Download 143 BBA 35 oo4 INTERACTION OF NEUROSPORA

143
BIOCHIMICA ET BIOPHYSICA ACTA
BBA 35 oo4
INTERACTION OF NEUROSPORA MITOCHONDRIAL STRUCTURAL
P R O T E I N W I T H O T H E R PROTEIN'S AND COENZYME NUCLEOTIDES
K E N N E T H D. 3,IUNKRES AND D O W O. W O O D W A R D
Department of Biological Sciences, Stanford U~dversity, StaJTford, Calif. (U.S.A.)
(Received February i4th, 1966)
(Revised manuscript received August 8th, 1966)
SUMMARY
Neurospora mitochondrial structural protein associates with Neurospora malate
dehydrogenase (EC 1.1.1.37) as evidenced by fluorimetric titration, enzyme inhibition,
immunodiffusion-precipitin reaction, and solubility tests. Association of the structural
protein with horse-heart myoglobin is revealed by sedimentation and immunodiffusion-precipitin tests. Evidence of association specificity of the structural protein
for other proteins is presented. Fluorimetric titration data indicate that I mole of
either of the coenzyme nucleotides, ATP or NADH, is bound per mole of structural
protein.
INTRODUCTION
Systematic investigation of the genetics of enzymes and proteins associated
with mitochondria may lend insight into not only the structure and function of this
organelle, but may also ultimately elucidate the nature of mitochondrial heredity
and protein synthesis 1,2. Some enzymes, such as malate dehydrogenase and aspartate
aminotransferasel, a-7, aconitase s, and cytochrome c (ref. 9), although localized exclusively or to a large degree in mitochondria, are encoded in primary structure by
nuclear DNA. Moreover, experiments suggest l°,n that at least malate dehydrogenase
and cytochrome c are synthesized elsewhere in the cell and later assembled, by
processes not well understood 1~, to form the complete mitochondrion. Once assembled,
tile mitochondrion undergoes autonomous replication during exponential growth13, H
and hereditary continuity is maintained by the mitochondrial DNA 15. This DNA
apparently encodes for the primary structure of at least one protein 2, a structural
protein, which comprises about one-third of the total mitochondrial protein~, 22.
Previous investigations of the interaction of Neurospora malate dehydrogenase
(EC 1.1.1.37 ) and mitochondrial structural protein indicated the importance of the
genetically determined structural integrity of both proteins in determining the
function of this enzyme when complexed with mitochondrial structural protein in vitro
and in vivo 2,3. Thus, studies of the interaction of mitochondrial structural protein
with malate dehydrogenase and other mitochondrial enzymes encoded by nuclear
genes may elucidate the molecular genetics of nucleocytoplasmic relationships 2.
Biochim. Biophys. Acla, t33 (1967) t43-15o
144
K. D, MUNKRES, D. O. WOODWARD
In the present paper, some aspects of the interaction of Neurospora mitochondrial structural protein with malate dehydrogenase, other proteins, and coenzyme
nucleotides are described.
MATERIALS AND METHODS
Chemicals
Special commercial chemicals used were: bovine serum albumin (Armour);
oxaloacetic acid, lysozyme, and yeast alcohol dehydrogenase (Calbiochem); EDTA
(Eastman Kodak Co.) ; sucrose, enzyme grade (Mann Res. Lab.) ; horse-heart myoglobin, cryst. (Nutritional Biochemical Corp.); NADH, ATP, andTris (Sigma Chem. Co.).
Neurospora malate dehydrogenase was purified and assayed as previously
described 16. Neurospora mitochondrial structural protein of wild-type and mutants
was prepared by a modification 2 of the method of CRIDDLE et al. 22. Both malate
dehydrogenase and mitochondrial structural protein were exhaustively dialyzed
against distilled water, lyophilized, and stored at room temperature in a desiccator
with CaC12. Samples of beef and yeast mitochondria were gifts from R. S. CRIDDLE.
Protein and coenzvme concentrations
Protein was determined by dry weight and by a microbiuret method 16. The
molar extinction coefficient of 6.22. IOa at 34 ° m~ was employed to determine N A D H
concentration 17. ATP concentrations were determined at p H 2 and 257 m~ with the
molar extinction coefficient, 14. 7. lO s (Pabst Laboratories, Circular OR-7).
Fluorimetric titrations
Fluorescence polarization was determined with a Farrand recording spectrofluorometer equipped with quartz polarizing filters in the activating monochromatic
beam and H N P ' B Polaroid film in the emitted beam is. Apparent polarization was
corrected for monochromator grating polarization is. Fluorimetric titration data were
corrected for volumetric changes and the intrinsic fluorescence of the titrant. With
NADH, the procedure described by WINER, SCHWERT AND MILLAR19 was employed.
Immunochemical analyses
Rabbit anti-Neurospora mitochondrial structural protein serum was prepared
by Antibodies, Inc. (Davis, Calif.). Globulin was purified and concentrated 5-fold by
precipitation with (NH4)2SO4 (50 % satn.) followed by exhaustive dialysis against
50 mM Tris-HC1 buffer (pH 8.0) at 4 °. Double diffusion-precipitin tests were performed by the method of OUCHTERLONY20.
Sedimentation
Sucrose density-gradient centrifugation was performed with a Spinco Model L-2
centrifuge by the method of MARTIN AND AMES~1.
RESULTS
Interaction of mitochondrial structural protein with malate dehydrogenase
In the presence of a large molar excess of structural protein, malate dehydrogenase m a y be completely inhibited; with half-maximal inhibition occurring near
Biochim. Biophys. Acta, i33 (I967) 143 15o
145
MITOCHONDRIAL STRUCTURAL PROTEIN INTERACTIONS
o. 9/~M (Fig. I). The integrity of the structural protein is apparently essential in the
inhibition reaction since structural proteins of 2 mutants with single amino acid
replacements yield significantly greater inhibition constants 2.
When wild-type malate dehydrogenase and structural protein are mixed in
equimolar amounts at pH 8, neither inhibition nor alteration of the Michaelis constant
for malate are found, but genetic amino acid replacement in either malate dehydrogenase or structural protein (or both) leads, in some instances, to marked alteration
of the Michaelis constant of the enzyme in the equimolar complex 3.
2.0
f._-----.-----9
g
t.5
/
g
~) 0 5 I
m
~c
z
w
1.0
om o
g
O 0.5
0~
3
L
S
Zw 0 2
g
0.1[
_a
du_
0
0
i
,
I
2
MSP
(/~M]
I
3
0 o
.
.
.
.
i
O.5
MOLE
MSP/MOLE
,
,
1.0
MDH
b'ig. 1. h l h i b i t i o n of N e u r o s p o r a m a l a t e d e h y d r o g e n a s e (MDH) b y N e u r o s p o r a m i t o c h o n d r i a l
s t r u c t u r a l p r o t e i n (MSP). M D H (o. 1 2 jiM) vvas i n c u b a t e d with 3{SP in 5 ° m M p o t a s s i u m p h o s p h a t e
buffer (pH 7.4) a t 25 ° for IO min. A o.o5-ml a l i q u o t of t h e m i x t u r e was diluted to I.O ml for
a s s a y at 35 ° in a reaction m i x t u r e c o n t a i n i n g : p o t a s s i u m p h o s p h a t e (pH 7.4), 5 ° m M ; N A D H ,
o.i m M ; a n d oxaloacetate, o.65 mM. T h e specific a c t i v i t y of M D H was 355 ° / , m o l e s N A D H
oxidized rain 1. (mg protein) 1. At h a l f - m a x i m a l inhibition (/,2~ = o.9 IBM), t h e m o l a r ratio of
M S P to M D H is 15o:1.
Fig. 2. Fluorescence polarization t i t r a t i o n of N e u r o s p o r a m a l a t e d e h y d r o g e n a s e (MDH) w i t h
N e u r o s p o r a m i t o c h o n d r i a l s t r u c t u r a l p r o t e i n (MSP). M D H (2.7 I,M) in a solution c o n t a i n i n g :
sucrose, o.44 M; E D T A , i raM; p o t a s s i u m p h o s p h a t e buffer (pH 8.2), xo m M was t i t r a t e d with
lO-/,1 aliquots of m i t o c h o n d r i a l s t r u c t u r a l protein (75 t*M) dissolved in t h e s a m e solution. T h e
fluorescence a c t i v a t i o n a n d emission w a v e l e n g t h s were 28o a n d 338 m,u, respectively. Slit width,
5 m/z- A m n l e t c r p o t e n t i o m e t e r , o.i x . T e m p . , 23-25 °.
Additional evidence for interaction of malate dehydrogenase and structural
protein is obtained by fluorescence polarization titration (Fig. 2). Approx. I mole of
structural protein associates with I mole of malate dehydrogenase. The sigmoidal
titration curve implies that several processes may be involved in the association.
Since mitochondrial structural protein tends to form large molecular weight aggregates at the pH of these experiments 2, conceivably deaggregation of this protein
accompanies the association with malate dehydrogenase. This interpretation is supported by the results of titration of structural protein with NADH discussed below.
Moreover, by a simple solubility test, structural protein is visibly deaggregated at
pH 6 by the addition of malate dehydrogenase. At this pH, in o.I M acetate buffer,
the structural protein exists as a flocculent precipitate that is readily solubilized by
the addition of malate dehydrogenase in near equimolar concentrations.
Biochim. Biophys. Acta, 133 (1967) 143-15o
146
K. D. MUNKRES, D. O. WOODWARD
Interaction of malate dehydrogenase and structural protein is also detected in
diffusion-precipitin tests against rabbit anti-Neurospora mitochondrial structural
protein globulin (Fig. 3A). In these tests, structural protein alone forms only a faint
diffuse precipitin band near the center well, but in mixture with malate dehydrogenase
a dense and slower diffusing band is found.
Fig. 3. Tracings of OUCHTERLONY diffusion precipitin tests against rabbit anti Neurospora mito
chondrial structural protein globulin. A. The center well contains antiscrmn. The outer wells
contain as follows: (I) structural protein; (2) structural p r o t e i n - - M b ; (3) structural protein
+ malate dehydrogenasc; (4) structural protein -- N A D H ; (5) structural protein + ATP. B. The
center well contains antiserum. The outer wells contain as follows: (i) yeast initochondria (sonicated) ; (2) yeast alcohol dehydrogenase; (3) beef-heart mitochondria (sonicated) ; (4) bovine serum
albumin; (5) lysozyme. Both Plates A and B contain 2 °'o agar buffered with o.ot M T r i s - H C l
(pH 8.o). Protein solutions were 5 mg/ml. ATP and N A D H concentrations were a b o u t 5 mg;ml.
()primal density of prccipitin lines appeared after 3 4 (lays at room temperature.
Dzteraction of Neuro@ora mitochondrial structural protein with horse-heart n~voglobin
Mitochondrial structural protein of beef heart characteristically forms stoichiometric complexes with heine proteins such as myoglobin or the cytochromes 22-24.
Several types of experiments yield evidence that Neurospora mitochondrial structural
protein also associates with myoglobin. An equal weight mixture of structural protein
and myoglobin in a sucrose density gradient at pH 8 formed a high molecular weight
aggregate that sedimented completely to the bottom of the tube (Fig. 4)- Structural
protein alone, on the contrary, sedimented as a single peak with a sedimentation
constant of 2.2. Myoglobin alone, although evidently existing in several states of
aggregation, did not sediment to the bottom of the tube.
Interaction of Neurospora mitochondrial structural protein with myoglobin is
also detected by diffusion-precipitin tests against rabbit anti-Neurospora mitochondrial structural protein globulin (Fig. 3A). In these tests, structural protein alone
formed only a faint diffuse precipitin band near the center well, but in mixture with
myoglobin, a sharp and slower diffusing precipitin line is observed.
In disc electrophoresis, an excess of structural protein added to myoglobin
complexes all of the myoglobin, resulting in the disappearance of the myoglobin bands
and the appearance of a new band which presumably represents the mitochondrial
13 iocl, im. Biophys. ~4cla, 133 (I967) I43 I5o
MITOCHONDRIAL STRUCTURAL PROTEIN INTERACTIONS
147
structural protein-myoglobin complex. Structural protein alone in disc electrophoresis appears as a broad diffuse band.
V°'~t~
c,l OH0
/rnyoglobin
~.
u 0.08
2:
0.06
o0o
yoglobin
0.02
o
-
2
~
6
i
I0
FRACTION
14
18
22
.
26
NUMBER
Fig. 4" Sucrose density gradient ceutrifugation (23 h at 38ooa rev./min) of N e u r o s p o r a mitochondriaI structural protein (MSP) and horse heart myoglobin, i n a 5 2o% sucrose gradient
buffered at p H 8.o with o.o z M Tris-HC1, myoglobin sediments in several peaks due to aggregation.
MSP sediments primarily as a single peak. The midpoint of this peak is calculated to be 2.2 S.
The m i x t u r e of s t r u c t u r a l protein + Mb is characterized by the disappearance of b o t h peaks due
to the heavier structural p r o t e i n - M b complex sedimenting to the b o t t o m of the tube and hence
n o t recovered.
Specificity of protein-protein interaction
The observation that single amino acid replacements in either structural protein
or malate dehydrogenase lead to functional and conformational alteration of malate
dehydrogenase-structural protein complexes indicates a high degree of specificity of
interaction 3. This conclusion is supported by the results of diffusion-precipitin tests
of mixtures of wild-type structural protein with yeast alcohol dehydrogenase, bovine
serum albumin, or lysozyme against rabbit anti-Neurospora mitochondrial structural
protein antibody (Fig. 3B). No trace of complex of these proteins with structural
protein is evident in these experiments. Similarly, extensive tests by CRIDDLE e! el. 22
for the association of beef-heart mitochondrial structural protein with various enzymes
and proteins also indicated association specificity.
The association of Neurospora mitochondrial structural protein with horse-heart
myoglobin probably reflects the specificity of structural proteins for heme proteins,
as well as a similarity in structure and function of Neurospora and mammalian
mitochondrial structural protein (see below).
Antigenic homology of mitochondrial structural protein of various genera
Previous analyses of the amino acid_ compositions of mitochondrial structural
protein of Neurospora yeast and beef heart indicated a marked similarity in amino
Biochim. Biophys. Acre, 133 (I967) I43-1.5o
i48
K. I). IVIUNKRES, D. O. WOODWARD
acid composition S. Similarity of these proteins is also indicated by cross-reaction with
rabbit anti-Neurospora mitochondrial structural protein globulin (Fig. 3B).
Association of Neurospora mitochondrial structural protein with N A D H and A TP
Fluorimetric polarization titration of structural protein with NADH indicates
that about I mole of nucleotide is bound per mole of protein (Fig. 5). The marked
change in fluorescence polarization of the p r o t e i n - N A D H complex may indicate that
a deaggregation of structural protein accompanies the association reaction, as noted
above in titrations with malate dehydrogenase. Approximate estimates of the dissociation constants of structural p r o t e i n - N A D H complexes yielded (in t,M) : o.6 for
wild-type and 2.1 and 16 for mutants mi-I and mi-3, respectively.
61 /..:.;
0.5
,4g
o
¢n
4
//
,/
g
o
0.2°
J
~
a
¢
<3
o
O.I
O
NADH/MOLE
I
O0
I
MOLE
j
MSP
./
/
/
I
MOLE
2
ATP/MOLE
MSP
Fig. 5. Fluorimetric titration of Neurospora mitochondrial s t r u c t u r a l protein (MSP) with
Protein (IO #M) in o.25 ml of 5 ° mM potassium p h o s p h a t e (pH 7-4) was titrated with 5-/.1
of a solution of N A D H in the same buffer. Activation and emission wavelengths were
44o m/*, respectively. Slit width, 3 ° m/*. A m m e t e r potentiometer, o.I × . Temp., 23-25 °.
fluorescence e n h a n c e m e n t ; O - - O, polarization.
NADH.
aliquots
34 ° and
O--O,
Fig. 6. Fluorimetric titration of Neurospora mitochondrial s t r u c t u r a l protein (MSP) with ATP.
Protein (2/*M) in o.25 ml of 5 ° mM p o t a s s i u m p h o s p h a t e (pH 7.4) was titrated with 5 #1 of ATP
(2o #M) in the same buffer. Activation and emission wavelengths were 28o and 34 ° m y , respectively. Slit width, 3 ° m#. A m m e t e r potentiometer, o.oi ×. Temp., 23-25 °.
Fluorescence quenching titration of structural protein with ATP also indicates
that approx. I mole of nucleotide is bound per mole of protein (Fig. 6). For ATP,
the apparent dissociation constants (in t~M) were: o. 4 (wild-type), 1. 4 (mi-I), and
o.6 (mi-3). Thus, mutant structural proteins not only exhibit a lower affinity for
malate dehydrogenase, but also for the coenzyme nucleotides. Qualitative evidence
for interaction of the nucleotides with structural protein is observed in diffusionprecipitin tests (Fig. 3A).
The association of phosphoryl compounds with mitochondrial structural protein
is apparently non-specific and a general property of such proteins. The association of
phosphate, pyrophosphate, various nucleotides ~a, and phospholipid 2~ with beef-heart
mitochondrial structural protein has been reported. Possibly, some of the anomalous
behavior of mitochondrial structural protein (e.g., apparent heterogeneity under
Biochim. Biophys. Acla, 133 (I967) ~43-I5O
MITOCHONDRIAL
STRUCTURAL PROTEIN
INTERACTIONS
149
certain conditions, and differences in solubility properties of different preparations)
can be attributed to binding of the structural protein to various non-protein components that are not completely removed by the purification procedure. This could
result in heterogeneity with respect to phosphoryl compounds, N A D H and other
non-protein components which bind tightly to mitochondrial structural protein.
DISCUSSION
Mitochondria, whether from fungi or animal cells, are composed of an outer
and inner membrane. The 2 membranes of beef-heart mitochondria differ in respect
to the nature of repeating units, enzymic composition, chemical composition and
function27, ". In particular, enzymes of the citric acid cycle, including malate dehydrogenase, are associated with the outer membrane27, ".
The exact three-dimensional arrangement of mitochondrial enzymes with
respect to one another, to the membranes and to structural protein is not yet well
understood. However, the existence of enzymes in structurally and functionally
organized macromolecular assemblies is well-established and aids in understanding
certain genetical and physiological problems. Thus, in the maternally inherited,
respiratory-deficient mutants of Neurospora, genetic amino acid replacement in the
lnitochondrial membrane structural protein 2 leads to a profound alteration in the
function of mitochondria, with a I6-fold accumulation of cytochrome c in a nonparticulate cell fraction 2~, a 2-fold excess of riboflavin 2~, and a I5-fold excess of longchain unsaturated fatty acids 3°. On the basis of these observations and the observed
decrease in affinity of m u t a n t structural protein for malate dehydrogenase and coenzyme nucleotides, it is conceivable that mutation decreases the affinity of mitochondrial structural protein for cytochrome c, riboflavin and phospholipid; hence,
disrupting the process of assembly of the electron-transfer chain.
Previous studies demonstrating locational specificity of Neurospora malate
dehydrogenase indicated that the association of this enzyme with the structural
protein of the mitochondrial membrane is of genetical and physiological importance s.
One m a y speculate that the modulation of the functional properties of malate dehydrogenase, myoglobin 2a, and cytochrome b (ref. 24) by association with mitochondrial structural protein and the mitochondrial membrane i~ vivo m a y be important
in the regulation of metabolic rates. The marked differences in the association constants of the oxidized and reduced forms of cytochrome b (ref. 24) or myoglobin sa
with mitochondrial structural protein is perhaps one aspect of a regulatory mechanism. Indeed, novel and hitherto unsuspected regulatory mechanisms may control
the activity of membrane-associated enzymes.
ACKNOWLEDGEMENTS
This investigation was supported by grants from the U.S. Public Health Service
and the National Science Foundation. The technical assistance of Miss A. PEARSON,
Miss G. HOLLAND, and Miss B. A~<DREWS is gratefully acknowledged.
* ID. ALLMANN, E . BACItMANN AND D . GREEN, p e r s o n a l c o m m u n i c a t i o n .
Biochim. Baophys. Acla, i 3 3 ( i 9 6 7 ) i 4 3 - i 5 3
150
K.D.
MUNKRES,
D. 0 . W O O D W A R D
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Biochim. Biophys. Acta, 133 (I967) 143- 15o