THE SOLUBLE BUT NOT MITOCHONDRIALLY BOUND HEXOKINASE IS A SUBSTRATE FOR THE ATP-DEPENDENT AND UBIQUITIN-DEPENDENT PROTEOLYTIC SYSTEM

Intracellular protein degradation is highly selective, however, the me chanism(s) underlying this selectivity are not fully understood. We ha ve previously shown that purified rabbit hexokinase type I, an enzyme present in mammalian brain both in soluble and mitochondrial bound for m, is conjugate to ubiquitin and then degraded by a rabbit reticulocyt e fraction II. In the present study we report that the mitochondrial b ound hexokinase is stable for several hours in the same proteolytic sy stem both in the presence or absence of ATP. E(1), E(2) and E(3), the enzymes of the ubiquitin conjugating system, are able to incorporate I -125- Or biotin-labelled ubiquitin in an ATP-dependent manner in solub le hexokinase as well as in a number of mitochondrial proteins. Furthe rmore, the mitochondria by themselves have a pronounced ATP-dependent ability to conjugate I-125-ubiquitin, However, Western blotting experi ments, using a specific antibody against hexokinase, or against ubiqui tin, showed that the mitochondrial bound enzyme is neither ubiquitinat ed nor degraded. This result has been confirmed by purification of bou nd hexokinase from the brain mitochondrial fraction or following the i ncubation of intact mitochondria with ATP, I-125-ubiquitin and E(1), E (2) and E(3). Thus, mitochondrial bound hexokinase is not recognized b y the ubiquitin conjugating system while the soluble enzyme is conjuga te to ubiquitin and then degraded. Furthermore, the soluble hexokinase from rabbit brain was isolated by immunoaffinity chromatography and s hown to be recognized by an anti-ubiquitin antibody. These results sug gest that the intracellular distribution of protein is an important fe ature of a protein which determines its susceptibility to ubiquitin-de pendent degradation.