Ubiquitination of soluble and membrane-bound tyrosine hydroxylase and degradation of the soluble form.
Tyrosine hydroxylase (TH) demonstrates by two-dimensional electrophoresis a microheterogeneity both as a soluble recombinant human TH (hTH1) and as a membrane-bound bovine TH (bTHmem). Part of the heterogeneity is likely due to deamidation of labile asparagine residues. Wild-type (wt)-hTH1 was found to be a substrate for the ubiquitin (Ub) conjugating enzyme system in a reconstituted in vitro system. When wt-hTH1 was expressed in a coupled transcription-translation TnT(R)-T7 reticulolysate system 35S-labelled polypeptides of the expected molecular mass of native enzyme as well as both higher and lower molecular mass forms were observed. The amount of high-molecular-mass forms increased by time and was enhanced in the presence of Ub and clasto-lactacystin beta-lactone. In pulse-chase experiments the amount of full-length hTH1 decreased by first-order kinetics with a half-time of 7.4 h and 2.1 h in the absence and presence of an ATP-regenerating system, respectively. The ATP-dependent degradation was inhibited by clasto-lactacystin beta-lactone. Our findings support the conclusion that hTH1 is ubiquitinated and at least partially degraded by the proteasomes in the reticulocyte lysate system. Finally, it is shown that the integral TH of the bovine adrenal chromaffin granule membrane (bTHmem) is ubiquitinated, most likely monoubiquitinated. Additional Ub-conjugates of this membrane, detected by Western blot analysis, have not yet been identified.
Døskeland
Anne P
AP
Department of Biochemistry and Molecular Biology, University of Bergen, Norway.
Flatmark
Torgeir
T
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D002417:Cattle; D002837:Chromaffin Granules / Q000378:metabolism; D015180:Electrophoresis, Gel, Two-Dimensional; D008565:Membrane Proteins / Q000378:metabolism; D051379:Mice; D011994:Recombinant Proteins / Q000378:metabolism; D012156:Reticulocytes / Q000378:metabolism; D014446:Tyrosine 3-Monooxygenase / Q000378:metabolism*; D025801:Ubiquitin / Q000378:metabolism*
D008565:Membrane Proteins; D011994:Recombinant Proteins; D025801:Ubiquitin; D000255:Adenosine Triphosphate; D014446:Tyrosine 3-Monooxygenase