419
Bioscience Reports 2, 419-426 (1982)
Printed in Great Britain
Does m o r e t h a n one m i t o c h o n d r i a l l y s y n t h e s i z e d p r o t e i n
in y e a s t have l a r g e r p r e c u r s o r s ?
3aved ASHRAF and 3. 3AYARAMAN ~
Department of Biochemistry, School of Biological Sciences,
Madurai Kamaraj University, Madurai 625 021, India
(Received 19 April 1982)
Y e a s t ceils u n d e r g o i n g d e r e p r e s s i o n (the phase of
mitochondriogenesis) were exposed to [ l ~ C ] f o r m a t e in
t h e p r e s e n c e of cycloheximide, the cytosolic protein
s y n t h e s i s i n h i b i t o r , and of 1 , 1 0 - p h e n a n t h r o l i n e , a
m e t a l l o - p r o t e a s e inhibitor.
Extensive labelling was
o b t a i n e d under such conditions.
Incubation of these
l a b e l l e d products with mitochondrial l y s a t e s released
small p e p t i d e s (mol. wt. 500-i000).
These results
indicate that mitochondria probably synthesize some of
t h e p r o t e i n s in t h e p r e c u r s o r form and they are
processed by a specific matrix-located protease before
proper integration.
G r o w i n g numbers of reports point out that many cytosolically
s y n t h e s i z e d mitochondrial proteins are synthesized in the form of
larger precursors: in yeast (Maccechini et al., 1979a,b; Cot4 et al.,
1979; Nelson & S c h a t z , 1979), in N e u r o s p o r a c r a s s a (Harmey &
Neupert, 1979), and in rat liver (Mori et al., 1980; Schmelzer &
Heinrich, 1980; Raymond & Shore, 1981).
These precursors are in
g e n e r a l post-translationally processed to their mature forms.
This
process is termed 'vectorial processing' (Schatz, 1979).
A matrixlocated metallo-protease in yeast mitochondria has been implicated in
t h e process (Bohni et al., 1980).
Sevarino and Poyton (1980)
presented evidence for the occurrence of a precursor of subunit II of
cytochrome oxidase which is synthesized by the mitochondria, but it is
not fully known whether the rest of such proteins in yeast are also
present in the form of precursors.
In order to l o c a t e and if possible identify any mitochondrially
synthesized
precursor proteins, we resorted to the use of [I~C]f o r m a t e for labelling the proteins. Two logical reasons for this choice
were (i) that the 'leader sequences' of precursor proteins are located
a t the N-terminal end (Blobel & Dobberstein, 1975) and (ii) that
[ l ~ C ] f o r m a t e can be incorporated in the initiator formyl-methionine of
mitochondrially synthesized proteins (Feldman & Mahler, 197t~).
A
similar approach has been used recently to identity the precursor of
subunit I of cytochrome oxidase in N. c r a s s a (Van't Sant et al.,
19Sl).
*To whom correspondence should be addressed.
01982
The Biochemical Society
420
ASHRAF
& 3AYARAMAN
If precursor proteins are processed by specific proteases, then in
t h e p r e s e n c e of p r o t e a s e inhibitors~ [ l # C ] f o r m a t e labelling and
accumulation of precursors should be maximal.
M a t e r i a l s and M e t h o d s
The system
The glucose repression-derepression system used in these studies
with Saccharomyces c e r e v i s i a e NCIM 3095 has been extensively
d e s c r i b e d in t he e a r l i e r p u b l i c a t i o n s ( 3 a y a r a m a n et al., 1966;
3ayaraman et al., 197#; Chandrasekaran et al.~ 1978~ 1980).
Incorporation of the radioactive label
Under our conditions, the ceils are maximally repressed at 2.3 h
a f t e r being inoculated into the repression medium.
Chloramphenicol
(CAP)~ 5 mg/ml~ was added at that point. Cells were washed free of
chloramphenicol a f t e r 2 h and then resuspended in the 'used medium'
(Chandrasekaran et al., 1980).
To this~ cycloheximide (CHI)~ 100
p g / m l , and protease inhibitors (0.5 mM) as indicated were added.
A f t e r 5 min of shaking, either [ t ~ C ] f o r m a t e (0.5 tJCi/ml, sp. activity
##.8 mCi/nmol, efficiency of the counter 89%) or [3H]leucine (0.5
tJCi/ml~ sp. activity 6.8 Ci/mmol, efficiency 20%) was added.
The
incorporation was carried out for 30 min and then the cells were
h a r v e s t e d and washed with salin% and mitochondria were isolated
( 3 a y a r a m a n et al., 1966).
Radioactivity was determined in acidprecipitable fractions.
M i t o c h o n d r i a l l y s y n t h e s i z e d proteins are
m a x i m a l l y l a b e l l e d a c c o r d i n g to t he a b o v e - m e n t i o n e d p r o t o c o l
(Tzagoloff~ 1971 ).
Assay of the protease activity
[l#C]formate-labelled
m i t o c h o n d r i a l proteins were prepared as
described above.
They were used as the substrate.
Mitochondrial
lysate obtained on lysing the mitochondria by vortexing in distilled
water was used as the source of protease. This crude lysate (0.25 M
sucrose being added to it af t e r lysis) was incubated for i h with the
l a b e l l e d s u b s t r a t e mitochondria in an incubation medium (pH 7.#)
c o n t a i n i n g 0.25 M sucrose~ 50 mM Tris/HCl~ and 0.5 mM phenylm e t h y l s u l f o n y l f l u o r i d e (PMSF).
PMSF was used to inhibit nonspecific proteolysis.
At suitable time intervals a f t e r incubating at
30~
th e m i x t u r e was spun down at 12 000 r.p.m, in a TH-12
centrifuge for 5 min. Aliquots of the supernatant were withdrawn and
radioactivity was determined. The ext ent of the release of the counts
was taken as an indication of proteolysis. (The counts released at the
start of the incubation plus the counts released from the labelled
mitochondria alone were subtracted from the total counts.)
Results
T a b l e 1 shows the results of one typical experiment in which
[ l # C ] f o r m a t e and [3H]leucine labelling of mitochondria proteins was
carried out under in vivo conditions.
PRECURSORS OF YEAST MITOCHONDRIAL PROTEINS
Table 1.
Treatment
CHI
CAP
CHI
CHI
CHI
421
Radioactive labelling of mitochondrial proteins
in vivo
[14C]formate (c.p.m./mg [3H]leucine (c.p.m./mg
mitochondrial protein) mitochondrial protein)
(control)
12
2
26
16
13
+ 1,10-phenanthroline
+ PMSF
+ EGTA
CAP) chloramphenicol;
sulfonyl fluoride.
CHI,
800
705
000
334
732
cycloheximide;
1075
210
3968
1518
Not tried
PMSF)
phenylmethyl-
The above-mentioned results point to the following:
(a) The i n c o r p o r a t i o n of the label was chloramphenicol-sensitive,
showing that the proteins synthesized were of mitochondrial origin (all
precautions having been taken to prevent microbial contamination). It
could be pointed out that [14C]formate could label purines also) but
the bulk of the nucleic acids precipitated by trichloroacetic acid is
r e m o v e d d u r i n g the course of the processing of the radioactive
samples, i.e. heating the samples with trichloroacetic acid at 70~ for
l0 min (Beattie, 1979).
(b) There was a 2.5- to 4-fold enhancement in the incorporation in
the presence of 1,10-phenanthroline (OP) but not significantly in the
presence of PMSF (some enhancement in the presence of PMSF may
be due to the prevention of non-specific degradation of the proteins.
( c ) T h e r e was not much enhancement in the presence of EGTA,
showing that the protease involved does not require calcium for its
a c t i v i t y . It has been subsequently found that the protease in question
is a zinc-containing enzyme (to be published separately).
In order to check whether this enhanced incorporation was of a
generalized nature or was confined to specific species, we labelled the
mitochondria with [1~C]formate in the presence of CHI and in the
presence of CH! and 1,10-phenanthroline.
The labelled mitochondria
were isolated and immunoprecipitation with specific antibodies against
subunits I and Il of cytochrome oxidase and cytochrome b was carried
out. The choice was dictated by the fact that these proteins are well
established to be synthesized by mitochondria.
The results presented in Table 2 show increased counts in the
immunoprecipitates from mitochondria treated with CHI + phenanthroline. However) only a very small fraction of counts was precipitated,
for reasons which are unclear.
P o l y a c r y l a m i d e - g e l analysis of mitochondrial proteins labelled with
[ I # C ] f o r m a t e in the p r e s e n c e ol CHI and of CHI + OP was next
c a r r i e d o u t , and r a d i o a c t i v e profiles are given in Fig. I.
Four
additional polypeptides were significantly labelled when 1,10-phenant h r o l i n e was included in the medium.
While the i d e n t i t y of t h e s e species is being worked out, we decided
to investigate
w h e t h e r El ~ C ] f o r m a t e - l a b e l l e d
m a t e r i a l could be
r e l e a s e d on t r e a t e m e n t with m i t o c h o n d r i a l Iysates.
The logic of this
ASHRAF
422
Table 2.
& ~AYARAMAN
Immunoprecipitation of labelled mitochondrial
proteins
C.p.m. in immunoprecipitates from
mitochondria treated with
Antibody
used
CHI + 0P
-Fold
increase
14 227
95
31 758
203
2.23
2.14
54
144
2.67
69
252
3.65
CHI
None
Cytochrome b
Subunit I of cytochrome
oxidase
Subunit II of cytochrome
oxidase
CHI~ chloramphenicol; OP~ 1,10-phenanthroline.
I
I
I
I
16
300
a_ 2 0 0
r8
4.3/l
~'
I00
9"
r
.....
0
\ ..'......;
i"..
,.,...., ..... ...~ .................... j
--.," ..-
'..,../',-." ".......r."
~'~
..... "...,...,.,..~"
~
I
I
I
I
20
40
60
80
SLICE
NUMBER
Fig. i. Slab gels were sliced (1 mm thick) and were
digested in H202 for 10 h 9 and radioactivity was
determined in the individual slices. The numbers on
the peaks denote the approx, mol. wts. of the
proteins (in kilodaltons).
CHl-treated
mitochondria.
Mitochondria treated with
CHI + OP.
PRECURSORS OF YEAST MITOCHONDRIAL PROTEINS
423
experiment has already been dealt with in the introduction.
As the
results in Fig. 2 show, such material was released in a time-bound
fashion and the release was sensitive to phenanthroline.
In an a t t e m p t to understand the nature of the released products,
m i t o c h o n d r i a labelled with l i n G ] f o r m a t e in the presence of CHI +
1,10-phenanthroline were incubated with the untabelled mitochondrial
lysates at 30~
for 1 h.
The incubation mixture was spun down to
remove mitochondria and the supernatant was fractionated in a small
Sephadex G-50 column (15 c m x t.5 cm).
Elution was carried out
with 10 mM Tris/HC1 buffer (pH 7.t~).
Fig. 3 shows that most of the radioactivity was Muted in the
molecular-weight range of 500-1000.
Discussion
Three major conclusions emanate from this communication (details
~o be published subsequently):
(a) Labelling of mitochondrial proteins with [ l ~ C ] f o r m a t e increased
about 3-fold in the presence of 1,10-phenanthroline, a metallo-protease
inhibitor.
More than 5096 of this label was present at the /V-terminal
ends of polypeptides as indicated by the mild acid hydrolysis procedure
of Fetdman and Mahler (1974) (unpublished data).
Considering the
f a c t t h a t f o r m y t - m e t h i o n i n e serves as the initiator amino acid in
mitochondria (Bianchetti et aI., 1971), the accumulation of formatelabelled proteins in the presence of 1,10-phenanthrolin% a metalloprotease inhibitor, assumes significance.
(b) M i t o c h o n d r i a l lysates can release the [ l ~ C ] f o r m a t e label into
soluble form, and this process is sensitive to Phenanthroline, but not to
PMSF.
All t h e s e r e s u l t s , when taken together, indicate that a
m a t r i x - l o c a t e d e n d o p r o t e a s e is involved in the processing of the
m i t o c h o n d r i a l proteins, and furthermore, that the processing takes
place from the /V-terminal end. The activity of this protease increases
p r o g r e s s i v e l y during the derepression phase of mitochondriogenesis,
i n d i c a t i n g its i n v o l v e m e n t in t h e process of mitochondriogenesis.
M o v e o v e r , the inclusion of phenanthroline in synchronously growing
y e a s t cultures also resulted in maximal [ l q C ] f o r m a t e incorporation
during the late S or early G 2 phase of the cell cycle (unpublished
data).
It was earlier shown that the maximum synthesis of the
mitochondrially synthesized proteins takes place during the late S and
early G 2 phases (Somasundaram & $ayaraman, 1981a,b).
( c ) Most of t h e p r o d u c t s released during proteolysis fall in the
molecular-weight range of 500-1000. Further, these smalI peptides are
more than 60% hydrophobic in nature (unpublished data) as judged by
t h e c r i t e r i a of Kadenbach and Hadvary (1973).
This would also
explain the high extent of labelling by [3H]leucine.
On the basis of the conclusions, we propose that some proteins
made by the mitochondria (identity pending) are nascently synthesized
in the form of precursors (the fact that the control mitochondria also
could incorporate a considerable amount of Et#C]formate explains that
some matured proteins also have f - m e t at the /V-terminal end, in other
words, t h e y do not u n d e r g o processing) and there exists in the
o r g a n e l l e a m e t a l l o - p r o t e a s e which p r o c e s s e s them before their
integration into the membrane.
ASHRAF
424
& 3AYARAMAN
KINETICS OF PROTEASE ACTIVITY
2,~
I,o
'0
m
~
2
s
E
1.5
r
i/)
A
~
A
n
0
0.~
I
0
I
I
I5
50
45
INCUBATION PERIOD (rain)
I
60
Fig.
2.
[14C]formate-labelled
precursors were
incubated
with
the isotonic
lysate (unlabelled)
containing protease in the ratio of i:I at 30~
for
1 h. The counts released at the start of incubation
were
subtracted
from the actual counts released.
Initial counts: 28 700 c.p.m./mg protein.
~Labelled
precursors + isotonic lysate of
the unlabelled mitochondria (protease);
V
~ l a b e l l e d precursors alone;
V------~labelled precursors + heat-killed protease;
O
Olabelled
precursors + protease + 0.5 mM
phenanthroline;
•
precursors + protease + 0.5 mM
PMSF;
~ labelled
precursors + intact unlabelled
mitochondria.
PRECURSORS OF YEAST MITOCHONDRIAL PROTEINS
425
II0
66ooo
200o
670
68
800
600
s
r
400
! ,! i
200
I
2
~
4
I
I
6
O
ELUENT
I
I
10
12
VOLUME
t
14
I
16
I
18
1
20
(ml)
Fig. 3.
The releasd products during proteolysis
were eluted through a Sephadex G-50 column (15 cm x
1.5 cm).
Fractions (250 ~i) were collected.
The
column was calibrated with BSA (mol. wt. 66 000),
cytochrome
c (12 000), B r o m o p h e n o l
blue (67),
labelled
amino acids (Ii0), and labelled formate
(68).
' labelled precursors + protease~
labelled precursors alone.
Added Note
I t has been c o n f i r m e d recently that cytochrome b in yeast is
n a s c e n t l y s y n t h e s i z e d in the form of a larger precursor which is
p o s t - t r a n s l a t i o n a l l y p r o c e s s e d to its matured form (Chen Y-S &
B e a t t i e DS [1981] Biochemistry 20, 7557).
Acknowledgem ents
3. A.
is grateful to the CSIR, New Delhi, for the award of the
S e n i o r R e s e a r c h Fellowship during the course of this study.
The
subunit-specific antibodies used in this study were kindly given to us
by Dr. G. Schatz, Basel, Switzerland.
426
ASHRAF & 3AYARAMAN
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