Ubiquitin-independent mechanisms of mouse ornithine decarboxylase degradation are conserved between mammalian and fungal cells.
The polyamine biosynthetic enzyme ornithine decarboxylase (ODC) is degraded by the 26 S proteasome via a ubiquitin-independent pathway in mammalian cells. Its degradation is greatly accelerated by association with the polyamine-induced regulatory protein antizyme 1 (AZ1). Mouse ODC (mODC) that is expressed in the yeast Saccharomyces cerevisiae is also rapidly degraded by the proteasome of that organism. We have now carried out in vivo and in vitro studies to determine whether S. cerevisiae proteasomes recognize mODC degradation signals. Mutations of mODC that stabilized the protein in animal cells also did so in the fungus. Moreover, the mODC degradation signal was able to destabilize a GFP or Ura3 reporter in GFP-mODC and Ura3-mODC fusion proteins. Co-expression of AZ1 accelerated mODC degradation 2-3-fold in yeast cells. The degradation of both mODC and the endogenous yeast ODC (yODC) was unaffected in S. cerevisiae mutants with various defects in ubiquitin metabolism, and ubiquitinylated forms of mODC were not detected in yeast cells. In addition, recombinant mODC was degraded in an ATP-dependent manner by affinity-purified yeast 26 S proteasomes in the absence of ubiquitin. Degradation by purified yeast proteasomes was sensitive to mutations that stabilized mODC in vivo, but was not accelerated by recombinant AZ1. These studies demonstrate that cell constituents required for mODC degradation are conserved between animals and fungi, and that both mammalian and fungal ODC are subject to proteasome-mediated proteolysis by ubiquitin-independent mechanisms.
Hoyt
Martin A
MA
Department of Microbiology and Immunology, University of California, San Francisco, California 94143-0414, USA.
Zhang
Mingsheng
M
Coffino
Philip
P
D000818:Animals; D002478:Cells, Cultured; D003546:Cysteine Endopeptidases / Q000378:metabolism; D019143:Evolution, Molecular; D015971:Gene Expression Regulation, Enzymologic; D008322:Mammals; D051379:Mice; D009097:Multienzyme Complexes / Q000378:metabolism; D009955:Ornithine Decarboxylase / Q000235:genetics / Q000378:metabolism*; D011073:Polyamines / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex; D011506:Proteins / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D025801:Ubiquitin / Q000378:metabolism
D009097:Multienzyme Complexes; D011073:Polyamines; D011506:Proteins; D011993:Recombinant Fusion Proteins; D025801:Ubiquitin; C031017:ornithine decarboxylase antizyme; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; D009955:Ornithine Decarboxylase