Localization to the proteasome is sufficient for degradation.
The majority of unstable proteins in eukaryotic cells are targeted for degradation through the ubiquitin-proteasome pathway. Substrates for degradation are recognized by the E1, E2, and E3 ubiquitin conjugation machinery and tagged with polyubiquitin chains, which are thought to promote the proteolytic process through their binding with the proteasome. We describe a method to bypass the ubiquitination step artificially both in vivo and in a purified in vitro system. Seven proteasome subunits were tagged with Fpr1, and fusion reporter constructs were created with the Fpr1-rapamycin binding domain of Tor1. Reporter proteins were localized to the proteasome by the addition of rapamycin, a drug that heterodimerizes Fpr1 and Tor1. Degradation of reporter proteins was observed with proteasomes that had either Rpn10 or Pre10 subunits tagged with Fpr1. Our experiments resolved a simple but central problem concerning the design of the ubiquitin-proteasome pathway. We conclude that localization to the proteasome is sufficient for degradation and, therefore, any added functions polyubiquitin chains possess beyond tethering substrates to the proteasome are not strictly necessary for proteolysis.
Janse
Daniel M
DM
Department of Genetics, Harvard Medical School, Boston, MA 02115, USA.
Crosas
Bernat
B
Finley
Daniel
D
Church
George M
GM
D003001:Cloning, Molecular; D003546:Cysteine Endopeptidases / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D009097:Multienzyme Complexes / Q000737:chemistry / Q000378:metabolism*; D009955:Ornithine Decarboxylase / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex; D011506:Proteins / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D025801:Ubiquitin / Q000737:chemistry / Q000378:metabolism*
D009097:Multienzyme Complexes; D011506:Proteins; D011994:Recombinant Proteins; D025801:Ubiquitin; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; D009955:Ornithine Decarboxylase