Journal of Bioenergetics and Biomembranes, Vol. 30, No. 3, 1998 Binding of Rat Brain Hexokinase to Recombinant Yeast Mitochondria: Effect of Environmental Factors and the Source of Porin1 Claude Aflalo2 and Heftsiba Azoulay2 Received October 14, 1997; accepted February 9, 1998 Heterologous binding of rat brain hexokinase to wild type, porinless, and recombinant yeast mitochondria expressing human porin was assessed, partially characterized, and compared to that in the homologous system (rat liver mitochondria). With porin-containing yeast mitochon- dria it is shown that (i) a significant, saturatable association occurs; (ii) its extent and apparent affinity, correlated with the origin of porin, are enhanced in the presence of dextran; (iii) the binding requires Mg ions and apparently follows a complex cooperative mechanism. This heterologous association does not seem to differ fundamentally from that in the homologous system and represents a good basis for molecular studies in yeast. With porinless yeast mitochondria, binding occurs at much lower affinity, but to many more sites per mitochondrion. The results indicating a major but not exclusive role for porin in the binding are discussed in terms of (i) the mode and mechanism of binding, and (ii) the suitability of the rat hexokinase- yeast mitochondria couple for the study of heterogeneous catalysis in reconstituted cellular model systems. KEY WORDS: Mitochondria; rat brain hexokinase; porin; VDAC; yeast; cooperative binding. INTRODUCTION Structural organization plays an important role in the control of metabolism. Biological catalytic systems evolved to either overcome—or take advantage of— physical limitations imposed by the intricate and con- centrated cellular environment. The binding of hexokinase to mitochondria in mammalian brain repre- sents a classical example for dynamic cellular organi- zation and rearrangement according to metabolic needs (Brdiczka, 1994; Bustamente et al., 1981; Inui and Ishibashi, 1979; McCabe, 1994). It is commonly accepted that bound hexokinase has exclusive access to mitochondrial ATP, and may in turn efficiently recycle limiting ADP to oxidative phosphorylation in the 1 Dedicated to the memory of Prof. Noun Shavit (deceased June 19. 1997). 2 Department of Life Sciences, The Ben Gurion University of the Negev, P.O. Box 653, Beer Sheva 84105, Israel. matrix (Arora and Pedersen, 1988; Beltran del Rio and Wilson, 1992;Laterveer et al., 1994; Saks et al., 1993). The reversible association between homologous puri- fied components has been demonstrated and character- ized in vitro (Gellerich et al., 1993; Kabir and Wilson, 1993; Polakis and Wilson, 1985; White and Wilson, 1989; Wicker et al., 1993; Xie and Wilson, 1988). Rat brain hexokinase (type I, 110 kDa) is com- posed of two distinct homologous domains, both struc- turally similar to yeast hexokinase (55 kDa), with the catalytic activity confined to the C-terminal domain (White and Wilson, 1989). Unlike in yeast, the mam- malian enzyme is sensitive to allosteric inhibition by glucose-6-phosphate, and binds to mitochondria. The current model (Wilson, 1995) for its association with the outer membrane involves primarily an N-terminal hydrophobic peptide inserted in the membrane (Xie and Wilson, 1988), oligomerization of the enzyme (Xie and Wilson, 1990), and stabilization through Mg bridges between the N-terminal domain and the nega- 245 0145-479X/98/0600-0245$15.00/0 C 1998 Plenum Publishing Corporation