Dual cytosolic/peroxisomal localization and function of NADP-specific isocitrate dehydrogenase from various species in yeast

NADP+-dependent isocitrate dehydrogenases (IDPs) provide NADPH, the essential cofactor for many enzymatic reactions in biosynthetic pathways and in thiol-dependent antioxidant systems. Saccharomyces cerevisiae cells have three similar but genetically distinct IDP isozymes in mitochondria, the cytosol, and peroxisomes. Mammalian cells have a mitochondrial isozyme (mamIDP1) and another genetically distinct isozyme (mamIDP2) that partitions between the cytosol and peroxisome. Goals of this dissertation were (a) to elucidate kinetic and other properties that may have evolved to maximize isozyme function in various cellular compartments and (b) to evaluate mechanisms for localization and function of peroxisomal isozymes.;As described in Chapter 2, analyses of affinity purified IDP isozymes from yeast and from mouse suggested a general correspondence between pH optima for catalysis and pI values with the pH values reported for resident cellular compartments. However, mamIDP2, which partitions between cytosolic and peroxisomal compartments in mammalian cells, exhibited a broad pH optimum and an intermediate pI value, suggesting an adaptation for function in both compartments. To study cellular influences on the dual distribution of the mammalian isozyme, mamIDP2 was expressed in yeast at levels comparable to those of endogenous yeast isozymes. MamIDP2 was found to localize in both the cytosol and peroxisomes in yeast cells, and complementation tests conducted with appropriate yeast mutants indicated that the mammalian enzyme was functional in both cellular compartments. The import of mamIDP2 into yeast peroxisomes increased significantly in response to use as a carbon source of an unsaturated fatty acid that requires a peroxisomal IDP activity for beta-oxidation. Since removal of the peroxisomal targeting signal of the mouse enzyme precluded localization in peroxisomes, an initial conclusion was that the mamIDP2 enzyme might normally contain a "cytosolic retention signal" to ensure partial localization in the cytosol.;As described in Chapter 3, to examine other requirements for peroxisomal localization and function of IDP, the peroxisomal targeting sequence from yeast IDP3 (a carboxyl-terminal Cys-Lys-Leu tripeptide) was attached to normally cytosolic IDP2. This IDP2CKL enzyme was only partially localized in peroxisomes and was able to function in lieu of either IDP2 or IDP3. In addition, the analogous IDP enzyme (IDPA) from Aspergillus nidulans was found to exhibit patterns of dual compartmental distribution and of dual function in yeast, similar to those observed for IDP2+CKL and for mamIDP2. These observations suggested that, rather than a cytosolic retention signal, there might be some limit to amounts of an IDP enzyme in peroxisomes. To test this, yeast peroxisomal IDP3, which is normally only located in peroxisomes, was over expressed. This also resulted in dual distribution and function of the enzyme in both the cytosol and in peroxisomes, supporting the possibility of a restriction on organellar amounts of IDP.;Since mamIDP2 can function in both peroxisomes and the cytosol in yeast cells, the enzyme in mammalian cells likely supplies NADPH for peroxisomal beta-oxidation (and other peroxisomal processes) and for cytosolic thiol-based antioxidant systems. We hypothesized that changes in environmental conditions impacting these functions might elicit changes in compartmental distribution patterns for mamIDP2, and examined this possibility using several cell lines as described in Chapter 4. We found that cellular levels and compartmental distribution patterns of mamIDP2 varied among cell lines. However, we were unable to find any effect upon levels or compartmental distribution of IDP2 as a function of addition of different fatty acids or of H2O2 to the medium. The only condition that caused a compartmental redistribution of IDP2 was a substantial reduction or removal of serum (normally 10%) from the medium, suggesting that factors in the serum may contribute to compartmentalization patterns.