Journal of Bioenergetics and Biomembranes, Vol. 29, No. 6, 1997 Localized Firefly Luciferase Probes ATP at the Surface of Mitochondria Claude Aflalo1 Received July 14, 1997 The concentration of ATP generated by yeast mitochondria and consumed by yeast hexokinase was monitored using native firefly luciferase in solution, or recombinant luciferase localized at the surface of mitochondria. In the absence of hexokinase, both probes perform similarly in detecting exogenous or mitochondrially-generated ATP. The steady-state concentrations of ATP can be reduced in a dose-dependent manner by hexokinase. With hexokinase added in large excess, the localized probe reports substantial ATP concentrations while none is detectable by soluble luciferase. Thus, ATP accumulates near the membrane where it appears, relatively to solution, and vice versa for ADP. The extent of nucleotide gradients is shown to be correlated with the specific activity of oxidative phosphorylation and with the viscosity of the medium, but independent of the concentration of the organelles. A simple model involving diffusional restrictions is presented to describe this behavior. The metabolic and evolutionary implications of cellular catalysis limitation by physical processes are discussed. INTRODUCTION Cellular metabolic processes in situ are highly coordinated through elaborate control mechanisms. In the standard analysis of cellular catalysis, homogene- ity of the internal environment is often implicitly assumed, in flagrant contradiction with the high degree of organization in cells (Clegg, 1984; Porter, 1987). Thus, metabolites, effectors, as well as cyto- plasmic enzymes and organelles are often treated as rapidly diffusible, homogeneously distributed compo- nents. Such an approximation precludes accounting for essential means of metabolic control via a dynamic (re)arrangement of catalytic units achieved through specific macromolecular interactions (Keleti et al., 1988; Masters, 1981; Srere, 1987). A critical appraisal of the role for enzyme location and the involvement of physical processes in cellular metabo- lism requires the assessment of local concentrations of metabolites in the intricate intracellular milieu. Hence, localized processes could only be assessed through indirect experimental evidence. Measurements with localized probes (Aflalo, 1991; Aflalo and DeLuca, 1987; Jones and Aw, 1990) should therefore con- tribute exclusive information on cryptic microscopic events, as opposed to the more readily assessed macro- scopic fluxes, and help to better understand the struc- tural basis of cellular function. Soluble firefly luciferase (FL2) can specifically monitor ATP in solution with a high sensitivity (DeLuca and McElroy, 1978). Thus, light emission from a localized enzyme should report the concentra- tion of ATP in its immediate vicinity. The first direct measurements of local [ATP] were conducted in arti- ficial model systems, with FL covalently immobilized within Sepharose beads, in the presence of coimmobi- 1 Department of Life Sciences, The Ben Gurion University of the Negev, P.O. Box 653, Beer Sheva 84105, Israel. 2 Abbreviations: BSA: bovine serum albumin; DTT: dithiothreitol; EDTA: ethylene diamine tetraacetic acid; FL: firefly luciferase; G6PDH: glucose-6-phosphate dehydrogenase; Hepes: 2-[4-(2- hydroxyethyl-1-piperazine)] ethanesulfonic acid; HK: hexoki- nase; IU: international unit of enzyme activity (= 1 umol product/min); PK: pyruvate kinase; PMSF: phenylmethylsul- fonyl fluoride; WT: wild type (mitochondria). 549 0145-479X/97/1200-0549$12.0/ C Plenum Publishing Corporation KEY WORDS: Luciferase; localized probe; heterogeneous coupled systems; mitochondria; hexokinase; nucleotide concentration gradients; cellular catalysis.