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Department of Biochemistry and Molecular Biology, Oregon Graduate Institute of Science and Technology, Beaverton, OR 97006-8921
3To whom correspondence should be addressed.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONThe role of the...Cloning and expression of...Structure-function studiesREFERENCES |
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KEY WORDS: • carnitine palmitoyltransferases • Pichia pastoris expression • malonyl CoA inhibition • binding residues
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONThe role of the...Cloning and expression of...Structure-function studiesREFERENCES |
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,
McGarry et al. 1989
). CPTI is an integral membrane
enzyme located on the outer mitochondrial membrane, and CPTII is a
membrane-associated enzyme that is loosely bound to the matrix side
of the inner mitochondrial membrane (Fig. 1
). A current model for the membrane topology of CPTI predicts exposure
of the CPTI N- and C-terminal domains crucial for activity and
malonyl CoA sensitivity on the cytosolic side of the;F2> outer
mitochondrial membrane (Fig. 2
) (Fraser et al. 1997
).
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, resulting in increased fatty acid oxidation (Brandt et al. 1998
, Mascaro et al. 1998,
Yu et al. 1998a
). Long-chain fatty acids are thus regulators of their
own metabolism. Understanding the molecular mechanism of the regulation
of the CPT system by malonyl CoA and long-chain fatty acids is
crucial in the design of drugs for control of excessive fatty acid
oxidation in diabetes mellitus (Prentki and Corkey 1996
), in myocardial ischemia in which accumulation of
acylcarnitines has been associated with arrhythmias (Corr and Yamada 1995
) and in human inherited CPT deficiency diseases
(Bonnefont et al. 1996
). |
The role of the CPT system in cellular fatty acid metabolism |
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TOPABSTRACTINTRODUCTIONThe role of the...Cloning and expression of...Structure-function studiesREFERENCES |
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, Weis et al. 1994,
Zhu et al. 1997b
). Compared with heart, liver has a much higher
affinity for carnitine but is less sensitive to malonyl CoA inhibition.
Metabolic conditions that increase cellular long-chain fatty acid
oxidation, such as starvation, diabetes and consumption of a high fat
diet, have been reported to increase L-CPTI activity and decrease
its malonyl CoA sensitivity (Bremer 1981
, Cook and Gamble 1987
).
Heart and adult testis express both the L- and M-forms of CPTI, but
only M-CPTI is expressed in skeletal muscle (Adams et al. 1998
, Zhu et al. 1997b
). In addition, two novel
CPTI isoforms generated by alternative splicing of the M-CPTI
transcript have been reported for M-CPTI (Yu et al. 1998b
). It is not yet known if these isoforms, which
are expressed at very low levels in most tissues compared with
M-CPTI, are catalytically active enzymes that play a role in fatty
acid oxidation.
In heart M-CPTI, the Km for carnitine is
~20-fold higher than that of L-CPTI, which is ascribed to the
higher tissue levels of carnitine (Brown et al. 1995
).
Heart M-CPTI is also much more sensitive to malonyl CoA inhibition
than liver, although there is no significant difference in the malonyl
CoA concentration between the two tissues. The activity and malonyl CoA
sensitivity of M-CPTI are also regulated by the fatty acid
composition of the diet (Power and Newsholme 1997
).
The important question of how fatty acid oxidation can proceed in heart
in the presence of high tissue levels of malonyl CoA appears to be
resolved in part by the more recent reports of the transcriptional
regulation of the M-CPTI gene expression by long-chain fatty
acids via the peroxisome proliferator-activated receptor
(PPAR) (Brandt et al. 1998
). High levels of
long-chain fatty acids stimulate their own metabolism by activation
of PPAR, which in turn induces fatty acid oxidation by
transcriptional activation of both the L- and M-CPTI genes. It is
estimated that ~60–80% of the energy requirement of the heart is
derived from fatty acid oxidation (Whitmer et al. 1978
).
In a normal nonlipogenic tissue such as heart, which is dependent
primarily on long-chain fatty acids as an energy source, high rates
of fatty acid oxidation should increase cellular long-chain fatty
acyl-CoA levels. Long-chain fatty acyl-CoA compete for the
malonyl CoA binding site on M-CPTI, effectively making M-CPTI
insensitive to malonyl CoA inhibition.
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Cloning and expression of the cDNAs for the CPT system |
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TOPABSTRACTINTRODUCTIONThe role of the...Cloning and expression of...Structure-function studiesREFERENCES |
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Rat and human L-CPTII cDNAs have been cloned and sequenced
(Finocchiaro et al. 1991
, Woeltje et al. 1990
). The cDNA sequences predicted proteins of 658 amino acid
residues (71 kDa) that had 82 and 85% identity at the amino acid and
nucleotide levels, respectively. The size of the mRNA in different rat
tissues was identical, and CPTII appears to be the product of a
single-copy gene that is expressed uniformly in every tissue
examined to date (McGarry and Brown 1997
). CPTII is a
catalytically active, malonyl CoA-insensitive, distinct enzyme
because a rat liver cDNA encoding CPTII synthesizes an active protein
when expressed in Escherichia coli (Brown et al. 1993
) and in the yeasts Saccharomyces cerevisiae
(Brown et al. 1994a
) and Pichia
pastoris (de Vries et al. 1997
), in baculovirus (Johnson et al. 1995
) or in COS cells (Woeltje et al. 1990
). Detergent-solubilized, yeast-expressed CPTII showed
saturation kinetics when the carnitine concentration was varied
relative to a constant second substrate, palmitoyl CoA. The calculated
Km for carnitine was 105
µmol/L, which is similar to the calculated
Km obtained for the S.
cerevisiae–expressed enzyme. However, the
yeast-expressed CPTII showed abnormal nonsaturation kinetics with
respect to palmitoyl CoA at a fixed albumin concentration of 1%
(wt/v). This was due to the high albumin concentration used to buffer
the detergent effect of palmitoyl CoA. With the S.
cerevisiae–expressed enzyme, mutations of the conserved
residues H372A, D376A and D464A resulted in complete loss of CPTII
activity, suggesting that these residues may be required for catalysis.
However, although chemical modification of P.
pastoris–expressed CPTII with the
histidine-specific reagent diethylpyrocarbonate resulted in >90%
loss of CPTII activity, it did not have any effect on the malonyl
CoA-sensitive L-CPTI and M-CPTI activity, suggesting that the
histidine residue in CPTI (H473) that corresponds to H372 of CPTII is
not essential for catalysis and/or may not be accessible to the reagent
(Shi and Woldegiorgis, unpublished).
L-CPTI.
The cDNAs for rat and human L-CPTI have been cloned and sequenced
(Britton et al. 1995
, Esser et al. 1993
).
The cDNAs predicted proteins of 773 amino acid residues (88 kDa) with
an overall identity of ~30% to CPTII. The rat and the human
L-CPT showed an 82 and 88% identity at the amino acid and
nucleotide levels, respectively. We were the first to report high level
expression of rat L-CPTI cDNA in P. pastoris,
a yeast with no endogenous CPT activity, and to demonstrate
conclusively that L-CPTI is a catalytically active, malonyl
CoA-sensitive, distinct enzyme that is reversibly inactivated by
detergents (de Vries et al. 1997
). Previous reports on the expression
of the L-CPTI cDNA concerned results either in COS cells
(Esser et al. 1993
), which have endogenous CPT activity,
or in S. cerevisiae (Brown et al. 1994b
). In the latter, the expression levels were too
low to study the enzyme and reach a definite conclusion whether CPTI is
a distinct, catalytically active, malonyl CoA-sensitive enzyme or a
regulatory protein that confers malonyl CoA sensitivity to CPTII.
P. pastoris–expressed L-CPTI was
localized to the mitochondria of the L-CPTI expression strain.
Immunoblots with L-CPTI–specific C-terminal polyclonal antibodies
showed the presence of an 88-kDa protein species corresponding to
L-CPTI in the mitochondria of the strain that expressed L-CPTI
and rat liver mitochondria (RLM), but not in the control strain with
the vector but without the L-CPTI cDNA insert. After our successful
expression of the L-CPTI cDNA in the yeast P.
pastoris, an improved method for expression of
L-CPTI cDNA in S. cerevisiae was
reported recently (Prip-Buus et al. 1998
). Furthermore,
the yeast-expressed L-CPTI, like RLM CPTI, is located in the outer
mitochondrial membrane of the expression strain.
Kinetic analysis of the P. pastoris,
S. cerevisiae and RLM L-CPTI showed
similar calculated Km for carnitine, but the
Vmax for the yeast-expressed L-CPTI were
higher than the corresponding RLM L-CPTI. The yeast-expressed
L-CPTI showed nonsaturable kinetics when palmitoyl CoA was varied
relative to a constant second substrate, i.e., carnitine at high
albumin concentration (1% wt/v). Normal saturation kinetics were
obtained when the fixed molar ratio of palmitoyl CoA to albumin was
raised from 0.53:1 to 6.1:1, suggesting that the decrease in substrate
availability due to the high albumin concentration was the cause of the
nonsaturation kinetic behavior observed. The calculated
Km and Vmax for
palmitoyl CoA were similar for the two yeast-expressed L-CPTI, but
were higher compared with the values obtained for RLM L-CPTI. This
could be due to the differences in the membrane lipid environment or to
one or more protein-protein interactions that occur in RLM but not
in yeast, and/or to higher expression levels in yeast.
Yeast-expressed L-CPTI is malonyl CoA sensitive, detergent
inactivated and reconstitutable (de Vries et al. 1997
).
Heart M-CPTI.
The rat brown adipose tissue (Yamazaki et al. 1995
) and
the human M-CPTI cDNAs have been cloned and sequenced (Adams et al. 1996
, Yamazaki et al. 1996
, Zhu et al. 1997b
). These cDNAs encode a protein of 772 amino acid
residues (88 kDa) with 62% identity to the rat L-CPTI at the amino
acid level. In human or rat tissues, M-CPTI is expressed only in
heart, skeletal muscle and testis. The genomic DNA for both the human
M-CPTI and the rat M-CPTI have also been isolated and
characterized (Adams et al. 1996
, Wang et al. 1998
). The genes for human muscle M-CPTI and L-CPTI are
located on chromosomes 22q and 11q, respectively (Adams et al. 1996
, Britton et al. 1995
). Thus, human liver
and skeletal muscle CPTI are different proteins encoded by separate
genes. Expression of the human M-CPTI cDNA in P.
pastoris, a yeast with no endogenous CPT activity, produced
an 80-kDa protein that was located in the mitochondria (Zhu et
al. 1997b). Isolated mitochondria from the M-CPTI
expression strain exhibited malonyl CoA-sensitive CPT activity that
was detergent labile. Furthermore, yeast-expressed M-CPTI had a
high Km for carnitine and low
I50 for malonyl CoA inhibition, characteristics similar to
those observed with rat heart mitochondrial CPTI, but different from
those for the yeast-expressed L-CPTI. This is the first report of
the expression of human heart M-CPTI in a system devoid of
endogenous CPT activity and the functional characterization of human
heart M-CPTI in the absence of the liver isoform and CPTII.
However, the level of expression of human heart M-CPTI was lower
than that obtained with rat L-CPTI. As a result, we were unable to
demonstrate restoration of malonyl CoA-sensitive CPT activity in
detergent-inactivated M-CPTI by reconstitution.
A high level of expression of human heart M-CPTI was obtained using
a multicopy P. pastoris expression strain
containing ~24 copies of the expression vector integrated into its
genome (Zhu et al. 1997a
). Levels of M-CPTI activity
were >10-fold higher than previously reported by us with a
single-copy strain and were sufficient to perform reconstitution
studies on the membrane protein, a key step in purification and
structural analysis of the enzyme. M-CPTI in the multicopy strain
was malonyl CoA sensitive and detergent labile. Immunoblots with the
M-CPTI–specific polyclonal antibodies showed the presence of an 80-kDa
protein species corresponding to M-CPTI in the mitochondria of the
multicopy transformant strain that expressed M-CPTI, but not in the
non-CPTI cDNA containing the control strain. The rat heart cDNA for
M-CPTI was also expressed in COS cells, which have endogenous
L-CPTI activity that may be induced (Esser et al. 1996
). Because of the high endogenous L-CPTI present in COS
cells, it was not possible to determine the I50 for malonyl
CoA inhibition of M-CPTI or the Km for
the substrates.
Reconstitution of yeast-expressed L- and M-CPTI.
Previous studies from our laboratory and others have demonstrated that
detergent solubilization of RLM and rat heart mitochondria, but not
CPTII, abolishes malonyl CoA-sensitive CPT activity, but not
malonyl CoA binding (Bremer et al. 1985
, Chung et al. 1992
, Lund and Woldegiorgis 1987
,
McGarry et al. 1989
, Woldegiorgis et al. 1985
and 1992
). Until recently, it was presumed that CPTI was
irreversibly inactivated by detergents and hence not recoverable as an
active enzyme; however, our reconstitution studies with
yeast-expressed L-CPTI and M-CPTI established for the first
time that detergent inactivation of CPTI is reversible (de Vries et al. 1997
, Zhu et al. 1997a
). Removal of CPTI from its membrane
environment inactivates the enzyme, and reconstitution via detergent
removal in the presence of phospholipids reactivates the enzyme,
suggesting that CPTI is active only in a membrane environment. The
ability to reconstitute yeast-expressed CPTI is critical to its
purification and further studies on the structure and function of this
important enzyme. Thus, P. pastoris
appears to be an excellent model system with which to investigate the
CPT enzymes.
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Structure-function studies |
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TOPABSTRACTINTRODUCTIONThe role of the...Cloning and expression of...Structure-function studiesREFERENCES |
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, Zhu et al. 1997a
and 1997b
). Amino acid sequence alignment of both human and rat
heart M-CPTI, L-CPTI and CPTII reveals the presence of a conserved
N-terminal sequence of 124 residues with two putative transmembrane
domains in all CPTI, which is absent from CPTII, the malonyl
CoA-insensitive enzyme (Fig. 3
). It has been hypothesized that malonyl CoA sensitivity of CPTI may
reside in these 124 N-terminal amino acid residues. By
systematically constructing a series of deletion mutants to the
N-terminus of L-CPTI, we showed that residues necessary for
malonyl CoA inhibition and binding are located within the conserved
first 18 N-terminal amino acids (Shi et al. 1998
).
To identify specific residue(s) involved in malonyl CoA binding and
inhibition of L-CPTI, we constructed two more deletion mutants 
12
and 6, that resulted in loss of malonyl CoA inhibition and binding
similar to that of the 18 mutant.
|
). A mutant L-CPTI with a
change of histidine 5 to alanine caused partial loss of malonyl CoA
inhibition, whereas a mutant L-CPTI with a change of glutamine 6 to
alanine had wild-type properties. Our results demonstrate that
glutamic acid 3 and histidine 5 are necessary for malonyl CoA
inhibition and binding to L-CPTI, but not catalytic activity
(Shi et al. 1999
). Similar deletion mutation analysis with human M-CPTI, which is 30-fold more sensitive to malonyl CoA inhibition, revealed loss of malonyl CoA inhibition and binding when the first 28 N-terminal residues were deleted (Shi et al., unpublished data). Unlike L-CPTI, deletion of the conserved first 18 N-terminal residues of the heart enzyme causes only moderate loss in malonyl CoA inhibition. To identify the specific residue(s) in this region of M-CPTI that are necessary in malonyl CoA inhibition and binding, we are currently constructing and testing substitution mutations of the N-terminal 19–28 amino acids in M-CPTI.
The N-terminal domain of L-CPTI (residues 1–150), which
contains two transmembrane segments, was recently shown to contain all
of the information required for mitochondrial targeting and insertion
into the outer mitochondrial membrane (Cohen et al. 1998
). When the N-terminal domain of L-CPTI was fused
to the N-terminal of malonyl CoA-insensitive CPTII, a
membrane-associated protein, the active chimeric CPTII was anchored
at the outer mitochondrial membrane but was insensitive to malonyl CoA
inhibition, indicating that malonyl CoA sensitivity is an intrinsic
property of L-CPTI and that its N-terminal domain cannot confer
malonyl CoA sensitivity to CPTII. Similarly, replacement of the
N-terminal domain of L-CPTI with the N-terminal domain of
M-CPTI and vice versa does not change the malonyl CoA sensitivity
of the chimeric L-CPTI or M-CPTI, suggesting that the amino
acid residues responsible for the differing sensitivity to malonyl CoA
are not located in this N-terminal region (Shi et al. unpublished
data, Swanson et al. 1998
). Replacement of the
N-terminal domain of L-CPTI by a specific outer mitochondrial
membrane signal anchor sequence (Cohen et al. 1998
) or
removal of the two transmembrane domains and the linker region (Zhu et
al., unpublished data) results in a less active protein that is malonyl
CoA insensitive. Thus, the N-terminal domain of L-CPTI is
essential to maintain an optimal conformation for both catalytic
function and malonyl CoA sensitivity. Future structure-function
studies of the CPT system should unravel the molecular architecture of
this important enzyme system in fatty acid metabolism.
In summary, our functional high level expression of rat L-CPTI and human M-CPTI in P. pastoris, a yeast with no endogenous CPT activity, establishes that CPTI is a catalytically active, malonyl CoA-sensitive, distinct enzyme that is reversibly inactivated by detergents. Our site-directed mutagenesis studies with the P. pastoris–expressed rat L-CPTI demonstrate for the first time that glutamic acid 3 and histidine 5 on the N-terminal domain of L-CPTI are necessary for malonyl CoA inhibition and binding to L-CPTI, but not for catalysis.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Supported by a grant from the National Institutes of Health (HL52571).
4 Abbreviations used: CPT, carnitine palmitoyltransferase; PPAR, peroxisome proliferator-activated receptor; RLM, rat liver mitochondria(l).
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