In this report, we demonstrate that mammalian cytosolic/peroxisomal NADP+
-specific isocitrate dehydrogenase (mamIDP2) is also localized in both cellular compartments when expressed in yeast cells. Furthermore, when expressed at levels comparable to the endogenous yeast enzymes, mamIDP2 can compensate for the absence of yeast IDP2 by permitting growth of an idp2Δidp3Δ zwf1Δ
yeast strain in medium with a fatty acid carbon source. Thus, the mammalian enzyme can produce sufficient NADPH for cytosolic antioxidant systems that detoxify the hydrogen peroxide produced during peroxisomal β-oxidation [9
]. The mamIDP2 enzyme can also compensate for loss of yeast IDP3 by permitting growth of an idp3Δ
yeast strain (and of the idp2Δidp3Δ zwf1Δ
strain) on carbon sources like petroselinate, i.e. on fatty acids with a double bond following an even-numbered carbon atom. Thus, partial peroxisomal localization of mamIDP2 results in production of sufficient NADPH for reduction of such double bonds during β-oxidation [5
In contrast, expression of the mamIDP2ΔAKL
enzyme in yeast at levels of the endogenous yeast eliminates peroxisomal localization. The cytosolic mamIDP2ΔAKL
enzyme, as might be predicted, can compensate for loss of yeast cytosolic IDP2 (e.g. for growth with oleate as the carbon source) but not for loss of peroxisomal IDP3 (e.g. for growth with petroselinate). We note that previous studies of complementation by the mammalian enzyme in yeast [12
] suggested that an enzyme lacking the peroxisomal targeting signal could still localize to peroxisomes and functionally substitute for yeast IDP3. However, those studies utilized multicopy vectors, and subsequent high levels of the heterologous enzyme presumably overwhelmed the peroxisomal import system. Thus, studies of compartmentalization and function in vivo
require near normal levels of expression of a catalytically competent heterologous enzyme as reported here.
An important outcome of the current study is evidence that, while the peroxisomal targeting sequence of mamIDP2 is necessary for peroxisomal localization in yeast cells (as it is in mammalian cells [13
]), this sequence does not ensure such localization. In other words, a substantial proportion of the enzyme is localized in the cytosol despite the presence of the carboxyl-terminal tripeptide signal. This suggests that some inherent property of the enzyme, potentially a cytosolic retention element, may prevent complete peroxisomal import both in mammalian and in yeast cells. This property will be examined in future experiments using hybrid fusion proteins.
The yeast expression system described here will be appropriate for assessing environmental and cellular factors that influence the distribution of mamIDP2 between cytosolic and peroxisomal locations. For example, we have some preliminary evidence that growth with a carbon source (petroselinate) that requires a functional peroxisomal activity may produce an increase in the proportion of mamIDP2 localized in the peroxisomes relative to growth with a carbon source (ethanol) that does not require a peroxisomal activity. Also, while the dual compartmental localization of mamIDP2 is unusual, at least one other human antioxidant enzyme (PMP20) is similarly located in the cytosol and in peroxisomes [33
]. It will be of interest to determine if similar factors affect distribution of both enzymes in yeast, and known mutant strains with defects in peroxisomal import and function [34
] can be applied to assess factors involved in the dual compartmental localization of these mammalian enzymes. Finally, while previous studies have demonstrated that levels of mamIDP2 vary in a tissue-specific manner [19
] and are dramatically elevated under certain conditions, e.g. in lactating bovine mammary gland [36
] and in rat ovary during gonadotropin-induced development [30
], nothing is known about differential compartmental localization of the enzyme under these conditions. Thus, our studies will be extended to mammalian cell lines and tissues.
A limitation of the yeast expression system is that mamIDP2 likely fulfills many cellular functions, particularly in peroxisomes, that may not be applicable in yeast cells. For example, while mammalian hepatocyte peroxisomes function in the β-oxidation of very long-chain fatty acids, and NADPH would be needed to reposition double bonds during β-oxidation of some of these fatty acids [37
], a peroxisomal source of NADPH is also required for steps in the biosynthesis of isoprenoids, ether phospholipids, and potentially cholesterol [39
]. With respect to antioxidant functions of cytosolically localized mamID2, most long- and short-chain fatty acids are degraded by β-oxidation in mammalian cell mitochondria without concomitant production of hydrogen peroxide [10
]. However, numerous mammalian peroxisomal oxidases produce hydrogen peroxide as a byproduct [37
]. Thus, while specific processes and reactions may not be evolutionarily conserved, basic requirements for both peroxisomal and cytosolic sources of NADPH appear to be common in eukaryotic cells. An obvious necessity for coordinate function of cytosolic and peroxisomal isocitrate dehydrogenases in fulfilling their cellular roles is an isocitrate/α-ketoglutarate shuttle system in the peroxisomal membrane to indirectly facilitate exchange of NADP(H). Such a system, a peroxisomal transporter for isocitrate and α-ketoglutarate, has recently been described for mammalian cells [18
] and is postulated to exist in lower eukaryotic cells as well [42
To our knowledge, the current report on kinetic properties is the first direct comparison of mammalian IDP1 and IDP2 enzymes from the same species. Despite some differences in pH optima and pI
values, the kinetic properties of the mouse IDP1 and IDP2 enzymes are quite similar. The mammalian IDP1 enzyme has been extensively characterized [22
], and our values for the mouse enzyme are similar to those reported for the purified recombinant human enzyme [44
]. In contrast, although the crystal structures of both mamIDP1 [45
] and mamIDP2 [46
] have been reported, there have been fewer kinetic studies of the mamIDP2 enzyme. Our values for the mouse IDP2 enzyme are quite similar to those previously reported for the rat IDP2 enzyme [47
]. However, they differ from those previously reported for the human enzyme [13
]. The latter report suggested substantially higher Km
values for isocitrate and NADP+
(76 μM and 112 μM, respectively) for the recombinant human enzyme than those we measured (~4 μM for both ligands) for the recombinant mouse IDP2. It seems unlikely that enzymes sharing identities of >95% would have such dissimilar properties. It is more likely that differences are due to affinity purification schemes (use of a maltose-binding fusion protein for the human enzyme versus
use of a histidine tag for the mouse enzyme) or to storage methods prior to kinetic analyses (ammonium precipitation of the human enzyme versus
short storage at 4°C for the mouse enzyme).
Our characterizations of purified yeast and mammalian IDPs are consistent with the concept that the enzymes have evolved for optimum function at least with respect to their cellular pH environments. The enzymes found in one cellular location (both mitochondrial IDPs, yeast peroxisomal IDP3, and yeast cytosolic IDP2) have pH optima for catalysis and pI
values in accordance with the pH value in each cellular compartment. The mamIDP2 enzyme, on the other hand, has a very broad neutral to basic pH optimum for catalysis and a pI
value intermediate to the pH values reported for the cytosol and peroxisomal matrix [31
]. In mammalian cells and in yeast cells, as shown in this study, the enzyme functions well in both cellular compartments. Interestingly, a previous study demonstrated that replacement of yeast cytosolic IDP2 with the yeast mitochondrial IDP1 or the peroxisomal IDP3 enzyme did not provide complementation for growth of an idp2Δzwf1Δ
strain with a fatty acid carbon source [48
]. This suggests that the mislocalized yeast isozymes may be compromised in vivo
, perhaps due to suboptimum catalytic pH values or pI
values for function in the cytosol. Clearly, it will be of interest to determine if yeast IDP2, which has a very narrow neutral pH optimum for catalysis and a slightly acidic pI
value, can be localized to peroxisomes and if it can fulfill the function of peroxisomal IDP3.
The existence of NADP-specific isocitrate dehydrogenase activity in multiple cellular compartments appears to be a general theme in eukaryotic organisms. However, as with yeast and mammalian cells, differential localization is accomplished by different mechanisms. In addition to proteins analogous to the three genetically distinct isozymes present in yeast cells, plants also have a chloroplast enzyme [49
]. In contrast, a single gene in Aspergillus sp.
encodes mitochondrial, cytosolic, and peroxisomal enzymes [50
]. In A. nidulans
, two different transcriptional start sites are used to produce a longer transcript encoding a protein with an amino-terminal mitochondrial targeting sequence or a shorter transcript lacking that sequence [50
]. The shorter transcript encodes a protein with a likely peroxisomal targeting signal (a carboxyl-terminal Ala-Arg-Leu tripeptide), and thus presumably is translated to produce both cytosolic and peroxisomal enzymes, as is the case for the non-mitochondrial mammalian isozyme. Our studies lead to a prediction that the genetically distinct enzymes have divergently evolved for function in their specific compartments, whereas enzymes like the A. nidulans
IDP may exhibit intermediate physical properties to permit function in different cellular environments.