The soluble but not mitochondrially bound hexokinase is a substrate for the ATP- and ubiquitin-dependent proteolytic system.
Intracellular protein degradation is highly selective, however, the mechanism(s) underlying this selectivity are not fully understood. We have previously shown that purified rabbit hexokinase type I, an enzyme present in mammalian brain both in soluble and mitochondrial bound form, is conjugate to ubiquitin and then degraded by a rabbit reticulocyte fraction II. In the present study we report that the mitochondrial bound hexokinase is stable for several hours in the same proteolytic system both in the presence or absence of ATP. E1, E2 and E3, the enzymes of the ubiquitin conjugating system, are able to incorporate 125I- or biotin-labelled ubiquitin in an ATP-dependent manner in soluble hexokinase as well as in a number of mitochondrial proteins. Furthermore, the mitochondria by themselves have a pronounced ATP-dependent ability to conjugate 125I-ubiquitin. However, Western blotting experiments, using a specific antibody against hexokinase, or against ubiquitin, showed that the mitochondrial bound enzyme is neither ubiquitinated nor degraded. This result has been confirmed by purification of bound hexokinase from the brain mitochondrial fraction or following the incubation of intact mitochondria with ATP, 125I-ubiquitin and E1, E2 and E3. Thus, mitochondrial bound hexokinase is not recognized by the ubiquitin conjugating system while the soluble enzyme is conjugate to ubiquitin and then degraded. Furthermore, the soluble hexokinase from rabbit brain was isolated by immunoaffinity chromatography and shown to be recognized by an anti-ubiquitin antibody. These results suggest that the intracellular distribution of protein is an important feature of a protein which determines its susceptibility to ubiquitin-dependent degradation.
Magnani
M
M
Istituto di Chimica Biologica, G. Fornaini, Università degli Studi, Urbino, Italy.
Crinelli
R
R
Antonelli
A
A
Casabianca
A
A
Serafini
G
G
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001921:Brain / Q000201:enzymology*; D002918:Chymotrypsin; D010450:Endopeptidases / Q000378:metabolism*; D006593:Hexokinase / Q000378:metabolism*; D008928:Mitochondria / Q000378:metabolism; D011817:Rabbits; D012156:Reticulocytes / Q000378:metabolism; D014452:Ubiquitins / Q000378:metabolism
D014452:Ubiquitins; D000255:Adenosine Triphosphate; D006593:Hexokinase; C080306:ATP-ubiquitin-dependent proteinase; D010450:Endopeptidases; D002918:Chymotrypsin