From the University of Cambridge, Department of Biochemistry, Old Addenbrooke's Site, 80 Tennis Court Road, Cambridge CB2 1GA, United Kingdom
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() |
---|
The enzymes of the tricarboxylic acid cycle in
the mitochondrial matrix are proposed to form a multienzyme complex, in
which there is channeling of substrates between enzyme active sites. However no direct evidence has been obtained in vivo for
the involvement of these enzymes in such a complex. We have labeled the
tricarboxylic acid cycle enzyme, citrate synthase 1, in the yeast
Saccharomyces cerevisiae, by biosynthetic incorporation of
5-fluorotryptophan. Comparison of the 19F NMR resonance
intensities from the labeled enzyme in the intact cell and in cell-free
lysates indicated that the enzyme is motionally restricted in
vivo, consistent with its participation in a multienzyme complex.
The protein concentration in the mitochondrial matrix is thought
to be very high, between 270 and 560 mg/ml depending on the functional
state of the organelle (1, 2). This very high protein concentration has
been proposed to restrict the diffusion of enzymes and metabolites and
to promote the association of matrix enzymes. This has led to the
suggestion that the reactions of the tricarboxylic acid cycle may occur
via channeling of metabolites between enzyme active sites in a
multienzyme complex (3-6) or metabolon (7).
There is a large body of data that supports the idea of a tricarboxylic
acid cycle metabolon. Specific associations have been demonstrated
in vitro between tricarboxylic acid cycle enzymes and also
between these enzymes and components of the respiratory chain and other
proteins in the inner mitochondrial membrane (3). Complexes able to
catalyze several consecutive steps of the tricarboxylic acid cycle have
been isolated following gentle disruption of both liver mitochondria
(8, 9) and a number of different microorganisms (10, 11). Coupled
reactions within complexes of tricarboxylic acid cycle enzymes have
been shown to have a kinetic advantage when compared with the
completely solubilized systems (3, 9). Molecular modeling studies on a
citrate synthase-malate dehydrogenase fusion protein, for which there
was experimental evidence of substrate channeling (12), showed that
there could be a very efficient, electrostatically based, channeling
mechanism for substrate transfer between the enzymes active sites (13).
Evidence has also been obtained in vivo for a tricarboxylic
acid cycle enzyme complex. Disruptions of the genes for citrate
synthase and malate dehydrogenase in yeast resulted in cells that were
unable to grow on acetate (14). This was despite the fact that there
are isozymes in the cytosol which could, in principle, bypass the
resulting blocks in the cycle. In the case of citrate synthase,
introduction of a structurally similar but catalytically inactive
mutant resulted in restoration of tricarboxylic acid cycle function and
growth on acetate (15). These studies have been interpreted as
indicating the presence of a complex of tricarboxylic acid cycle
enzymes in which the enzymes have structural as well as catalytic
roles. There is also evidence for substrate channeling in
vivo. Analysis of 13C labeling patterns in metabolites
derived from tricarboxylic acid cycle intermediates indicated
channeling of succinate and fumarate in the cycle (16, 17).
Demonstration of these weak enzyme complexes in situ has
been difficult, however, because many of them are dissociated during isolation due to dilution effects. There is also no direct evidence for
their presence in an intact cell. We show here, using NMR measurements
on a minimally derivatized enzyme of the tricarboxylic acid cycle, that
it exists in a motionally restricted form in the yeast mitochondrial
matrix in vivo, consistent with its participation in a
multienzyme complex.
Materials--
Saccharomyces cerevisiae strain BJ2168
(a, gal2, ura3-52, leu2-3, leu2-112, trp1=, pep4-3,
prb1-1122, prc1-407) was used in these studies (18). Growth media
were obtained from Difco. Oligonucleotide linkers were supplied by New
England Biolabs. All other reagents were obtained from Sigma or from
Boehringer Mannheim. Protein concentrations were determined with a dye
binding assay (19) kit from Bio-Rad, with bovine serum albumin as a standard.
Plasmid Construction--
The coding sequence for yeast
mitochondrial citrate synthase 1 (CIT1) (20) was cloned into
the Ecl136II site of the plasmid pYES2.0 (Invitrogen Corp.)
to generate the plasmid pYESYCS1. This plasmid was linearized by
digestion with KpnI, the resultant cohesive termini removed
with T4 DNA polymerase and the blunt-ended molecule religated with
BamHI linkers. This plasmid was digested with
BamHI and the 1.5-kilobase pair fragment containing the
CIT1 coding sequence was ligated into the unique
BglII expression site of the LEU2-expressing plasmid pKV49
(21). These manipulations generated the plasmid pBF208, which expressed
yeast mitochondrial citrate synthase 1 (CIT1, EC 4.1.3.7) under the
control of a galactose-inducible version of the yeast phosphoglycerate
kinase promoter.
Enzyme Labeling--
Cells were transformed with the plasmid
pBF208 by the method of Hinnen et al. (22). CIT1 was
fluorine-labeled by inducing enzyme expression in stationary phase
cells in the presence of 5-fluoro-DL-tryptophan
(5-FTrp).1 The labeling
protocol employed was similar to that used previously to label
phosphoglycerate kinase (23). Briefly, 2.5 × 109
cells were used to inoculate a 500-ml culture containing 2% glucose, 2% bactopeptone, and 1% yeast extract. This culture was grown for
24 h, by which time the cells were in stationary phase at a
density of approximately 2 × 108 cells/ml. The cells
were washed and resuspended in 500 ml of medium containing 2%
galactose, 0.67% yeast nitrogen base, and a mixture of amino acids
lacking tryptophan and leucine. After 2 h, 50 ml of a 0.2%
solution of 5-FTryp was added and the culture incubated for a further
24 h prior to cell harvesting.
Enzyme Assay--
Cells were disrupted by vigorous agitation in
extraction buffer (50 mM sodium phosphate, 5 mM
EDTA, 1% Triton X-100, pH 7.0). Citrate synthase activity was assayed
as described in (24). Enzyme activities are expressed per milliliter of
cell water, assuming that 1.67 g of cells contain 1 ml of cell
water (25). All means are quoted with their S.E.
Cell Immobilization and Perifusion--
Cells were immobilized
and perifused as described previously (26). The cells (6 g wet weight
with 6 ml of 1.8% agarose) were perifused with an oxygenated buffer
that had the same composition as that used for protein labeling, except
that it was supplemented with 0.002% tryptophan instead of 5-FTrp.
Protein Purification--
Mitochondria were isolated as
described in Ref. 27. All procedures were performed at 4 °C.
Isolated mitochondria were disrupted by sonication, after the addition
of Triton X-100 to 0.01% (v/v), and the debris and unlysed
mitochondria removed by centrifugation (10 min, 15,000 × g). The extract was brought to 85% saturation with
(NH4)2SO4 and stirred for 30 min
before centrifugation for 30 min at 27,000 × g. The
resulting pellet was dissolved in a buffer containing 1.7 M
(NH4)2SO4, 50 mM
Tris-HCl, 5 mM EDTA, and 2 mM dithiothreitol,
pH 8.0, and applied to a column of octyl-Sepharose (Amersham Pharmacia
Biotech) pre-equilibrated with the same buffer. Citrate synthase was
eluted from the column with a linear
(NH4)2SO4 gradient ranging from 1.7 to 0 M. Fraction purity was assessed by SDS-polyacrylamide
gel electrophoresis. Fractions that contained essentially pure CIT1
were pooled and concentrated using an Amicon stirred ultrafiltration
cell (YM30 membrane) until the enzyme reached a concentration of
greater than 1 mg/ml. CIT1 was stored at 4 °C as an
(NH4)2SO4 precipitate.
For NMR measurements the purified enzyme was desalted by gel
filtration, using NAP-5 columns (Amersham Pharmacia Biotech), into NMR
buffer (50 mM HEPES, 130 mM potassium acetate,
and 2 mM dithiothreitol, pH 7.2) and then concentrated
using Amicon centricon 10 microconcentrators. Samples contained 10%
v/v 2H2O for a field frequency lock. pH
measurements were not corrected for any deuterium isotope effect.
Sucrose was added to give solutions of higher viscosity where required,
and the final protein concentration was typically 15-25 mg/ml.
NMR Measurements--
NMR experiments were performed at a
19F resonance frequency of 376.29 MHz, as described
previously (26). Fluorine-19 chemical shifts are quoted relative to
p-fluorophenylalanine standards, either a 100 µM internal standard in solutions of the purified enzyme
or an external standard contained in a coaxial capillary with cell and
lysate preparations. Longitudinal relaxation time (T1) measurements on the purified protein were
performed using an inversion recovery sequence. Peak integrals in these
experiments were fit iteratively to a three parameter single
exponential function.
Viscosity measurements on solutions of the purified enzyme were made on
the same samples as used for the 19F NMR experiments. The
diffusion coefficient of water in the samples was measured from proton
spectra by pulsed-gradient spin echo techniques (28). The diffusion
coefficient was taken to be inversely and linearly proportional to the
viscosity of the solution (29, 30).
The NMR visibility of the labeled protein in the cell was assessed by
comparing its signal intensity in the cell with that in diluted cell
extracts. Extracts were prepared by disrupting 6 g of cells by
vigorous agitation with glass beads in chilled extraction buffer. The
lysates were then dialyzed against extraction buffer without Triton
X-100 and their volumes adjusted to 27 ml, which was the sample volume
used in cellular perifusions. Enzyme activity was assayed at this point
in order to determine the degree of enzyme extraction. Typically the
amount of enzyme in a dialyzed lysate was between 60 and 80% of the
amount of enzyme in the original cell preparation. Where we have quoted
concentrations of fluorine label in lysates, these have been corrected
for loss of enzyme during the extraction procedure. Therefore the
enzyme was diluted by a factor of 7.5 or more, depending on the degree
of cell extraction, compared with its concentration in the cell.
The equations used to calculate the theoretical
T1 values and line widths for CIT1 have been
described previously (26). The relaxation times were calculated
assuming that the protein tumbles as a sphere and that the label is
held rigidly within the molecule. The structure of pig citrate synthase
has been used previously to model the structure of CIT1 (13, 31). Using this approach we calculated the hydrated radius of CIT1 to be 33.3 Å and the rotational correlation time to be 29.5 ns in a solution of
aqueous viscosity.
Protein Labeling--
A mitochondrial isoform of citrate synthase
(CIT1) was selectively fluorine-labeled in vivo by inducing
its synthesis, in stationary phase cells, in the presence of 5-FTrp.
The addition of labeled or unlabeled tryptophan, following galactose
induction of enzyme expression, resulted in an approximately 10-fold
increase in the activity of the enzyme. The increase was from 163 ± 7 units/ml cell water (n = 9) to 1630 ± 60 units/ml cell water (n = 10) in the presence of 5-FTryp
and from 170 ± 10 units/ml cell water (n = 6) to
2000 ± 100 units/ml cell water (n = 3) in the
presence of unlabeled tryptophan. In the absence of additional
tryptophan there was only an approximately 1.5-fold increase in enzyme
activity to 266 ± 15 units/ml cell water (n = 9).
In the presence of 5-FTryp the enzyme concentration after induction was
166 ± 6 µM, assuming a specific activity for CIT1
of 100 units/mg and a molecular mass for the dimer of 98 kDa (32). The
comparable increase in enzyme activity that occurred following
induction of expression in the presence of 5-FTrp or unlabeled
tryptophan indicated that the specific activity of the labeled enzyme
was not significantly different from that of the unlabeled form. A
similar observation has been made previously for the glycolytic enzymes
phosphoglycerate kinase, hexokinase, and pyruvate kinase labeled with
5-FTrp (26).
S. cerevisiae contains three isoforms of citrate synthase,
CIT1 (20) and CIT3 (33) are mitochondrial, whereas CIT2 is peroxisomal
(34). Labeled CIT1 was localized predominantly in the mitochondria as
mitochondria prepared from the induced cells contained 91.1 ± 0.6% (n = 6) of the total citrate synthase activity in
the cell. The protein labeling procedure had little effect on cell
viability. The number of cells that grew on agar plates at the end of
the procedure was 81 ± 2% of those that grew prior to the start
of the procedure (n = 4). The labeling procedure increases the enzyme concentration by a factor of 10. This level of
overexpression of CIT1, however, has been shown to have no significant
effect on mitochondrial function (35).
19F NMR Measurements on the Purified Enzyme--
Yeast
CIT1 is a homodimer of molecular mass 98 kDa and has six tryptophans
per subunit (20, 32). Four of the six 5-FTrp resonances were resolved
in the spectrum of the purified protein (Fig.
1). The longitudinal relaxation time
constant (T1) of the well resolved downfield
resonance (resonance 1, Fig. 1), at near aqueous viscosity,
was 1.1 ± 0.2 s, in agreement with the value expected from
theory of 1.0 s. The T1 of the envelope of
resonances (Fig. 1, resonances 2-6) was also near to 1 s, at 1.1 ± 0.1 s. These data indicate that the tryptophans
are relatively immobile and have a correlation time similar to that of
the whole protein. The location of the tryptophans within the protein
structure is consistent with this apparent lack of mobility. Mapping of
the CIT1 sequence onto the structure of the bovine heart and chicken heart enzymes and investigation of the solvent accessibility of the
tryptophan residues, with the programs MODELLER and NACCESS (36, 37),
indicate that all are buried or constrained within the protein
structure. The T1 of the envelope of resonances
increased with solvent viscosity (Fig.
2), and this increase showed reasonable agreement with theoretically calculated values (26). The line widths
also showed good agreement with theory. The line width of resonance 1 (Fig. 1) was measured at 60 Hz in a solution of near-aqueous viscosity,
and the calculated value was 52 Hz.
19F NMR Measurements in Vivo--
Spectra were
obtained from cells that had been immobilized in agarose gel threads
and maintained in a metabolic steady state, during NMR data
acquisition, by perifusion with oxygenated medium (Fig.
3). The metabolic status of the cells was
confirmed by 31P NMR spectra (38) acquired before and after
the 19F NMR experiments. There was no significant loss of
citrate synthase activity in the cells during this period. The
19F NMR data were acquired under fully relaxed conditions,
assuming that the T1 values of the CIT1
resonances were similar to those of the purified enzyme in a solution
of near-aqueous viscosity i.e. approximately 1 s.
Unresolved 19F resonances were observed in cells that had
been induced to express CIT1 in the presence of 5-FTrp (Fig.
3C). However similar resonances were also observed in
control cells, which had been transformed with the empty vector, pKV49,
and taken through the same protein labeling protocol (Fig.
3A). The intensities of these unresolved resonances in the
two sets of cells were similar. The concentration of detectable
fluorine in the cells overexpressing citrate synthase was 300 ± 30 µM (mean ± S.E., n = 3) as
compared with 180 ± 20 µM (mean ± S.E.,
n = 2) in the control cells. The concentration of
detectable fluorine in dialyzed lysates prepared from the control
cells, when expressed on a cell water basis, was 285 ± 45 µM (mean ± S.E., n = 3) (Fig.
3B), showing that these resonances were due predominantly to
nonspecific incorporation of 5-FTrp into cell proteins and that these
proteins were fully visible in the 19F spectra of the
cells. However spectra of dialyzed lysates prepared from cells
overexpressing CIT1 (Fig. 3D) were very similar to those
obtained from the purified enzyme (Fig. 1), in particular the resolved
downfield resonance (resonance 1, Fig. 1), which was never
observed in spectra of cells, was clearly visible. Furthermore the
concentration of detectable fluorine in these lysates, expressed on a
cell water basis, was much higher at 850 ± 110 µM
(mean ± S.E., n = 3). Thus the labeled enzyme,
which was undetectable in 19F spectra of the cells, was
visible in the diluted extracts prepared from these same cells.
There is a considerable body of evidence for the organization of
the enzymes of the tricarboxylic acid cycle in a multienzyme complex,
in which there is channeling of cycle intermediates (reviewed in (3,
4)). The concept of a tricarboxylic acid cycle metabolon, however, has
remained controversial as it has been difficult to isolate an intact
complex from the cell. The relatively weak interactions between the
enzymes, which are favored in the cell by the very high protein
concentrations in the mitochondrial matrix (39, 40), are disrupted by
the dilution that occurs during cell extraction.
In this study we have investigated the rotational mobility of a
tricarboxylic acid cycle enzyme, CIT1, in the matrix of yeast mitochondria in vivo using 19F NMR measurements
on a fluorine-labeled enzyme. We have used this technique previously to
measure the rotational correlation times of three glycolytic enzymes in
yeast. Phosphoglycerate kinase and hexokinase were found to be tumbling
in a cytoplasm with a viscosity approximately twice that of water (26),
in good agreement with fluorescence measurements of cytoplasmic
viscosity in mammalian cells (see for example (41, 42)). Pyruvate
kinase, however, yielded no detectable NMR signals in vivo,
indicating that there was some degree of motional restriction of this
enzyme in the cell. This was thought to be due to binding of the enzyme
to other cellular macromolecules.
CIT1, like pyruvate kinase, showed no detectable fluorine resonances in
the intact cell, although it was readily detectable in diluted cell
extracts. The 19F signals that were observed in the cell
could be assigned to nonspecific labeling of cell proteins (see Fig.
3). There are several possible explanations for this lack of NMR
visibility of CIT1 in the cell.
NMR and fluorescence measurements have indicated a mitochondrial matrix
viscosity of between 25 and 37 times that of water (43, 44). Such a
high viscosity would lead to substantial broadening of the
19F resonances of labeled CIT1 and an increase in their
T1 relaxation times, resulting in signal
saturation under the NMR acquisition conditions employed. For example
at a viscosity of 25 centipoise (centipoise = 10 The lack of NMR visibility of labeled CIT1 in vivo could be
due to broadening of its resonances by paramagnetic ions present in the
mitochondrial matrix. This, however, seems unlikely as the tryptophan
residues are at least partially buried within the protein and should
thus be inaccessible to paramagnetic ions. Furthermore there is no
evidence for paramagnetic ions significantly affecting the relaxation
rates of resonances from intramitochondrial metabolites, including the
31P resonances of ATP (46-49) and inorganic phosphate (48)
and the 1H resonance of water (44).
The most likely explanation, therefore, for the NMR invisibility of
CIT1 is that its 19F resonances are broadened by an
increase in its correlation time, due to binding to other matrix
proteins. A complex containing five tricarboxylic acid cycle enzymes
has been isolated (8-11). Assuming that there is one molecule of each
enzyme in the complex then this would have a molecular mass of
approximately 600 kDa. If the complex behaves as a hard sphere and CIT1
has the same correlation time as the whole complex, then the line width
of the fluorine resonances of CIT1 would be increased to 210 Hz. Even
with this degree of line broadening the protein could still be
detectable in the cell. However since we have overexpressed the enzyme
by a factor of 10, it is unlikely that much of the labeled enzyme could
participate in such a stoichiometric complex. The enzyme has also been
shown to bind, with other tricarboxylic acid cycle enzymes, to the
mitochondrial membrane (3, 4, 14). This would lead to a much larger
increase in the enzyme's correlation time and thus the 19F
resonance line widths. Under these circumstances the NMR signals would
be broadened beyond detection and therefore membrane binding is a much
better candidate as an explanation for the invisibility of CIT1.
Immobilization of a GFP-tagged matrix enzyme was observed recently by
Partikian et al. (45). By contrast, fluorescence recovery
after photobleaching measurements on free GFP showed that its diffusion
was relatively rapid, being only three to four times slower than in
water (45). In order to explain the rapid diffusion of GFP it was
proposed that the matrix proteins are organized peripherally in
membrane-associated complexes, thus creating a central aqueous region
with relatively low protein density and low viscosity. Such a domain
would allow the rapid and unrestricted diffusion of solutes. This model
is consistent with the data presented here demonstrating immobilization
of CIT1 in vivo and with previous studies showing that it
binds with other tricarboxylic acid cycle enzymes to the mitochondrial membrane.
INTRODUCTION
Top
Abstract
Introduction
References
EXPERIMENTAL PROCEDURES
RESULTS
View larger version (9K):
[in a new window]
Fig. 1.
19F NMR spectrum of purified
5-fluorotryptophan-labeled CIT1. The peak numbering is used for
reference in the text. The spectrum was acquired with a spectral window
of 12 kHz into 16,000 data points. The pulse flip angle was 90° and
the interpulse delay 8 s. An exponential line broadening of 30 Hz
was applied.
View larger version (12K):
[in a new window]
Fig. 2.
Dependence of the
T1 values of the 5-fluorotryptophan
resonances from CIT1 on the viscosity of the medium. The symbols
show experimentally measured T1 values for the
envelope of 5-fluorotryptophan resonances from the labeled enzyme in
media of specified viscosity. The T1 values of
the resonances are expected to be linearly dependent upon the viscosity
of the medium, and therefore the solid line through these
points was obtained by linear regression. The dashed line
shows the expected variation of T1 with
viscosity based upon theory (26).
View larger version (8K):
[in a new window]
Fig. 3.
19F NMR spectra of
5-fluorotryptophan-labeled CIT1 in intact cells and in cell
lysates. Spectra from control cells (A) and
CIT1-expressing cells (C) and from lysates of control cells
(B) and CIT1-expressing cells (D). Spectra were
acquired with a spectral window of 12 kHz into 16,000 data points. The
pulse flip angle was 90° and the interpulse delay 6.7 s.
Spectra A and B are the sum of 10,000 transients, and spectra C and D are the sum of
4000 transients. An exponential line broadening of 60 Hz was
applied.
DISCUSSION
2
poise), the calculated line width of the 19F resonances
would be 1200 Hz and the T1 22 s. With this
T1 the signals detected in the cell would be
substantially saturated, and their intensities would be only 26% of
the fully relaxed value. However recent time resolved fluorescence
measurements on mitochondrially targeted GFP (45) showed the matrix
viscosity to be close to that of an aqueous solution. The high apparent
viscosity estimated from the earlier fluorescence anisotropy
measurements (43) was shown to be explicable by probe binding.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Paul Srere for the CIT1 purification protocol and for his encouragement, Balazs Sumegi for the plasmid pYESYCS1+, and Dr. Simon Williams and Dr. William Broadhurst for their assistance during this work. We are also grateful to the BBSRC for provision of NMR facilities.
![]() |
FOOTNOTES |
---|
* This work was supported by the Wellcome Trust.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Supported by a Studentship from the Biotechnology and Biological
Sciences Research Council.
§ To whom correspondence should be addressed: University of Cambridge, Dept. of Biochemistry, Old Addenbrooke's Site, 80 Tennis Court Rd., Cambridge CB2 1GA, UK. E-mail: k.m.brindle{at}bioc.cam.ac.uk.
The abbreviations used are: 5-FTrp, 5-fluoro-DL-tryptophan; CIT1, yeast mitochondrial citrate synthase 1; CIT2, yeast peroxisomal citrate synthase 2; CIT3, yeast mitochondrial citrate synthase 3; GFP, green fluorescent protein.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() |
---|