STRUCTURAL DETERMINANTS FOR THE INTRACELLULAR-LOCALIZATION OF THE ISOZYMES OF MAMMALIAN HEXOKINASE - INTRACELLULAR-LOCALIZATION OF FUSION CONSTRUCTS INCORPORATING STRUCTURAL ELEMENTS FROM THE HEXOKINASE ISOZYMES AND THE GREEN FLUORESCENT PROTEIN

Fusion constructs incorporating structural elements from mammalian iso zymes of hexokinase, Types I-IV, in frame with sequence encoding the g reen fluorescent protein (GFP) have been made and expressed in hexokin ase-deficient M + R 42 cells. Fusion proteins incorporating catalytica lly active regions from the Type II isozyme, or the entire Type IV seq uence, were expressed in catalytically active form. The intracellular localization of the fusion proteins was determined using confocal micr oscopy. Fusion proteins including the N-terminal halves of the Type I or Type II isozymes were targeted to mitochondria, while the N-termina l half of the Type III isozyme did not confer mitochondrial targeting. The mitochondrial targeting signal was represented by the hydrophobic sequence at the extreme N-termini (''binding domain'') of the Type I and Type II isozymes. Inclusion of the binding domain from the Type I isozyme was sufficient to confer mitochondrial binding on GFP itself a s well as on constructs including the N-terminal half of Type III hexo kinase. However, the Type I hexokinase binding domain was not sufficie nt to cause mitochondrial targeting of a construct containing the Type IV sequence. These results suggest that, although the binding domain is critical for mitochondrial targeting, other interactions involving an adjacent structure might also play a role. Fusion proteins includin g the N-terminal half of Type I hexokinase became dissociated from mit ochondria under conditions favorable for accumulation of intracellular Glc-6-P. The 2-deoxy analog was much less effective than Glc in causi ng mitochondrial dissociation of the fusion construct, in accord with previous studies showing 2-deoxy-Glc-6-P to be much less effective tha n Glc-6-P at promoting release of Type I hexokinase from mitochondria. Dissociation, induced by formation of Glc-6-P or 2-deoxy-Glc-6-P, did not occur with the fusion protein including only the binding domain o f Type I hexokinase. This is consistent with previous studies indicati ng that Glc-6-P-dependent dissociation results from binding of this li gand to a site in the N-terminal half of the enzyme, but which is not likely to be present in the small segment represented by the binding d omain. These studies demonstrate the usefulness of this approach in de fining structural elements involved in targeting hexokinase isozymes t o specific subcellular locations and modulation of that intracellular location by perturbations of metabolic status. (C) 1997 Academic Press .