mitochondria.)
action
and disrupted
was used directly
(Such
particles.
ribonuclease
using
to yield
more rigorously
necessary
to add pol-y-
submitochondrlal in the legends
partito the
text.
800 -
600 z. P 5, *400-
A
B
Fraction Number 14
with C-uridine (0.2 )rc/O.Ol umole Legend to Figure 1. Cells were labeled /ml, 18 hrs,) and a crude mitochondrial pellet was obtained, as described This pellet was treated at 5' with 2% Triton X-100 in buffer in Methods. containing 0.5 NaCl, 0.01 g Tris, pH 7.4, 0.0015 E MgC12 and 20 ug/ml polyvinylsulfate. The preparation was then layered onto a gradient of 5-20X sucrose in the same buffer, and centrifuged for 90 min. at 56,000 Samples were obtained and assayed rev /min. at 5O in a Spinco SW56 rotor. as previously described (2).
459
BIOCHEMICAL
Vol. 41, No. 2, 1970
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
RJlSULTSANDDISCUSSION Figure 1 shows the pattern chondrial pellet
obtained when a 14C-uridine-labeled
was disrupted with 2% Triton
a sucrose density gradient,
X-100, and sedimented through
both in the presence of 0.5N NaCl.
of the labeled RNAsedimented as two fairly
mito-
discrete
About 30%
peaks, whose estimated
sedimentation constants were 25s and 33s (range, 24-26s and 32-34s). markers, either
28s RNAin "acetate-NaCl"
buffer
(7) or 50s cytoplasmic
ribosomal subunits in 0.02 M EDTA (8), were run in parallel The yield of labeled
RNA
in these particles
13s and 17s RWAobtained when mitochondrial Similar particles
For
tubes.
approximated the yield of
pellets were lysed In 1% SDS.
were obtained when mitochondria were disrupted and
sedimented in buffer
containing
a low concentration
However, under these conditions the yield
(0.01 N) of NaCl.
of particles
was greatly
Et
18s 200 .E E Y) E "2 100 xxx k 2 I
‘0
20 , Fraction Number
lines of Figure 1 Legend to Figure 2. The fractions within the vertical The resulting were pooled, and precipitated wfth 2 volumes of ethanol. pellets were dissolved in buffer containing 0.5% SDS, 0.01 E NaCl, 0.01% Tris, pH 7.4 and centrifuged through a 5-20X sucrose gradient containing the same0.5% SDSbuffer at 27,000 rev/min. for 17 hours in an SW41rotor at 23'C. The position of the 18s RNAmarker is indicated by the arrow.
Abbreviation:
SDS, sodium dodecylsulfate 460
Vol. 41, No. 2, 1970
BIOCHEMICAL
diminished,with density
increased
gradient
density
particle
labeled
low
ment is
some
tein.
and the
just
or high
this
prior salt
shown in Fig.
these,
than
3.
of,the
were
poss$bUty,
to or just
after
free
amounts
addition
A representative
The added standard
(ref.
RNA (cf. -
ref.
RNA did 7)
RNA
and protein
mitochondrial of Triton-X, "high
conditions,
perhaps association
and using
salt"
somewhat
proportion
but
of 31+-uridine-
sediment
9) or limited
a significant
The 25s
structures,
to 14 C-uridine-labeled
added
in no case did
native
between trace
2.
17's, RNA.
not
association
conditions.
under
aggregation However,
after
25s and 33s peaks
shown in Fig.
33s particle
the particles
13s and 17s RNA were
either
under
13s,
that
To test
either
in the pellet
to the
such as that
from artifactual
8).
pellets,
corresponding
patterns
largely
resulted ref.
fractions
gradient
was possible
rather (cf. -
from
yielded It
of RNA appearing
centrifugation.
RNA extracted yielded
amounts
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
experi-
faster because with
of the added
of proRNA
500
f
400 0 E E
2 s s
300
200
100
Fraction Number
Legend to chondrial Immediately plus 13s fractionated 100 min.
Figure 3. Cells were labeled with 14C-uridine, and a crude mitopellet was obtained and treated with Triton-X, as for Figure 1. after addition of the Triton-X, trace amounts of purified 17s RNA, labeled with 3H-uridine, were added and the mixture was as described for Fig, 1, except that centrifugation was for 14c,
(x-x)
; 3H,
(-e-o-),
461
Vol. 41, No. 2, 1970
sediment
with
trates
BIOCHEMICAL
the 25s or 33s particles.
another
property
of our
25s and 33s particles Although
is not
are
system.
Namely,
somewhat
33s particle
situation
several
from
of Fig.
the relative
one experiment
The reason
3 also
illus-
amounts
of
to another.
was the more heavily
was reversed.
described
"pulsed"
in liver
radioactive
labeled
(Fig.
this
variability
for
subunits. Xenopis
Swanson
l),
results,
about
95s,
as well
as "45s"
and ours
In summary, are
mately
we have
intimately
Together
that,
the mitochondrial
compared
cell
with
mitochondria
to ribosomal
(14-19). 33s particles
that
is
at present
shown
other
particles
evidence
in
sediment
reasonable (13) that the bulk
agreement have
described
sedimented
at
of their
mito-
relationship
between
clear.
the 13s and 17s ENA's of hamster tith
that recent rfbosomal
have
A more detailed is
that
con-
presented
and subunits
contained
not
particle
of our putative
and Penman
RNA; the
were
low sedimentation
monomer
Perlman
at about
this
in HeLa mitochondria
assoctated
subunits,
animal
hand,
its
also
(11)
mitochondria
have recently
--values
particles
25s and 33s respectively,
like
(12)
high-molecular-weight
particles
chondrfa
respectively
sedimented
low values
ribosomal
structure"
chondrion-specific these
and Dawid
On the other
a "protein-synthesizing
monomer,
particles
and Work
as was suggested,
unusually
the mitochondrial
at 6Os, and 43s and 329, our
If,
the
that
when isolated
ribosomal with
ribosome-like
and Ashwell
mitochondria
leucine.
be in accord
(10)
labeled
the mitochondrial
ribosomal
of distinctive
and Kalf
was preferentially
would
in
reports
Obrien
a particle
with
represents stant
other
mitochondria.
55s and that
with
The pattern
known,
in animal
that
the
this
There
have
varied
generally
occasionally
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
partfcles, appear
sedfmentfng
to be analogous these
RNA (3),
the ri,bosomal
low
sedimentation
from mitochondria
physicochemical
of lower
characterization
in progress,
462
at approxito rrbosomal
observations,
unusually
mito-
findtngs
suggest
partLcles
values,
of
even
organisms of the
25s and
Vol. 4 1, No. 2, 1970
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
This work was supported by grant No. GM 14957 of the National Institute of General Medical Sciences, U.S. Public Health Service,
and a grant
from the Moy Mel1 Foundation.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19.
Dubin, D. T. and Brown, R.E., Biochem. Biophys. Acta 145:538 (1967). Dubin, D. T. and Montenecourt, B. S., J. Mol. Biol. 48:279 (1970). Montenecourt, B. S., Langsam, M., and Dubin, D. T., J. Cell Biol. 46:241 (1970). Vesco, C. and Penman, S., Proc. Natl. Acad. Sci., Wash. 62:218 (1969). Dubin, D. T., Biochem. Biophys. Res. Commun.29:655 (1967). Rifkin, M. R., Wood, D. D. and Luck, D. 3. L., Proc. Natl. Acad. Sci., Wash. 58:1025 (1967). Dubin, D. T., J. Mol. Biol. 8:749 (1964). Girard, M. and Baltimore, D., Proc. Natl. Acad. Sci., Wash. 56:999 (1966). Vaughan, M. H., Warner, J. R., and Darnell, J. E., J. Mol. Biol. 25:235 (1967). Obrien, T. W. and Kalf, G. F., J. Biol. Chem. 242:2180 (1970). Ashwell, M. A. and Work, T. S., Biochem. Biophys. Res. Commun.39:204 (1970). Swanson, R. F. and Dawid, I. B., Proc. Natl. Acad. Sci., Wash. 66:117 (1970). Perlman, S., and Penman, S., Nature 227:133 (1970). Kiintzel, H. and Nell, H., Nature 215:1340 (1967). Kiintzel, H., J. Mol. Biol. 40:315 (1967). Vignais, P. V., Huet, J., and Andre, J., FEBSLetters 3:177 (1969). Schmitt, H., FEBSLetters 4:234 (1969). Stegeman, W. J., Cooper, C. S. and Avers, C. J., Biochem. Biophys. Res. Commun.39:69 (1970). Edelman, M., Verma, I. M. and Littauer, U. Z., J. Mol. Biol, 49:67 (1970).
463